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The Reconstruction of Life Processes requires both Genomics and Metabolomics to explain Phenotypes and Phylogenetics

Posted in Amino acids, Ca2+ triggered activation, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Curation, Cytoskeleton, De novo synthesis, Developmental biology, Dialectic, Discovery process, Disease Biology, Embryology, Epigenetics and Cardiovascular Risks, Evolutionary cognition, Explanatory, Gene Regulation and Evolution, Genome Biology, Historical relevance, Innovations in Neurophysiology & Neuropsychology, Metabolism, Metabolomics, Mutant Gene Expression, Na-K-ATPase, Nitric Oxide in Health and Disease, Nutrition, Pentose monophosphate shunt, Proteins, Proteomics, RNA Biology, Signaling, Signaling & Cell Circuits, tagged adaptiaton, Evolution, habitats, metabolites, oligomers, oligonucleotides, phenotypes, predators, protein conformation, protein interactions, protein synthesis, respiration, temperature, trrain, water salinity on February 25, 2015| Leave a Comment »

The Reconstruction of Life Processes requires both Genomics and Metabolomics to explain Phenotypes and Phylogenetics

Writer and Curator: Larry H. Bernstein, MD, FCAP

phylogenetics

http://upload.wikimedia.org/wikipedia/commons/thumb/1/12/CollapsedtreeLabels-simplified.svg/200px-CollapsedtreeLabels-simplified.svg.png

This discussion that completes and is an epicrisis (summary and critical evaluation) of the series of discussions that preceded it.

Innervation of Heart and Heart Rate
Action of hormones on the circulation
Allogeneic Transfusion Reactions
Graft-versus Host reaction
Unique problems of perinatal period
High altitude sickness
Deep water adaptation
Heart-Lung-and Kidney
Acute Lung Injury

The concept inherent in this series is that the genetic code is an imprint that is translated into a message. It is much the same as a blueprint, or a darkroom photographic image that has to be converted to a print. It is biologically an innovation of evolutionary nature because it establishes a simple and reproducible standard for the transcription of the message through the transcription of the message using strings of nucleotides (oligonucleotides) that systematically transfer the message through ribonucleotides that communicate in the cytoplasm with the cytoskeleton based endoplasmic reticulum (ER), composing a primary amino acid sequence. This process is a quite simple and convenient method of biological activity. However, the simplicity ends at this step. The metabolic components of the cell are organelles consisting of lipoprotein membranes and a cytosol which have particularly aligned active proteins, as in the inner membrane of the mitochondrion, or as in the liposome or phagosome, or the structure of the ER, each of which is critical for energy transduction and respiration, in particular, for the mitochondria, cellular remodeling or cell death, with respect to the phagosome, and construction of proteins with respect to the ER, and anaerobic glycolysis and the hexose monophosphate shunt in the cytoplasmic domain. All of this refers to structure and function, not to leave out the membrane assigned transport of inorganic, and organic ions (electrolytes and metabolites).

I have identified a specific role of the ER, the organelles, and cellular transactions within and between cells that is orchestrated. But what I have outlined is a somewhat limited and rigid model that does not reach into the dynamics of cellular transactions. The DNA has expression that may be old, no longer used messages, and this is perhaps only part of a significant portion of “dark matter”. There is also nuclear DNA that is enmeshed with protein, mRNA that is a copy of DNA, and mDNA is copied to ribosomal RNA (rRNA). There is also rDNA. The classic model is DNA to RNA to protein. However, there is also noncoding RNA, which plays an important role in regulation of transcription.

This has been discussed in other articles. But the important point is that proteins have secondary structure through disulfide bonds, which is determined by position of sulfur amino acids, and by van der Waal forces, attraction and repulsion. They have tertiary structure, which is critical for 3-D structure. When like subunits associate, or dissimilar oligomers, then you have heterodimers and oligomers. These constructs that have emerged over time interact with metabolites within the cell, and also have an important interaction with the extracellular environment.

When you take this into consideration then a more complete picture emerges. The primitive cell or the multicellular organism lives in an environment that has the following characteristics – air composition, water and salinity, natural habitat, temperature, exposure to radiation, availability of nutrients, and exposure to chemical toxins or to predators. In addition, there is a time dimension that proceeds from embryonic stage to birth in mammals, a rapid growth phase, a tapering, and a decline. The time span is determined by body size, fluidity of adaptation, and environmental factors. This is covered in great detail in this work. The last two pieces are in the writing stage that completes the series. Much content has already be presented in previous articles.

The function of the heart, kidneys and metabolism of stressful conditions have already been extensively covered in http://pharmaceuticalintelligence.com in the following and more:

The Amazing Structure and Adaptive Functioning of the Kidneys: Nitric Oxide – Part I

http://pharmaceuticalintelligence.com/2012/11/26/the-amazing-structure-and-adaptive-functioning-of-the-kidneys/

Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II

http://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-and-inos-have-key-roles-in-kidney-diseases/

The pathological role of IL-18Rα in renal ischemia/reperfusion injury – Nature.com

http://pharmaceuticalintelligence.com/2014/10/24/the-pathological-role-of-il-18r%CE%B1-in-renal-ischemiareperfusion-injury-nature-com/

Summary, Metabolic Pathways

http://pharmaceuticalintelligence.com/2014/10/23/summary-metabolic-pathways/

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Depth Underwater and Underground

Posted in Amino acids, Best evidence, Biochemical pathways, Biological Networks, Biological Networks, Gene Regulation and Evolution, Biomedical Measurement Science, Cell Biology, Cell Biology, Signaling & Cell Circuits, Curation, Curation methodology, Developmental biology, Diabetes Mellitus, Dialectic, Discovery process, Electrophysiology, Evolutionary cognition, Explanatory, Fatty acids, Gene Regulation, Gene Regulation and Evolution, Genome Biology, Hematopoiesis, Historical relevance, Innovations, Lipid metabolism, Metabolism, Mutant Gene Expression, Myocardial Ischemia, Myocardial Metabolism, Na-K transport, Na-K-ATPase, Neurological Diseases, Nutrition, Proteins, Proteomics, Signaling & Cell Circuits, Theoretic convergence, Translational Research, Translational Science, tagged Biochemistry and Molecular Biology, Bioinformatics, Biology, blind mole rat, Cell Biology, cellular stress, climate, decompression, Hemoglobin, human diving, mtDNA, myoglobin, nitrogen sickness, oxygen delivery, sea lion, seals and otters, whales on February 24, 2015| Leave a Comment »

Depth Underwater and Underground

Writer and Curator: Larry H. Bernstein, MD, FCAP

Introduction

Deep diving for mammals is dangerous for humans and land based animals for too long, and it has dangerous consequences, most notable in nitrogen emboli with very deep underwater diving. Other mammals live in water and have adapted to a water habitat. This is another topic that needs further exploration.

Deep diving has different meanings depending on the context. Even in recreational diving the meaning may vary:

In recreational diving, a depth below about 30 metres (98 ft), where nitrogen narcosis becomes a significant hazard for most divers, may be considered a “deep dive”

In technical diving, a depth below about 60 metres (200 ft) where hypoxic breathing gas becomes necessary to avoid oxygen toxicity may be considered a “deep dive”.

Early experiments carried out by Comex S.A. (Compagnie maritime d’expertises) using hydrox and trimix attained far greater depths than any recreational technical diving. One example being the Comex Janus IV open-sea dive to 501 metres (1,644 ft) in 1977. The open-sea diving depth record was achieved in 1988 by a team of Comex divers who performed pipe line connection exercises at a depth of 534 metres (1,752 ft) in the Mediterranean Sea as part of the Hydra 8 program. These divers needed to breathe special gas mixtures because they were exposed to very high ambient pressure (more than 50 times atmospheric pressure).

Then there is the adaptation to the water habitat as a living environment. The two main types of aquatic ecosystems are marine ecosystems and freshwater ecosystems.

http://en.wikipedia.org/wiki/Deep_diving

Marine ecosystems are part of the earth’s aquatic ecosystem. The habitats that make up this vast system range from the productive nearshore regions to the barren ocean floor. The marine waters may be fully saline, brackish or nearly fresh. The saline waters have a salinity of 35-50 ppt (= parts per thousand). The freshwater has a salinity of less than 0.5 ppt. The brackish water lies in between these 2. Marine habitats are situated from the coasts, over the continental shelf to the open ocean and deep sea. The ecosystems are sometimes linked with each other and are sometimes replacing each other in other geographical regions. The reason why habitats differ from another is because of the physical factors that influence the functioning and diversity of the habitats. These factors are temperature, salinity, tides, currents, wind, wave action, light and substrate.

Marine ecosystems are home to a host of different species ranging from planktonic organisms that form the base of the marine food web to large marine mammals. Many species rely on marine ecosystems for both food and shelter from predators. They are very important to the overall health of both marine and terrestrial environments. Coastal habitats are those above the spring high tide limit or above the mean water level in non-tidal waters. They are close to the sea and include habitats such as coastal dunes and sandy shores, beaches , cliffs and supralittoral habitats. Coastal habitats alone account for approximately 30% of all marine biological productivity.

http://www.marbef.org/wiki/marine_habitats_and_ecosystems

All plant and animal life forms are included from the microscopic picoplankton all the way to the majestic blue whale, the largest creature in the sea—and for that matter in the world. It wasn’t until the writings of Aristotle from 384-322 BC that specific references to marine life were recorded. Aristotle identified a variety of species including crustaceans, echinoderms, mollusks, and fish.
Today’s classification system was developed by Carl Linnaeus external link as an important tool for use in the study of biology and for use in the protection of biodiversity. Without very specific classification information and a naming system to identify species’ relationships, scientists would be limited in attempts to accurately describe the relationships among species. Understanding these relationships helps predict how ecosystems can be altered by human or natural factors.

Preserving biodiversity is facilitated by taxonomy. Species data can be better analyzed to determine the number of different species in a community and to determine how they might be affected by environmental stresses. Family, or phylogenetic, trees for species help predict environmental impacts on individual species and their relatives.

http://marinebio.org/oceans/marine-taxonomy/

For generations, whales and other marine mammals have intrigued humans. 2,400 years ago, Aristotle, a Greek scientist and philosopher, recognized that whales are mammals, not fish, because they nurse their young and breathe air like other mammals. There are numerous myths and legends surrounding marine mammals. The Greeks believed that killing a dolphin was as bad as murdering a human. An Amazon legend said that river dolphins came to shore dressed as men to woo pretty girls during fiestas. During the Middle Ages, there were numerous legends surrounding the narwhals’ amazing tusk, which was thought to have come from the unicorn.

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Marine mammals evolved from their land dwelling ancestors over time by developing adaptations to life in the water. To aid swimming, the body has become streamlined and the number of body projections has been reduced. The ears have shrunk to small holes in size and shape. Mammary glands and sex organs are not part of the external physiology, and posterior (hind) limbs are no longer present.

Mechanisms to prevent heat loss have also been developed. The cylindrical body shape with small appendages reduces the surface area to volume ratio of the body, which reduces heat loss. Marine mammals also have a counter current heat exchange mechanism created by convergent evolution external link where the heat from the arteries is transferred to the veins as they pass each other before getting to extremities, thus reducing heat loss. Some marine mammals also have a thick layer of fur with a water repellent undercoat and/or a thick layer of blubber that can’t be compressed. The blubber provides insulation, a food reserve, and aids with buoyancy. These heat loss adaptations can also lead to overheating for animals that spend time out of the water. To prevent overheating, seals or sea lions will swim close to the surface with their front flippers waving in the air. They also flick sand onto themselves to keep the sun from directly hitting their skin. Blood vessels can also be expanded to act as a sort of radiator.

One of the major behavioral adaptations of marine mammals is their ability to swim and dive. Pinnipeds swim by paddling their flippers while sirenians and cetaceans move their tails or flukes up and down.

Some marine mammals can swim at relatively high speeds. Sea lions swim up to 35 kph and orcas can reach 50 kph. The fastest marine mammal, however, is the common dolphin, which reaches speeds up to 64 kph. While swimming, these animals take very quick breaths. For example, fin whales can empty and refill their huge lungs in less than 2 seconds. Marine mammals’ larynx and esophagus close automatically when they open their mouths to catch prey during dives. Oxygen is stored in hemoglobin in the blood and in myoglobin in the muscles. The lungs are also collapsible so that air is pushed into the windpipe preventing excess nitrogen from being absorbed into the tissues. Decreasing pressure can cause excess nitrogen to expand in the tissues as animals ascend to shallower depths, which can lead to decompression sickness, aka “the bends.” Bradycardia, the reduction of heart rate by 10 to 20%, also takes place to aid with slowing respiration during dives and the blood flow to non-essential body parts. These adaptations allow sea otters to stay submerged for 4 to 5 minutes and dive to depths up to 55 m. Pinnipeds can often stay down for 30 minutes and reach average depths of 150-250 m. One marine mammal with exceptional diving skills is the Weddell seal, which can stay submerged for at least 73 minutes at a time at depths up to 600 m. The length and depth of whale dives depends on the species. Baleen whales feed on plankton near the surface of the water and have no need to dive deeply so they are rarely seen diving deeper than 100 m external link. Toothed whales seek larger prey at deeper depths and some can stay down for hours at depths of up to 2,250 m external link.

http://marinebio.org/oceans/marine-mammals/

Human Experience

Albert Behnke: Nitrogen Narcosis

Casey A. Grover and David H. Grover
The Journal of Emergency Medicine, 2014; 46(2):225–227
http://dx.doi.org/10.1016/j.jemermed.2013.08.080

As early as 1826, divers diving to great depths noted that descent often resulted in a phenomenon of intoxication and euphoria. In 1935, Albert Behnke discovered nitrogen as the cause of this clinical syndrome, a condition now known as nitrogen narcosis. Nitrogen narcosis consists of the development of euphoria, a false sense of security, and impaired judgment upon underwater descent using compressed air below 34 atmospheres (99 to 132 feet). At greater depths, symptoms can progress to loss of consciousness. The syndrome remains relatively unchanged in modern diving when compressed air is used. Behnke’s use of non-nitrogencontaining gas mixtures subsequent to his discovery during the 1939 rescue of the wrecked submarine USS Squalus pioneered the use of non-nitrogencontaining gas mixtures, which are used by modern divers when working at great depth to avoid the effects of nitrogen narcosis.

Behnke’s first duty station as a licensed physician was as assistant medical officer for Submarine Division 20 in San Diego, which was then commanded by one of the Navy’s rising stars, Captain Chester W. Nimitz of World War II fame.
In this setting, Dr. Behnke spent his free time constructively by learning to dive, using the traditional ‘‘hard-hat’’ gear aboard the USS Ortalon, a submarine rescue vessel to which he also rotated. Diving was not a notable specialty of the Navy at the time, and the service was slow in developing the infrastructure for it. Dr. Behnke devoted his efforts to research on the topic of diving medicine, as well as developing a more sound understanding of the biophysics of diving. In 1932, he wrote a letter to the Surgeon General describing some of his observations on arterial gas embolism, which earned him some accolades from the Navy and resulted in his transfer to Harvard’s School of Public Health as a graduate fellow. After 2 years at Harvard, the Navy assigned duty to Dr. Behnke at the Navy’s submarine escape training tower at Pearl Harbor. He worked extensively here on developing techniques for rescuing personnel from disabled submarines on the sea floor. In 1937, he was one of three Navy physicians assigned to the Navy’s Experimental Diving Unit. This team worked on improving the rescue system, plus updating the diving recompression tables originally developed by the British in 1908.

The intoxicating effects of diving were first described by a French physician named Colladon in 1826, who reported that descent in a diving bell resulted in his feeling a ‘‘state of excitement as though I had drunk some alcoholic liquor’’.
The etiology of this phenomenon remained largely unknown until the 1930s, when the British military researcher Damant again highlighted the issue, and reported very unpredictable behavior in his divers during descents as deep as 320 feet during the British Admiralty Deep Sea diving trials. Two initial theories arose as to the etiology for this effect, the first being from psychological causes by Hill and Phillip in 1932, and the second being from oxygen toxicity by Haldane in 1935.

Dr. Behnke and his colleagues at the Harvard School of Public Health had another idea as to the etiology of this phenomenon. In 1935, based on observation of individuals in experiments with a pressure chamber, Dr. Behnke published an article in the American Journal of Physiology in which he posited that nitrogen was the etiology of the intoxicating effects of diving.

Nitrogen narcosis, described as ‘‘rapture of the deep’’ by Jacques Cousteau, still remains a relatively common occurrence in modern diving, despite major advances in diving technology since Behnke’s initial description of the pathophysiologic cause of the condition in 1935. The development of symptoms of this condition varies from diver to diver, but usually begins when a depth of 4 atmospheres (132 feet) is reached in divers using compressed air. More sensitive divers can develop symptoms at only 3 atmospheres (99 feet), and other divers may not be affected up to depths as high as 6 atmospheres (198 feet). Interestingly, tolerance to nitrogen narcosis can be developed by frequent diving and exposure to the effects of compressed air at depth.

Acott C. A brief history of diving and decompression illness. SPUMS J 1999;29:98–109.
2. Bornmann R. Dr. Behnke, founder of UHMS, dies. Pressure 1992; 21:14.
3. Behnke AR, Thomson RM, Motley P. The psychologic effects from breathing air at 4 atmospheric pressures. Am J Physiol 1935; 112:554–8.
4. Behnke AR, Johnson FS, Poppen JR, Motley P. The effect of oxygen on man at pressures from 1 to 4 atmospheres. AmJ Physiol 1934; 110:565–72.

Exhaled nitric oxide concentration and decompression-induced bubble formation: An index of decompression severity in humans?

J.-M. Pontier, Buzzacott, J. Nastorg, A.T. Dinh-Xuan, K. Lambrechts
Nitric Oxide 39 (2014) 29–34
http://dx.doi.org/10.1016/j.niox.2014.04.005

Introduction: Previous studies have highlighted a decreased exhaled nitric oxide concentration (FE NO) in divers after hyperbaric exposure in a dry chamber or following a wet dive. The underlying mechanisms of this decrease remain however unknown. The aim of this study was to quantify the separate effects of submersion, hyperbaric hyperoxia exposure and decompression-induced bubble formation on FE NO after a wet dive.
Methods: Healthy experienced divers (n = 31) were assigned to either

a group making a scuba-air dive (Air dive),
a group with a shallow oxygen dive protocol (Oxygen dive) or

a group making a deep dive breathing a trimix gas mixture (deep-dive).
Bubble signals were graded with the KISS score. Before and after each dive FE NO values were measured using a hand-held electrochemical analyzer.
Results: There was no change in post-dive values of FE NO values (expressed in ppb = parts per billion) in the Air dive group (15.1 ± 3.6 ppb vs. 14.3 ± 4.7 ppb, n = 9, p = 0.32). There was a significant decrease in post-dive values of FE NO in the Oxygen dive group (15.6 ± 6 ppb vs. 11.7 ± 4.7 ppb, n = 9, p = 0.009). There was an even more pronounced decrease in the deep dive group (16.4 ± 6.6 ppb vs. 9.4 ± 3.5 ppb, n = 13, p < 0.001) and a significant correlation between KISS bubble score >0 (n = 13) and percentage decrease in post-dive FE NO values (r = -0.53, p = 0.03). Discussion: Submersion and hyperbaric hyperoxia exposure cannot account entirely for these results suggesting the possibility that, in combination, one effect magnifies the other. A main finding of the present study is a significant relationship between reduction in exhaled NO concentration and dive-induced bubble formation. We postulate that exhaled NO concentration could be a useful index of decompression severity in healthy human divers.

Brain Damage in Commercial Breath-Hold Divers

Kiyotaka Kohshi, H Tamaki, F Lemaıtre, T Okudera, T Ishitake, PJ Denoble
PLoS ONE 9(8): e105006 http://dx.doi.org:/10.1371/journal.pone.0105006

Background: Acute decompression illness (DCI) involving the brain (Cerebral DCI) is one of the most serious forms of diving related injuries which may leave residual brain damage. Cerebral DCI occurs in compressed air and in breath-hold divers, likewise. We conducted this study to investigate whether long-term breath-hold divers who may be exposed to repeated symptomatic and asymptomatic brain injuries, show brain damage on magnetic resonance imaging (MRI).
Subjects and Methods: Our study subjects were 12 commercial breath-hold divers (Ama) with long histories of diving work in a district of Japan. We obtained information on their diving practices and the presence or absence of medical problems, especially DCI events. All participants were examined with MRI to determine the prevalence of brain lesions.
Results: Out of 12 Ama divers (mean age: 54.965.1 years), four had histories of cerebral DCI events, and 11 divers demonstrated ischemic lesions of the brain on MRI studies. The lesions were situated in the cortical and/or subcortical area (9 cases), white matters (4 cases), the basal ganglia (4 cases), and the thalamus (1 case). Subdural fluid collections were seen in 2 cases. Conclusion: These results suggest that commercial breath-hold divers are at a risk of clinical or subclinical brain injury which may affect the long-term neuropsychological health of divers.

Decompression illness

Richard D Vann, Frank K Butler, Simon J Mitchell, Richard E Moon
Lancet 2010; 377: 153–64

Decompression illness is caused by intravascular or extravascular bubbles that are formed as a result of reduction in environmental pressure (decompression). The term covers both arterial gas embolism, in which alveolar gas or venous gas emboli (via cardiac shunts or via pulmonary vessels) are introduced into the arterial circulation, and decompression sickness, which is caused by in-situ bubble formation from dissolved inert gas. Both syndromes can occur in divers, compressed air workers, aviators, and astronauts, but arterial gas embolism also arises from iatrogenic causes unrelated to decompression. Risk of decompression illness is
affected by immersion, exercise, and heat or cold. Manifestations range from itching and minor pain to neurological symptoms, cardiac collapse, and death. First aid treatment is 100% oxygen and definitive treatment is recompression to increased pressure, breathing 100% oxygen. Adjunctive treatment, including fluid administration and prophylaxis against venous thromboembolism in paralyzed patients, is also recommended. Treatment is, in most cases, effective although residual deficits can remain in serious cases, even after several recompressions.

Bubbles can have mechanical, embolic, and biochemical effects with manifestations ranging from trivial to fatal. Clinical manifestations can be caused by direct effects from extravascular (autochthonous) bubbles such as mechanical distortion of tissues causing pain, or vascular obstruction causing stroke-like signs and symptoms. Secondary effects can cause delayed symptom onset up to 24 h after surfacing. Endothelial damage by intravascular bubbles can cause capillary leak, extravasation of plasma, and haemoconcentration. Impaired endothelial function, as measured by decreased effects of vasoactive compounds, has been reported in animals and might occur in man. Hypotension can occur in severe cases. Other effects include platelet activation and deposition, leucocyte-endothelial adhesion, and possibly consequences of vascular occlusion believed to occur in thromboembolic stroke such as ischaemia-reperfusion injury, and apoptosis.

Classification of initial and of all eventual manifestations of decompression illness in 2346 recreational diving accidents reported to the Divers Alert Network from 1998 to 2004 For all instances of pain, 58% consisted of joint pain, 35% muscle pain, and 7% girdle pain. Girdle pain often portends spinal cord involvement. Constitutional symptoms included headache, lightheadedness, inappropriate fatigue, malaise, nausea or vomiting, and anorexia. Muscular discomfort included stiffness, pressure, cramps, and spasm but excluded pain. Pulmonary manifestations included dyspnoea and cough.

Other than depth and time, risk of decompression sickness is affected by other factors that affect inert gas exchange and bubble formation, such as immersion (vs dry hyperbaric chamber exposure), exercise, and temperature. Immersion decreases venous pooling and increases venous return and cardiac output. Warm environments improve peripheral perfusion by promoting vasodilation, whereas cool temperatures decrease perfusion through vasoconstriction. Exercise increases both peripheral perfusion and temperature. The effect of environmental conditions on risk of decompression sickness is dependent on the phase of the pressure exposure. Pressure, exercise, immersion, or a hot environment increase inert gas uptake and risk of decompression sickness. During decom-pression these factors increase inert gas elimination and therefore decrease the risk of decompression sickness. Conversely, uptake is reduced during rest or in a cold environment, hence a diver resting in a cold environment on the bottom has decreased risk of decompression sickness. Rest or low temperatures during decompression increase the risk. If exercise occurs after decompression when super-saturation is present, bubble formation increases and risk of decompression sickness rises.

Exercise at specific times before a dive can decrease the risk of serious decompression sickness in animals and incidence of venous gas emboli in both animals and man. The mechanisms of these effects are unknown but might involve modulation of nitric oxide production and effects on endothelium. Venous gas emboli and risk of decompression sickness increase slightly with age and body-mass index.

Arterial gas embolism should be suspected if a diver has a new onset of altered consciousness, confusion, focal cortical signs, or seizure during ascent or within a few minutes after surfacing from a compressed gas dive.

If the diver spends much time at depth and might have absorbed substantial inert gas before surfacing, arterial gas embolism and serious decompression sickness can coexist, and in such cases, spinal cord manifestations can predominate. Other organ systems, such as the heart, can also be affected, but the clinical diagnosis of gas embolism is not reliable without CNS manifestations. Arterial gas embolism is rare in altitude exposure; if cerebral symptoms occur after altitude exposure, the cause is usually decompression sickness.

Nondermatomal hypoaesthesia and truncal ataxia are common in neurological decompression sickness and can be missed by cursory examination. Pertinent information includes level of consciousness and mental status, cranial nerve function, and motor strength. Coordination can be affected disproportionately, and abnormalities can be detected by assessment of finger-nose movement, and, with eyes open and closed, ability to stand and walk and do heel-toe walking backwards and forwards. Many of these simple tests can be done on the scene by untrained companions.

Panel: Differential diagnosis of decompression illness

Inner-ear barotrauma

Middle-ear or maxillary sinus overinfl ation

Contaminated diving gas and oxygen toxic effects

Musculoskeletal strains or trauma sustained before, during, or after diving

Seafood toxin ingestion (ciguatera, pufferfish, paralytic shellfish poisoning)

Immersion pulmonary edema

Water aspiration

Decompression chamber

Decompression chamber. fluidic or pneumatic ventilator is shown at the left. The infusion pump is contained within a plastic cover, in which 100% nitrogen is used to decrease the fi re risk in the event of an electrical problem. The monitor screen is outside the chamber and can be seen through the viewing port. Photo from Duke University Medical Center, with permission.

Long-term outcomes of 69 divers with spinal cord decompressionsickness, by manifestation
	n	%
No residual symptoms	34	49·3
Any residual symptom	35	50·7
Mild paraesthesias, weakness, or pain	14	20·3
Some impairment of daily activities	21	30·4
Difficulty walking	11	15·9
Impaired micturition	13	18·8
Impaired defecation	15	21·7
Impaired sexual function	15	21·7

Decompression illness occurs in a small population but is an international problem that few physicians are trained to recognise or manage. Although its manifestations are often mild, the potential for permanent injury exists in severe cases, especially if unrecognised or inadequately treated. Emergency medical personnel should be aware of manifestations of decompression illness in the setting of a patient with a history of recent diving or other exposure to substantial pressure change, and should contact an appropriate consultation service for advice.

Diving Medicine: Contemporary Topics and Their Controversies

Michael B. Strauss and Robert C. Borer, Jr
Am J Emerg Med 2001; 19:232-238
http://dx.doi.org:/10.1053/ajem.2001.22654

SCUBA diving is a popular recreational sport. Although serious injuries occur infrequently, when they do knowledge of diving medicine and/or where to obtain appropriate consultation is essential. The emergency physician is likely to be the first physician contact the injured diver has. We discuss 8 subjects
in diving medicine which are contemporary, yet may have controversies associated with them. From this information the physician dealing primarily with the injured diver will have a basis for understanding and managing, as
well as where to find additional help, for his/her patients’ diving injuries.

Over the past 10 years, new knowledge and equipment improvements have made diving safer and more enjoyable. Estimates of actively participating sports divers show a striking increase over this time interval while the number of SCUBA diving deaths annually has remained nearly level at approximately 100. A further indicator of recreational diving safety is that reflected in the nearly constant number of diving injuries (1000 per annum) over the most recent 5 reported years, or approximately 0.53 to 3.4 incidents/10,000 dives.

Divers Alert Network.
The Divers Alert Network (DAN) is a nonprofit organization directed and staffed by experts in the specialty of diving medicine.6 DAN provides immediate consultation for both divers and physicians in the diagnosis and initial management of diving injuries. This 24-hour service is available free world-wide through a dedicated emergency telephone line: 1-919-684-4326. The DAN staff will also identify the nearest appropriate recompression treatment facility and knowledgeable physicians for an expedient referral. General diving medical inquiries can be answered during normal weekday hours either through an information telephone line: 1-919-684-2948 or through an interactive web site http://www.diversalertnetwork.org.

Use of 100% Oxygen for Initial, on the Scene, Management of Diving Accidents
The breathing of pure oxygen is crucial for the initial management of the diving related problems of arterial gas embolism (AGE), decompression sickness (DCS), pulmonary barotrauma (thoracic squeeze), aspiration pneumonitis, and hypoxic encephalopathy associated with near drowning. In 1985, Dick reported that in many cases the neurologic symptoms of AGE and DCS were resolved with the immediate breathing of pure oxygen on the surface. The breathing of pure oxygen reduces bubble size by increasing the differential pressure for the inert gas to diffuse out of the bubble and it also speeds the washout of inert gas from body tissues. The early elimination of the bubble prevents hypoxia and the interaction of the bubble with the blood vessel lining. This interaction leads to secondary problems of capillary leak, bleeding, inflammation, ischemia, and cell death. These secondary problems are the reasons not all DCS symptoms resolve with recompression chamber treatment. The immediate use of pure oxygen for the medical management of these diving problems is analogous to the use of cardiopulmonary resuscitation for the witnessed cardiac arrest; the sooner initiated the better the results.

Diving Education

Medical Fitness for Diving

Asthma has the potential risk for AGE. Neuman reviewed the subject of asthma and diving. He and his coauthors recommend that asthmatics who are asymptomatic, not on medications and have no exercised induced abnormality on pulmonary function studies be allowed to dive.

Conditions leading to loss of consciousness, such as insulin dependent diabetes and epilepsy, can result in drowning. Carefully controlled diving studies in diabetics, who are free from complications, are now defining the safe requirements for diving. Epilepsy remains as a disqualification except in individuals with a history of febrile seizures ending prior to 5 years of age.

Availability of Hyperbaric Oxygen Treatment Facilities

The availability of these chambers makes it possible for divers who become symptomatic after SCUBA diving to readily receive recompression treatment. This is important because the closer the initiation of recompression treatment to the onset of DCS (and AGE) signs and symptoms, the greater the likelihood of full recovery.

Improved Diving Equipment

Mixed and Rebreather Gas Diving
Mixed gas diving involves changing the breathing gas from air which has 20% oxygen to higher oxygen percentages (nitrox). As the amount of oxygen is increased in the gas mixture, the amount of the inert gas (nitrogen) is reduced. With oxygen enriched air there is less tissue deposition of inert gas per unit of time under water for any given depth. However, because of increased oxygen partial pressures, the seizure threshold for oxygen toxicity is lowered. For normal sports diving activities, oxygen toxicity with mixed gas diving is only a theoretical concern.

Decompression Illness is More Than Bubbles

When AGE occurs, DCS symptoms may be concurrent or appear during or after recompression treatment even though the decompression tables were not violated on the dive. When DCS occurs in this situation it appears resistant to recompression treatment (Neuman) perhaps because of the inflammatory reaction generated by the bubble-blood vessel interaction from the AGE. In cases of DCI where components of both DCS and AGE are suspected, the diver should be observed for a minimum of 24 hours after the recompression treatment is completed for the delayed onset of DCS.

No theory of DCS discounts the primary role of bubbles in this condition. However, new information suggests that there are precursors to bubble formation and post-bubbling events that occur as a consequence of the bubbles. As mentioned earlier, venous gas emboli are a common occurrence diving ascent and ordinarily are filtered out harmlessly by the lungs. Precursors to DCS include stasis, dehydration and too rapid of ascents. These conditions allow the ubiquitous VGE to enlarge, coalesce and occlude the venous side of the circulation. Massive venous bubbling to the lungs can cause pulmonary vessel obstruction described as the chokes. If right to left shunts occur in the heart, VGE can become AGE to the brain. If the arterial flow is slow enough and/or the gradients large enough, autochthonus (ie, spontaneous) bubbles can form in the arterial circulation and lead to any of the consequences of AGE. In such situations it could be difficult to determine whether the DCI event was from AGE or DCS even after careful analysis of the dive profile. Hollenbeck’s model for diving paraplegia includes the setting of venous stasis (Batson’s plexus of veins) in the spinal canal, bubble formation, bubble enlargement possibly from off gassing of the spinal cord, blood vessel occlusion, and venous side infarctions of the spinal cord.
Contemporary Management of DCS

Problem	Intervention	Effect
Bubble	Recompression with HBO	Reduce bubble size 1. Washout inert gas. 2. Change bubble composition by diffusion.
Stasis and dehydration	Hydration: oral fluids if alert, IV fluids otherwise.	Improve blood flow.
InflammationCell Ischemia	? Anti-inflammatory medicationsHBO	Reduce interaction between bubble and blood vessel endothelium. Improve oxygen availability to hypoxic tissues, reduce edema and also reduces the interaction between bubble and blood vessel endothelium.

Conclusions

We anticipate that in the future there will be further improvements for the safety and enjoyment of the recreational SCUBA diver. For example, the dive computer of the future will be able to individualize dive profiles for different personal medical parameters such as age, body composition and fitness level. Diver locators could quickly target a missing diver and save time and gas consumption as well as prevent serious diving mishaps. Drugs may be developed that would minimize the effect of bubbles interacting with body tissues and prevent DCS and AGE.

Extracorporeal membrane oxygenation therapy for pulmonary decompression illness

Yutaka Kondo, Masataka Fukami and Ichiro Kukita
Kondo et al. Critical Care 2014; 18:438 http://ccforum.com/content/18/3/438/10.1186/cc13935

Pulmonary decompression illness is rarely observed in clinical settings, and most patients die prior to hospitalization. We administered ECMO therapy to rescue a patient, even though this therapy has rarely been reported with good outcome in patients with decompression illness. In addition, we had to select venovenous ECMO even with the patient showing right ventricular failure. A lot of physicians may select venoarterial ECMO if the patient shows right ventricular failure, but the important physiological mechanism of pulmonary decompression illness is massive air embolism in the pulmonary arteries, and the bubbles diminish within the first 24 hours. The management of decompression illness therefore differs substantially from the usual right-sided heart failure.

Extremes of barometric pressure

Jane E Risdall, David P Gradwell
Anaesthesia and Intensive Care Medicine 16:2
Ascent to elevated altitude, commonly achieved through flight, by climbing or by residence in highland regions, exposes the individual to reduced ambient pressure. Although there are physical manifestations of this exposure as a consequence of Boyle’s law, the primary physiological challenge is of hypobaric hypoxia. The acute physiological and longer-term adaptive responses of the cardiovascular, respiratory, hematological and neurological systems to altitude are described, together with an outline of the presentation and management of acute mountain sickness, high-altitude pulmonary edema and high-altitude cerebral edema. While many millions experience modest exposure to altitude as a result of flight in pressurized aircraft, fewer individuals are exposed to increased ambient pressure. The pressure changes during diving and hyperbaric exposures result in greater changes in gas load and gas toxicity. Physiological effects include the consequences of increased work of breathing and redistribution of circulating volume. Neurological manifestations may be the direct result of pressure or a consequence of gas toxicity at depth. Increased tissue gas loads may result in decompression illness on return to surface or subsequent ascent in flight.

understand the physical effects of changes in ambient pressure and the physiological consequences on the cardiovascular respiratory and neurological systems
gain an awareness that exposure to reduced ambient pressure produces both acute and more chronic effects, with differing signs, symptoms and time to onset at various altitudes
develop an awareness of the toxic effects of ‘inert’ gases at increased ambient pressures and the pathogenesis and management of decompression illness

Decompression illness According to Henry’s law, at a constant temperature the amount of gas which dissolves in a liquid is proportional to the pressure of that gas or its partial pressure, if it is part of a mixture of gases. Breathing gases at increased ambient pressure will increase the amount of each gas dissolved in the fluid phases of body tissues. On ascent this excess gas has to be given up. If the ascent is controlled at a sufficiently slow rate, elimination will be via the respiratory system. If the ascent is too fast, excess gas may come out of solution and form free bubbles in the tissues or circulation. Bubbles may contain any of the gases in the breathing mixture, but it is the presence of inert gas bubbles (nitrogen or helium) that are thought most likely to give rise to problems, since the elimination of excess oxygen is achieved by metabolism as well as ventilation. These bubbles may act as venous emboli or may trigger inflammatory tissue responses giving rise to symptoms of decompression illness (DCI). Signs and symptoms of DCI may appear up to 48 hours after exposure to increased ambient pressure and include joint pains, motor and sensory deficits, dyspnoea, cough and skin rashes.

Neurological effects of deep diving

Marit Grønning, Johan A. Aarli
Journal of the Neurological Sciences 304 (2011) 17–21
http://dx.doi.org:/10.1016/j.jns.2011.01.021

Deep diving is defined as diving to depths more than 50 m of seawater (msw), and is mainly used for occupational and military purposes. A deep dive is characterized by the compression phase, the bottom time and the decompression phase. Neurological and neurophysiologic effects are demonstrated in divers during the compression phase and the bottom time. Immediate and transient neurological effects after deep dives have been shown in some divers. However, the results from the epidemiological studies regarding long term neurological effects from deep diving are conflicting and still not conclusive.

Possible immediate neurological effects of deep diving
Syndrome	Pressure
Hyperoxia/oxygen seizures	>152 kPa (5 msw)
HypoxiaHypercapnia
Nitrogen narcosis	>354 kPa (25 msw)
High pressure nervous syndrome	>1.6 MPa (150 msw)
Neurological decompression sickness

Neurological effects have been demonstrated, both clinically and neurophysiologically in divers during the compression phase and the bottom time. Studies of divers before and after deep dives have shown immediate and transient neurological effects in some divers. However, the results from the epidemiological and clinical studies regarding long term neurological effects from deep diving are conflicting and still not conclusive. Prospective clinical studies with sufficient power and sensitivity are needed to solve this important issue.

Today deep diving to more than 100 msw is routinely performed globally in the oil- and gas industry. In the North Sea remote underwater intervention and maintenance is performed by the use of remotely operated vehicles (ROV), both in conjunction to and as an alternative to manned underwater operations. There will, however, always be a need for human divers in the technically more advanced underwater operations and for contingency repair operations.

P300 latency indexes nitrogen narcosis

Barry Fowler, Janice Pogue and Gerry Porlier
Electroencephalography, and clinical Neurophysiology, 1990, 75:221-229

This experiment investigated the effects of nitrogen narcosis on reaction time (RT) and P300 latency and amplitude, Ten subjects breathed either air or a non-narcotic 20% oxygen-80% helium (heliox) mixture in a hyperbaric chamber at 6.5, 8.3 and 10 atmospheres absolute (ATA), The subjects responded under controlled accuracy conditions to visually presented male or female names in an oddball paradigm. Single-trial analysis revealed a strong relationship between RT and P300 latency, both of which were slowed in a dose-related manner by hyperbaric air but not by heliox. A clear-cut dose-response relationship could not be established for P300 amplitude. These results indicate that P300 latency indexes nitrogen narcosis and are interpreted as support for the slowed processing model of inert gas narcosis.

Adaptation to Deep Water Habitat

Effects of hypoxia on ionic regulation, glycogen utilization and antioxidative ability in the gills and liver of the aquatic air-breathing fish Trichogaster microlepis

Chun-Yen Huang, Hui-Chen Lina, Cheng-Huang Lin
Comparative Biochemistry and Physiology, Part A 179 (2015) 25–34
http://dx.doi.org/10.1016/j.cbpa.2014.09.001

We examined the hypothesis that Trichogaster microlepis, a fish with an accessory air-breathing organ, uses a compensatory strategy involving changes in both behavior and protein levels to enhance its gas exchange ability. This compensatory strategy enables the gill ion-regulatory metabolism to maintain homeostasis during exposure to hypoxia. The present study aimed to determinewhether ionic regulation, glycogen utilization and antioxidant activity differ in terms of expression under hypoxic stresses; fish were sampled after being subjected to 3 or 12 h of hypoxia and 12 h of recovery under normoxia. The air-breathing behavior of the fish increased under hypoxia. No morphological modification of the gills was observed. The expression of carbonic anhydrase II did not vary among the treatments. The Na+/K+-ATPase enzyme activity did not decrease, but increases in Na+/K+-ATPase protein expression and ionocyte levels were observed. The glycogen utilization increased under hypoxia as measured by glycogen phosphorylase protein expression and blood glucose level, whereas the glycogen content decreased. The enzyme activity of several components of the antioxidant system in the gills, including catalase, glutathione peroxidase, and superoxidase dismutase, increased in enzyme activity. Based on the above data, we concluded that T. microlepis is a hypoxia-tolerant species that does not exhibit ion-regulatory suppression but uses glycogen to maintain energy utilization in the gills under hypoxic stress. Components of the antioxidant system showed increased expression under the applied experimental treatments.

Divergence date estimation and a comprehensive molecular tree of extant cetaceans

Michael R. McGowen , Michelle Spaulding, John Gatesy
Molecular Phylogenetics and Evolution 53 (2009) 891–906
http://dx.doi.org/10.1016/j.ympev.2009.08.018

Cetaceans are remarkable among mammals for their numerous adaptations to an entirely aquatic existence, yet many aspects of their phylogeny remain unresolved. Here we merged 37 new sequences from the nuclear genes RAG1 and PRM1 with most published molecular data for the group (45 nuclear loci, transposons, mitochondrial genomes), and generated a supermatrix consisting of 42,335 characters. The great majority of these data have never been combined. Model-based analyses of the supermatrix produced a solid, consistent phylogenetic hypothesis for 87 cetacean species. Bayesian analyses corroborated odontocete (toothed whale) monophyly, stabilized basal odontocete relationships, and completely resolved branching events within Mysticeti (baleen whales) as well as the problematic speciose clade Delphinidae (oceanic dolphins). Only limited conflicts relative to maximum likelihood results were recorded, and discrepancies found in parsimony trees were very weakly supported. We utilized the Bayesian supermatrix tree to estimate divergence dates among lineages using relaxed-clock methods. Divergence estimates revealed rapid branching of basal odontocete lineages near the Eocene–Oligocene boundary, the antiquity of river dolphin lineages, a Late Miocene radiation of balaenopteroid mysticetes, and a recent rapid radiation of Delphinidae beginning [1]10 million years ago. Our comprehensive, time calibrated tree provides a powerful evolutionary tool for broad-scale comparative studies of Cetacea.

Mitogenomic analyses provide new insights into cetacean origin and evolution

Ulfur Arnason, Anette Gullberg, Axel Janke
Gene 333 (2004) 27–34
http://dx.doi.org:/10.1016/j.gene.2004.02.010

The evolution of the order Cetacea (whales, dolphins, porpoises) has, for a long time, attracted the attention of evolutionary biologists. Here we examine cetacean phylogenetic relationships on the basis of analyses of complete mitochondrial genomes that represent all extant cetacean families. The results suggest that the ancestors of recent cetaceans had an explosive evolutionary radiation 30–35 million years before present. During this period, extant cetaceans divided into the two primary groups, Mysticeti (baleen whales) and Odontoceti (toothed whales). Soon after this basal split, the Odontoceti diverged into the four extant lineages, sperm whales, beaked whales, Indian river dolphins and delphinoids (iniid river dolphins, narwhals/belugas, porpoises and true dolphins). The current data set has allowed test of two recent morphological hypotheses on cetacean origin. One of these hypotheses posits that Artiodactyla and Cetacea originated from the extinct group Mesonychia, and the other that Mesonychia/Cetacea constitutes a sister group to Artiodactyla. The current results are inconsistent with both these hypotheses. The findings suggest that the claimed morphological similarities between Mesonychia and Cetacea are the result of evolutionary convergence rather than common ancestry.

The order Cetacea traditionally includes three suborders: the extinct Archaeoceti and the recent Odontoceti and Mysticeti. It is commonly believed that the evolution of ancestral cetaceans from terrestrial to marine (aquatic) life was accompanied by a fast and radical morphological adaptation. Such a scenario may explain why it was, for a long time, difficult to morphologically establish the position of Cetacea in the mammalian tree and even to settle whether Cetacea constituted a monophyletic group.

Biochemical analyses in the 1950s and 1960s had shown a closer relationship between cetaceans and artiodactyls (even-toed hoofed mammals) than between cetaceans and any other eutherian order and karyological studies in the late 1960s and early 1970s unequivocally supported cetacean monophyly (Arnason, 1969, 1974). The nature of the relationship between cetaceans and artiodactyls was resolved in phylogenetic studies of mitochondrial (mt) cytochrome b (cytb) genes (Irwin and Arnason, 1994; Arnason and Gullberg, 1996) that placed Cetacea within the order Artiodactyla itself as the sister group of the Hippopotamidae (see also Sarich, 1993). The Hippopotamidae/ Cetacea relationship was subsequently supported in studies of nuclear data (Gatesy et al., 1996; Gatesy, 1997) and statistically established in analysis of complete mt genomes (Ursing and Arnason, 1998). The relationship has also been confirmed in analyses of combined nuclear and mt sequences (Gatesy et al., 1999; Cassens et al., 2000) and in studies of short interspersed repetitive elements (SINEs). Artiodactyla and Cetacea are now commonly referred to as Cetartiodactyla.

Previous analyses of the complete cytb gene of more than 30 cetacean species (Arnason and Gullberg, 1996) identified five primary lineages of recent cetaceans, viz., Mysticeti and the four odontocete lineages Physeteridae (sperm whales), Platanistidae (Indian river dolphins), Ziphiidae (beaked whales) and Delphinoidea (iniid river dolphins, porpoises, narwhals and dolphins). However, these studies left unresolved the relationships of the five lineages as well as those between the three delphinoid families Monodontidae (narwhals, belugas), Phocoenidae (porpoises) and Delphinidae (dolphins). Similarly, the relationships between the four mysticete families Balaenidae (right whales), Neobalaenidae (pygmy right whales), Eschrichtiidae (gray whales) and Balaenopteridae (rorquals) were not conclusively resolved in analyses of cytb genes.

Fig. (not shown). Cetartiodactyl relationships and the estimated times of their divergences. The tree was established on the basis of maximum likelihood analysis of the concatenated amino acid (aa) sequences of 12 mt protein-coding genes. Length of alignment 3610 aa. Support values for branches A–H are shown in the insert.
Cetruminantia (branch A) receives moderate support and Cetancodonta (B) strong support. Cetacea (C) splits into monophyletic Mysticeti (baleen whales) and monophyletic Odontoceti (toothed whales). Odontoceti has four basal lineages, Physeteridae (sperm whales: represented by the sperm and pygmy sperm whales), Ziphiidae (beaked whales: bottlenose and Baird’s beaked whales), Platanistidae (Indian river dolphins: Indian river dolphin) and Delphinoidea. Delphinoidea encompasses the families Iniidae (iniid river dolphins: Amazon river dolphin, La Plata dolphin), Monodontidae (narwhals/belugas: narwhal), Phocoenidae (porpoises: harbour porpoise) and Delphinidae (dolphins: white-beaked dolphin). The common odontocete branch and the branches separating the four cetacean lineages are short. These relationships are therefore somewhat unstable (cf. Section 3.1 and Table 1). Iniid river dolphins (F) are solidly nested within the Delphinoidea (E). Thus, traditional river dolphins (Platanistidae + Iniidae) do not form a monophyletic unit. Molecular estimates of divergence times (Sanderson 2002) were based on two calibration points, A/C-60 and O/M-35 (cf. Section 3.2). Due to the short lengths of internal branches, some estimates for these divergences overlap. NJ: neighbor joining; MP: maximum parsimony; LBP: local bootstrap probability; QP: quartet puzzling. The bar shows the number of aa substitutions per site.

The limited molecular resolution among basal cetacean lineages has been known for some time. Studies of hemoglobin and myoglobin (Goodman, 1989; Czelusniak et al., 1990) have either joined Physeteridae and Mysticeti to the exclusion of Delphinoidea (myoglobin data) or Mysticeti and Delphinoidea to the exclusion of Physeteridae (hemoglobin data). Thus, neither of the data sets identified monophyletic Odontoceti by joining the two odontocete lineages (Physeteridae and Delphinoidea) to the exclusion of Mysticeti. A similar instability was recognized and cautioned against in analyses of some mt data, notably, sequences of rRNA genes (Arnason et al., 1993b). The suggestion (Milinkovitch et al., 1993) of a sister group relationship between Physeteridae and the mysticete family Balaenopteridae (rorquals) was based on a myoglobin data set (which joins Physeteridae and Mysticeti to the exclusion of Delphinoidea) that was complemented with partial data of the mt 16S rRNA gene.

The cetancodont divergence times calculated using A/C-60 and O/M-35 as references have been included in Fig. 1. As a result of the short branches separating several cetacean lineages, the estimates of these divergences overlap. The same observation has been made in calculations based on SINE flanking sequences (Nikaido et al., 2001). There is a general consistency between the current and the flanking sequence datings, except for those involving the Balaenopteridae, which are somewhat younger in our analysis than in the SINEs study. The currently estimated age of the divergence between Hippopotamus and Cetacea (c53.5 MYBP) is consistent with the age (>50 MY) of the oldest archaeocete fossils identified so far (Bajpai and Gingerich, 1998). This suggests that the ages allocated to the two references, A/C-60 (the divergence between ruminant artiodactyls and cetancodonts) and O/M-35 (the divergence between odontocetes and mysticetes) are reasonably accurate.

The dating of the divergence between the blue and fin whales is of interest regarding hybridization between closely related mammalian species. Previous molecular analyses (Arnason et al., 1991b; Spilliaert et al., 1991) demonstrated the occurrence of hybridization between these two species. These studies, which were based on three hybrids (one female and two males), showed that either species could be the mother or father in these hybridizations. The two male hybrids had rudimentary testes, whereas the female hybrid was in her second pregnancy. This suggests that the blue and fin whales may be close to the limit for permissible species hybridization among mammals.

The current data set has allowed examination of the coherence between the molecular results and two prevalent morphological hypotheses related to cetacean evolution. The first hypothesis, which in essence originates from Van Valen (1966, 1968), postulates that monophyletic Artiodactyla and monophyletic Cetacea evolved separately from the extinct Palaeocene group Mesonychia. This hypothesis was recently reinforced in a morphological study (Thewissen et al., 2001) that included mesonychians, two archaeocete taxa (Ambuloocetus and Pakicetus) and some extant and fossil artiodactyls. The study of Thewissen et al. (2001) showed a sister group relationship between monophyletic Artiodactyla and monophyletic Cetacea, with Mesonychia as the basal sister group of Artiodactyla/Cetacea, a conclusion consistent with the palaeontological age of Mesonychia relative to that of Artiodactyla and Cetacea. The second hypothesis favours a sister group relationship between Mesonychia and Cetacea with the Mesonychia/Cetacea clade as the sister group of monophyletic Artiodactyla (O’Leary and Geisler, 1999; see also Gatesy and O’Leary, 2001).

Although the position of Mesonychia differs in the two morphological hypotheses, both correspond to a sister group relationship between Cetacea and monophyletic Artiodactyla among extant cetartiodactyls. Thus, both hypotheses can be tested against the current data set. The result of such a test has been included in Table 1, topology (m)(not shown). As evident, both these morphological hypotheses are incongruent with the mitogenomic findings.

Morphological studies have not provided an answer to the question whether mysticetes and odontocetes had separate origins among the archaeocetes (Fordyce and de Muizon, 2001). However, the long common cetacean branch and the short branches separating the five extant cetacean lineages strongly suggest an origin of modern cetaceans from the same archaeocete group (probably the Dorudontidae).

The limbs of Ambulocetus constitute somewhat of an evolutionary enigma. As evident in Thewissen et al.’s (1994) paper, Ambulocetus has very large hind limbs compared to its forelimbs, a difference that is less pronounced in later silhouette drawings of the animal. It is nevertheless evident that evolution from the powerful hindlimbs of Ambulocetus to their rudimentation in archaeocetes constitutes a remarkable morphological reversal if Ambulocetus is connected to the cetacean branch after the separation of the hippopotamid and cetacean lineages.

For natural reasons, systematic schemes have traditionally been based on external morphological characteristics. The rates of morphological and molecular evolution are rarely (if ever) strictly correlated, however, and this may give rise to inconsistency between traditional systematics and molecular findings. The emerging consensus that the order Cetacea resides within another traditional order, Artiodactyla, makes apparent the incongruity in cetartiodactyl nomenclature (Graur and Higgins, 1994). In this instance, a possible solution for maintaining reasonable consistency between nomenclature and phylogeny would be to recognize Cetartiodactyla as an order with three suborders: Suina, Tylopoda and Cetruminantia. According to such a scheme, Cetacea would (together with the Hippopotamidae) constitute a parvorder within the infraorder Cetancodonta.

Cytochrome b and Bayesian inference of whale phylogeny

Laura May-Collado, Ingi Agnarsson
Molecular Phylogenetics and Evolution 38 (2006) 344–354
http://dx.doi.org//10.1016/j.ympev.2005.09.019

In the mid 1990s cytochrome b and other mitochondrial DNA data reinvigorated cetacean phylogenetics by proposing many novel

and provocative hypotheses of cetacean relationships. These results sparked a revision and reanalysis of morphological datasets, and the collection of new nuclear DNA data from numerous loci. Some of the most controversial mitochondrial hypotheses have now become benchmark clades, corroborated with nuclear DNA and morphological data; others have been resolved in favor of more traditional views. That major conflicts in cetacean phylogeny are disappearing is encouraging. However, most recent papers aim specifically to resolve higher-level conflicts by adding characters, at the cost of densely sampling taxa to resolve lower-level relationships. No molecular study to date has included more than 33 cetaceans. More detailed molecular phylogenies will provide better tools for evolutionary studies. Until more genes are available for a high number of taxa, can we rely on readily available single gene mitochondrial data? Here, we estimate the phylogeny of 66 cetacean taxa and 24 outgroups based on Cytb sequences. We judge the reliability of our phylogeny based on the recovery of several deep-level benchmark clades. A Bayesian phylogenetic analysis recovered all benchmark clades and for the Wrst time supported Odontoceti monophyly based exclusively on analysis of a single mitochondrial gene. The results recover the monophyly, with the exception of only one taxa within Cetacea, and the most recently proposed super- and subfamilies. In contrast, parsimony never recovered all benchmark clades and was sensitive to a priori weighting decisions. These results provide the most detailed phylogeny of Cetacea to date and highlight the utility of both Bayesian methodology in general, and of Cytb in cetacean phylogenetics. They furthermore suggest that dense taxon sampling, like dense character sampling, can overcome problems in phylogenetic reconstruction.

Some long standing debates are all but resolved: our understanding of deeper level cetacean phylogeny has grown strong. However, the strong focus of most recent studies, aiming specifically to resolve these higher level conflicts by adding mostly characters rather than taxa, has left our understanding of lower level relationships among whale species lagging behind. Mitogenomic data, for example, is available only for 16 cetacean species, and no molecular study to date has included more than 33 cetaceans. It seems timely to focus on more detailed (genus, and species level) molecular phylogenies. These will provide better tools for detailed evolutionary studies, and are necessary to test existing morphological phylogenetic hypotheses, and current cetacean classification.

We judge the reliability of our phylogeny based on the recovery of the previously mentioned benchmark clades, in addition to the less controversial clades Perissodactyla, Euungulata (sensu Waddell et al., 2001; Perissodactyla+ Cetartiodactyla), Cetacea, and Mysticeti. Because Cytb is thought to be most reliable at lower taxonomic levels (due to high substitution rates), recovering ‘known’ deeper clades gives credibility to these new findings which have not been addressed by studies using few taxa. We compare the performance of Bayesian analyses versus parsimony under four different models, and briefly examine the sensitivity of the results to taxon sampling. We use our results to discuss agreement and remaining conflict in cetacean phylogenetics, and provide comments on current classification.

The Bayesian analysis recovered all seven benchmark clades. Support for five of the benchmark clades is high (100 posterior probabilities) but rather low for Cetancodonta (79) and marginal for the monophyly of Odontoceti. The analysis also recovered all but one family level, and most sub- and superfamily level cetacean taxa. The results broadly corroborate current cetacean classiffcation, while also pointing to some lower-level groups that may need redefinition.

Many recent cetacean phylogenetic studies include relatively few taxa, in part due to a focus on generating more characters to resolve higher level phylogenetics. While addressing crucial questions and providing the backbone for lower level phylogenies, such studies have limited utility for classification, and for comparative evolutionary studies. In some cases sparse taxon sampling may also confound the results. Of course, taxon sampling is usually simply constrained by the availability of character data, but for some reason many studies have opted to include only one, or a few outgroup taxa, even if many are available.

We find that as long as outgroup taxon sampling was extensive, Bayesian analyses of Cytb recovered all the a priori identified benchmark clades. When only a few outgroups were chosen, however, the Bayesian analysis negated Odontoceti monophyly, as have many previous parsimony analyses of mitochondrial DNA. Furthermore, in almost every detailed comparison possible our results mirror the findings O’Leary et al. (2004), the most ‘character-complete’ (but including relatively few cetacean taxa) analysis to date (37,000 characters from morphology, SINE, and 51 gene fragments). This result gives credibility to our findings, including previously untested lower level clades.

Monophyly and placement of Mysticeti (baleen whales).
Monophyly of Odontoceti (toothed whales)
Delphinoids
River Dolphins
Beaked and sperm whales

A major goal of phylogenetics is a phylogeny of life (i.e., many taxa), based on multiple lines of evidence (many characters of many types). However, when phylogenies based on relatively few characters can be judged reliable based on external evidence (taxonomic congruence with other phylogenies using many characters, but few taxa), they seem like very promising and useful ‘first guess’ hypotheses. The evolution of sexual dimorphism, echolocation, social behavior, and whistles and other communicative signals, and major ecological shifts (e.g., transition to fresh water) are among the numerous interesting questions in cetacean biology that this phylogeny can help answer.

Deep-diving sea lions exhibit extreme bradycardia in long duration dives

Birgitte I. McDonald1, and Paul J. Ponganis
The Journal of Experimental Biology (2014) 217, 1525-1534 http://dx.doi.org:/10.1242/jeb.098558

Heart rate and peripheral blood flow distribution are the primary determinants of the rate and pattern of oxygen store utilization and ultimately breath-hold duration in marine endotherms. Despite this, little is known about how otariids (sea lions and fur seals) regulate heart rate (fH) while diving. We investigated dive fH in five adult female California sea lions (Zalophus californianus) during foraging trips by instrumenting them with digital electrocardiogram (ECG) loggers and time depth recorders. In all dives, dive fH (number of beats/duration; 50±9 beats min⁻¹) decreased compared with surface rates (113±5 beats min⁻¹), with all dives exhibiting an instantaneous fH below resting (<54 beats min⁻¹) at some point during the dive. Both dive fH and minimum instantaneous fH significantly decreased with increasing dive duration. Typical instantaneous fH profiles of deep dives (>100 m) consisted of:

(1) an initial rapid decline in fH resulting in the lowest instantaneous fH of the dive at the end of descent, often below 10 beats min⁻¹ in dives longer than 6 min in duration;
(2) a slight increase in fH to ~10–40 beats min⁻¹ during the bottom portion of the dive; and
(3) a gradual increase in fH during ascent with a rapid increase prior to surfacing.

Thus, fH regulation in deep-diving sea lions is not simply a progressive bradycardia. Extreme bradycardia and the presumed associated reductions in pulmonary and peripheral blood flow during late descent of deep dives should

(a) contribute to preservation of the lung oxygen store,
(b) increase dependence of muscle on the myoglobin-bound oxygen store,
(c) conserve the blood oxygen store and
(d) help limit the absorption of nitrogen at depth.

This fH profile during deep dives of sea lions may be characteristic of deep-diving marine endotherms that dive on inspiration as similar fH profiles have been recently documented in the emperor penguin, another deep diver that dives on inspiration.

The resting ƒH measured in this study (54±6 beats min⁻¹) was lower than predicted for an animal of similar size (~80 beats min⁻¹ for an 80 kg mammal). In part, this may be due to the fact that the sea lions were probably sleeping. The resting ƒH in our study was also lower than previous measurements in captive juvenile California sea lions (87±17 beats min⁻¹, average mass 30 kg) and wild Antarctic fur seals (78±5 beats min⁻¹, body mass 30–50 kg). However, we found a significant negative relationship between mass and resting ƒH even with our small sample size of five sea lions (resting ƒH = –0.58 Mb +100.26, r²=0.81, F_1,3=12.37, P=0.039). For a 30 kg sea lion, this equation predicts a resting ƒH of 83 beats min⁻¹, which is similar to what was measured previously in juvenile sea lions, suggesting this equation may be useful in estimating resting ƒH in sea lions.

The sea lions exhibited a distinct sinus arrhythmia fluctuating between a minimum of 42±9 and a maximum of 87±12 beats min⁻¹, comparable to the sinus arrhythmias described in other diving birds and mammals, including sea lions. The minimum instantaneous ƒH during the sinus arrhythmia was similar to the mean minimum ƒH in dives less than 3 min (37±7 beats min⁻¹), indicating that in dives less than 3 min (estimated cADL), ƒH only decreased to levels observed during exhalation at rest. This is consistent with observations in emperor penguins and elephant seals, where it was proposed that in dives shorter than the aerobic dive limit (ADL) the reduction in ƒH is regulated by a mechanism of cardiorespiratory control similar to that governing the respiratory sinus arrhythmia, with a further reduction only occurring in dives longer than the ADL.

Fig. 3. (not shown) Instantaneous fH and dive depth profiles of a California sea lion (CSL12_2). Data are from (A) a short, shallow dive (1.3 min, 45 m), (B) a mid-duration dive (4.8 min, 239 m) and (C) a long-duration dive (8.5 min, 305 m). Minimum instantaneous fH reached 37 beats min⁻¹ in the short dive
(A) 19 beats min⁻¹ in the mid-duration dive
(B) and 7 beats min⁻¹ in the long duration dive
(C) Prominent features typical of mid- and long-duration dives include

a surface interval tachycardia (pre- and post-dive);
a steady rapid decrease in fH during initial descent;
a gradual decline in fH towards the end of descent with the lowest fH of the dive at the end of descent;
a slight increase and sometimes variable fH during the bottom portion of the dive; and
a slow increase in fH during ascent,
often ending in a rapid increase just before surfacing.

We obtained the first diving ƒH data from wild sea lions on natural foraging trips, demonstrating how they regulate ƒH over a range of dive durations. Sea lions always decreased dive ƒH from surface ƒH values; however, individual sea lions exhibited different dive ƒH, accounting for a significant amount of the variation in the relationship between dive duration and ƒH (intra-individual correlation: 75–81%)). The individual differences in dive ƒH exhibited in this study suggest that different dive capacities of individual sea lions may partially account for the range of dive strategies exhibited in a previous study (Villegas-Amtmann et al., 2011). Despite the individual differences in ƒH, the pattern of the dive ƒH response was similar in all the sea lions. As predicted, sea lions only consistently displayed a true bradycardia on mid- to long- duration dives (>4 min) (Fig. 5A). Additionally, as seen in freely diving phocids, dive ƒH and minimum ƒH were negatively related to dive duration, with the longest duration dives having the lowest dive ƒH and displaying the most intense bradycardia, often below 10 beats min⁻¹ (Fig. 5A,B).

Profiles of mean fH at 10 s intervals of dives

Fig 4. Profiles of mean fH at 10 s intervals of dives for (A) six duration categories and (B) five depth categories. Standard error bars are shown. Data were pooled from 461 dives performed by five sea lions. The number of dives in each category and the number of sea lions performing the dives in each category are provided in the keys.

The mild bradycardia and the dive ƒH profiles observed in the shorter duration dives (<3 min) were similar to those observed in trained juvenile California sea lions and adult Stellar sea lions, but much more intense than ƒH observed in freely diving Antarctic fur seals. Surprisingly, although dive ƒH of trained Steller sea lions was similar, Steller sea lions regularly exhibited lower minimum ƒH, with minimum ƒH almost always less than 20 beats min⁻¹ in dives less than 2 min in duration. In the wild, California sea lions rarely exhibited a minimum ƒH less than 20 beats min⁻¹ in similar duration dives (Fig. 5B), suggesting greater blood oxygen transport during these natural short-duration dives.

Fig. 5. (not shown) fH decreases with increasing dive duration. Dive duration versus (A) dive fH (total number of beats/dive duration), (B) minimum instantaneous fH and (C) bottom fH (total beats at bottom of dive/bottom time) for California sea lions (461 dives from five sea lions).

Although California sea lions are not usually considered exceptional divers, they exhibited extreme bradycardia, comparable to that of the best diving phocids, during their deep dives. In dives greater than 6 min in duration, minimum ƒH was usually less than 10 beats min⁻¹ and sometimes as low as 6 beats mins⁻¹ (Fig. 5B), which is similar to extreme divers such as emperor penguins (3 beats min−1), elephant seals (3 beats min⁻¹), grey seals (2 beats min−1) and Weddell seals (<10 beats min⁻¹), and even as low as what was observed in forced submersion studies. Thus, similar to phocids, the extreme bradycardia exhibited during forced submersions is also a routine component of the sea lion’s physiological repertoire, allowing them to perform long-duration dives.

While the degree of bradycardia observed in long dives of California sea lions was similar to the extreme bradycardia observed in phocids, the ƒH profiles were quite different. In general, phocid ƒH decreases abruptly upon submergence. The intensity of the initial phocid bradycardia either remains relatively stable or intensifies as the dive progresses, and does not start to increase until the seal begins its ascent. In contrast, the ƒH profiles of sea lions were more complex, showing a more gradual decrease during descent, with the minimum ƒH of the dive usually towards the end of descent (Figs 3, 6). There was often a slight increase in ƒH during the bottom portion of the dive, and as soon as the sea lions started to ascend, the ƒH slowly started to increase, often becoming irregular during the middle of ascent, before increasing rapidly as the sea lion approached the surface.

Fig. 6. (not shown) Instantaneous fH and dive depth profiles of the longest dive (10.0 min, 385 m) from a California sea lion (CSL12_1). During this dive, instantaneous fH reached 7 beats min⁻¹ and was less than 20 beats min⁻¹ for over 5.5 min. Post-dive fH was high in the first 0.5–1 min after surfacing, but then declined to ~100 beats min⁻¹ towards the end of the surface interval.

Implications for pulmonary gas exchange

The moderate dive ƒH in short, shallow dives compared with the much slower ƒH of deep long-duration dives suggests more pulmonary blood flow and greater potential for reliance on lung O2. Most of these dives were to depths of less than 100 m (well below the estimated depth of lung collapse near 200 m), so maintenance of a moderate ƒH during these dives may allow sea lions to maximise use of the potentially significant lung O2 stores (~16% of total body O2 stores) throughout the dive. This is supported by venous blood O2 profiles, where, occasionally, there was no decrease in venous blood O2 between the beginning and end of the dive; this can only occur if pulmonary gas exchange continues throughout the dive. Greater utilization of the lung O2 store in sea lions is consistent with higher dive ƒH in other species that both dive on inspiration and typically perform shallow dives (dolphins, porpoises, some penguin species), and in deeper diving species when they perform shallow dives (emperor penguins).

In deeper dives of sea lions, although ƒH was lower and bradycardia more extreme, the diving ƒH profiles suggest that pulmonary gas exchange is also important. In long-duration dives, even though ƒH started to decrease upon or shortly after submergence, the decrease was not as abrupt as in phocids. Additionally, in long deep dives, despite having overall low dive ƒH, there were more heart beats before resting ƒH was reached compared with short, shallow dives. In dives less than 3 min in duration, there were ~10–15 beats until instantaneous ƒH reached resting values. In longer duration dives (>3 min), there were usually ~30–40 beats before instantaneous ƒH reached resting values. We suggest the greater number of heart beats early in these deeper dives enables more gas exchange and blood O2 uptake at shallow depths, thus allowing utilisation of the postulated larger respiratory O2 stores in deeper dives The less abrupt decline in ƒH we observed in sea lions is similar to the more gradual declines documented in emperor penguins and porpoises, where it has also been proposed that the gradual decrease in ƒH allows them to maximise pulmonary gas exchange at shallower depths. However, as sea lions swam deeper, ƒH decreased further (Figs 3, 6), and by 200 m depth (the approximate depth of lung collapse, instantaneous ƒH was 14 beats min⁻¹. Such an extreme decline in ƒH in conjunction with increased pulmonary shunting due to lung compression at greater depths will result in minimization of both O2 and N2 uptake by blood, even before the depth of full lung collapse (100% pulmonary shunt) is reached.

Implications for blood flow

ƒH is often used as a proxy to estimate blood flow and perfusion during diving because of the relative ease of its measurement. This is based on the assumption that stroke volume does not change during diving in sea lions, and, hence, changes in ƒH directly reflect changes in cardiac output. As breath-hold divers maintain arterial pressure while diving, changes in cardiac output should be associated with changes in peripheral vascular resistance and changes in blood flow to tissues. In Weddell seals, a decrease in cardiac output of ~85% during forced submersions resulted in an 80–100% decrease in tissue perfusion in all tissues excluding the brain, adrenal glands and lung. Sea lions exhibited extremely low instantaneous ƒH values that often remained low for significant portions of the dive (Figs 4, 6), suggesting severe decreases in tissue perfusion in dives greater than 5 min in duration. In almost all dives greater than 6 min in duration, instantaneous ƒH reached 10 beats min⁻¹, and stayed below 20 beats min⁻¹ for more than a minute. At a ƒH of 20 beats min⁻¹, cardiac output will be ~36% of resting cardiac output and only about 18% of average surface cardiac output. At these levels of cardiac suppression, most of this flow should be directed towards the brain and heart.

Conclusions

We successfully obtained diving ƒH profiles from a deep-diving otariid during natural foraging trips. We found that

(1) ƒH decreases during all dives, but true and more intense bradycardia only occurred in longer duration dives and
(2) in the longest duration dives, ƒH and presumed cardiac output were as low as 20% of resting values.

We conclude that, although initial high ƒH promotes gas exchange early in deep dives, the extremely low ƒH in late descent of deep dives (a) preserves lung O2, (b) conserves blood O2, (c) increases the dependence of muscle on myoglobin-bound O2 and (d) limits N2 absorption at depth. This ƒH profile, especially during the late descent/early bottom phase of deep dives is similar to that of deep-diving emperor penguins, and may be characteristic of deep diving endotherms that dive on inspiration.

Dive duration was the fixed effect in all models, and to account for the lack of independence caused by having many dives from the same individual, individual (sea lion ID) was included as a random effect. Covariance and random effect structures of the full models were evaluated using Akaike’s information criterion (AIC) and examination of residual plots. AICs from all the tested models are presented with the best model in bold.

Additionally, dives were classified as short-duration (less than 3 min, minimum cADL), mid-duration (3–5 min, range of cADLs) or long-duration (>5 min) dives. Differences in pre-dive ƒH, dive ƒH, minimum ƒH, post-dive ƒH, and heart beats to resting between the categories were investigated using mixed effects ANOVA, followed by post hoc Tukey tests. In all models, dive duration category was the fixed effect and individual (sea lion ID) was included as a random effect. Model fit was accessed by examination of the residuals. All means are expressed ±s.d. and results of the Tukey tests were considered significant at P<0.05. Statistical analysis was performed in R.

Investigating Annual Diving Behaviour by Hooded Seals (Cystophora cristata) within the Northwest Atlantic Ocean

Julie M. Andersen, Mette Skern-Mauritzen, Lars Boehme
PLoS ONE 8(11): e80438. http://dx.doi.org:/10.1371/journal.pone.0080438

With the exception of relatively brief periods when they reproduce and molt, hooded seals, Cystophora cristata, spend most of the year in the open ocean where they undergo feeding migrations to either recover or prepare for the next fasting period. Valuable insights into habitat use and diving behavior during these periods have been obtained by attaching Satellite Relay Data Loggers (SRDLs) to 51 Northwest (NW) Atlantic hooded seals (33 females and 18 males) during icebound fasting periods (200422008). Using General Additive Models (GAMs) we describe habitat use in terms of First Passage Time (FPT) and analyze how bathymetry, seasonality and FPT influence the hooded seals’ diving behavior described by maximum dive depth, dive duration and surface duration. Adult NW Atlantic hooded seals exhibit a change in diving activity in areas where they spend .20 h by increasing maximum dive depth, dive duration and surface duration, indicating a restricted search behavior. We found that male and female hooded seals are spatially segregated and that diving behavior varies between sexes in relation to habitat properties and seasonality. Migration periods are described by increased dive duration for both sexes with a peak in May, October and January. Males demonstrated an increase in dive depth and dive duration towards May (post-breeding/pre-molt) and August–October (post-molt/pre-breeding) but did not show any pronounced increase in surface duration. Females dived deepest and had the highest surface duration between December and January (post-molt/pre-breeding). Our results suggest that the smaller females may have a greater need to recover from dives than that of the larger males. Horizontal segregation could have evolved as a result of a resource partitioning strategy to avoid sexual competition or that the energy requirements of males and females are different due to different energy expenditure during fasting periods.

Novel locomotor muscle design in extreme deep-diving whales

P. Velten, R. M. Dillaman, S. T. Kinsey, W. A. McLellan and D. A. Pabst
The Journal of Experimental Biology 216, 1862-1871
http://dx.doi.org:/10.1242/jeb.081323

Most marine mammals are hypothesized to routinely dive within their aerobic dive limit (ADL). Mammals that regularly perform deep, long-duration dives have locomotor muscles with elevated myoglobin concentrations that are composed of predominantly large, slow-twitch (Type I) fibers with low mitochondrial volume densities (Vmt). These features contribute to extending ADL by increasing oxygen stores and decreasing metabolic rate. Recent tagging studies, however, have challenged the view that two groups of extreme deep-diving cetaceans dive within their ADLs. Beaked whales (including Ziphius cavirostris and Mesoplodon densirostris) routinely perform the deepest and longest average dives of any air-breathing vertebrate, and short-finned pilot whales (Globicephala macrorhynchus) perform high-speed sprints at depth. We investigated the locomotor muscle morphology and estimated total body oxygen stores of several species within these two groups of cetaceans to determine whether they

(1) shared muscle design features with other deep divers and
(2) performed dives within their calculated ADLs.

Muscle of both cetaceans displayed high myoglobin concentrations and large fibers, as predicted, but novel fiber profiles for diving mammals. Beaked whales possessed a sprinterʼs fiber-type profile, composed of ~80% fast-twitch (Type II) fibers with low Vmt. Approximately one-third of the muscle fibers of short-finned pilot whales were slow-twitch, oxidative, glycolytic fibers, a rare fiber type for any mammal. The muscle morphology of beaked whales likely decreases the energetic cost of diving, while that of short-finned pilot whales supports high activity events. Calculated ADLs indicate that, at low metabolic rates, both beaked and short-finned pilot whales carry sufficient onboard oxygen to aerobically support their dives.

Serial cross-sections of the m. longissimus dorsi of Mesoplodon densirostris

Fig. Serial cross-sections of the m. longissimus dorsi of Mesoplodon densirostris (A–D) and Globicephala macrorhynchus (E–H). Scale bars, 50μm. Muscle sections stained for the alkaline (A,E) and acidic (B,F) preincubations of myosin ATPase were used to distinguish Type I and II fibers. Muscle sections stained for succinate dehydrogenase (C,G) and α-glycerophosphate dehydrogenase (D,H) were used to distinguish glycolytic (gl), oxidative (o) and intermediate (i) fibers.

Previous studies of the locomotor muscles of deep-diving marine mammals have demonstrated that these species share a suite of adaptations that increase onboard oxygen stores while slowing the rate at which these stores are utilized, thus extending ADL. Their locomotor muscles display elevated myoglobin concentrations and are composed predominantly of large Type I fibers. Vmt are also lower in deep divers than in shallow divers or athletic terrestrial species. The results of this study indicate that beaked whales and short-finned pilot whales do not uniformly display these characteristics and that each possesses a novel fiber profile compared with those of other deep divers.

The phylogeny of Cetartiodactyla: The importance of dense taxon sampling, missing data, and the remarkable promise of cytochrome b to provide reliable species-level phylogenies

Ingi Agnarsson, Laura J. May-Collado
Molecular Phylogenetics and Evolution 48 (2008) 964–985
http://dx.doi.org:/10.1016/j.ympev.2008.05.046

We perform Bayesian phylogenetic analyses on cytochrome b sequences from 264 of the 290 extant cetartiodactyl mammals (whales plus even-toed ungulates) and two recently extinct species, the ‘Mouse Goat’ and the ‘Irish Elk’. Previous primary analyses have included only a small portion of the species diversity within Cetartiodactyla, while a complete supertree analysis lacks resolution and branch lengths limiting its utility for comparative studies. The benefits of using a single-gene approach include rapid phylogenetic estimates for a large number of species. However, single-gene phylogenies often differ dramatically from studies involving multiple datasets suggesting that they often are unreliable. However, based on recovery of benchmark clades—clades supported in prior studies based on multiple independent datasets—and recovery of undisputed traditional taxonomic groups, Cytb performs extraordinarily well in resolving cetartiodactyl phylogeny when taxon sampling is dense. Missing data, however, (taxa with partial sequences) can compromise phylogenetic accuracy, suggesting a tradeoff between the benefits of adding taxa and introducing question marks. In the full data, a few species with a short sequences appear misplaced, however, sequence length alone seems a poor predictor of this phenomenon as other taxa.

The mammalian superorder Cetartiodactyla (whales and eventoed ungulates) contains nearly 300 species including many of immense commercial importance (cow, pig, and sheep) and of conservation interest and aesthetic value (antelopes, deer, giraffe, dolphins, and whales) (MacDonald, 2006). Certain members of this superorder count among the best studied organisms on earth, whether speaking morphologically, behaviorally, physiologically or genetically. Understanding the interrelationships among cetartiodactyl species, therefore, is of obvious importance with equally short sequences were not conspicuously misplaced. Although we recommend awaiting a better supported phylogeny based on more character data to reconsider classification and taxonomy within Cetartiodactyla, the new phylogenetic hypotheses provided here represent the currently best available tool for comparative species-level studies within this group. Cytb has been sequenced for a large percentage of mammals and appears to be a reliable phylogenetic marker as long as taxon sampling is dense. Therefore, an opportunity exists now to reconstruct detailed phylogenies of most of the major mammalian clades to rapidly provide much needed tools for species-level comparative studies.

Our results support the following relationship among the four major cetartiodactylan lineages (((Tylopoda ((Cetancodonta (Ruminantia + Suina))), with variable support. This arrangement has not been suggested previously, to our knowledge (see review in O’Leary and Gatesy, 2008 and discussion).

Relationships among clades within Cetancodonta are identical to those found by May-Collado and Agnarsson (2006).

Within Ruminantia all our analyzes suggest the following relationships among families: (((((Tragulidae((((Antilocapridae(((Giraffidae(( Cervidae(Moschidae + Bovidae))))) with relatively high support, supporting the subdivision of Ruminantia into Tragulina and Pecora.
In the rare cases where our results are inconsistent with benchmark clades, ad hoc explanations seem reasonable. The placement of M. meminna (Tragulidae) within Bovidae is likely an artifact of missing data, although remarkably it is the only conspicuous misplacement of a species across the whole phylogeny at the family level (while three species appear to be misplaced at the subfamily level within Cervidae in the full analysis, see Fig. 5a). This is supported by the fact that the placement of Moschiola receives low support, and the removal of Moschiola prior to analysis increases dramatically the support for clades close to where it nested (not shown, analysis available from authors), suggesting it had a tendency to ‘jump around’. Two other possibilities cannot be ruled out, however. One, that possibly the available sequence in Genbank may be mislabeled. And second, it should be kept in mind that the validity of Tragulidae has never been tested with molecular data including more than two species.

Oxygen and carbon dioxide fluctuations in burrows of subterranean blind mole rats indicate tolerance to hypoxic–hypercapnic stresses

Imad Shams, Aaron Avivi, Eviatar Nevo
Comparative Biochemistry and Physiology, Part A 142 (2005) 376 – 382
http://dx.doi.org:/10.1016/j.cbpa.2005.09.003

The composition of oxygen (O2), carbon dioxide (CO2), and soil humidity in the underground burrows from three species of the Israeli subterranean mole rat Spalax ehrenbergi superspecies were studied in their natural habitat. Two geographically close populations of each species from contrasting soil types were probed. Maximal CO2 levels (6.1%) and minimal O2 levels (7.2%) were recorded in northern Israel in the breeding mounds of S. carmeli in a flooded, poor drained field of heavy clay soil with very high volumetric water content. The patterns of gas fluctuations during the measurement period among the different Spalax species studied were similar. The more significant differentiation in gas levels was not among species, but between neighboring populations inhabiting heavy soils or light soils: O2 was lower and CO2 was higher in the heavy soils (clay and basaltic) compared to the relatively light soils (terra rossa and rendzina). The extreme values of gas concentration, which occurred during the rainy season, seemed to fluctuate with partial flooding of the tunnels, animal digging activity, and over-crowded breeding mounds inhabited by a nursing female and her offspring. The gas composition and soil water content in neighboring sites with different soil types indicated large differences in the levels of hypoxic–hypercapnic stress in different populations of the same species. A growing number of genes associated with hypoxic stress have been shown to exhibit structural and functional differences between the subterranean Spalax and the aboveground rat (Rattus norvegicus), probably reflecting the molecular adaptations that Spalax went through during 40 million years of evolution to survive efficiently in the severe fluctuations in gas composition in the underground habitat.

map of the studied sites

Schematic map of the studied sites: S. galili (2n =52): 1— Rehania (chalk); 2— Dalton (basaltic); S. golani (2n =54): 3— Majdal Shams (terra tossa); 4—Masa’ada (basaltic soils); S. carmeli (2n =58): 5— Al-Maker (heavy clay); 6— Muhraqa (terra rossa).

Comparison of gas composition (O2 and CO2) and water content between light and heavy soils inhabited by S. carmeli

Comparison of gas composition (O2 and CO2) and water content between light and heavy soils inhabited by S. carmeli, Al-Maker (heavy soil) and Muhraqa (light soil). AverageTSD of measurements in the burrows of approximately 10 animals at a given date is presented. **p <0.01, T-test and Mann– Whitney test).

Subterranean mammals, which live in closed underground burrow systems, experience an atmosphere that is different from the atmosphere above-ground. Gas exchange between these two atmospheres depends on diffusion through the soil, which in turn, depends on soil particle size, water content, and burrow depth. Heavy soils (clay and basaltic), hold water and have little air space for gas diffusion. A large deviation from external gas composition is found in the burrows of Spalax living in these soil types. The maximal measured concentration of CO2 was 6.1% in Spalax breeding mounds, which is one of the highest concentrations among studied mammals in natural conditions. At the same time 7.2% O2 was measured in water saturated heavy clay soil

seasonal variation from August to March in mean O2, CO2, and soil water content

Example of seasonal variation from August to March in mean O2, CO2, and soil water content (VWC) in the Al-Maker population (2n =58, heavy soil). Values are presented as mean TSD.

In this study new data were presented for a wild mammal that survives in an extreme hypoxic–hypercapnic environment. Interestingly, the very low concentrations of O2 experienced by Spalax are correlated with the expression pattern of hypoxia related genes. So far, we have shown higher and longer-term mRNA expression of erythropoietin, the main factor that regulates the level of circulating red blood cells, in subterranean Spalax compared to the above-ground rat in response to hypoxic stress, as well as differences in the response of erythropoietin to hypoxia in different populations of Spalax experiencing different hypoxic stress in nature. We also demonstrated that erythropoietin pattern of expression is different in Spalax than in Rattus throughout development, a pattern suggesting more efficient hypoxic tolerance in Spalax starting as early as in the embryonic stages. Furthermore, vascular endothelial growth factor (VEGF), which is a critical angiogenic factor that responds to hypoxia, is constitutively expressed at maximal levels in Spalax muscles, the most energy consuming tissue during digging. This level is 1.6-fold higher than in Rattus muscles and is correlated with significantly higher blood vessel concentration in the Spalax muscles compared to the Rattus muscles. Likewise, myoglobin the globin involved in oxygen homeostasis in skeletal muscles, exhibits different expression pattern under normoxia and in response to hypoxia in Spalax muscles compared to rat muscles as well as between different populations of Spalax exposed to different hypoxic stress in nature (unpublished results). Similarly, neuroglobin, a brain-specific globin involved in reversible oxygen binding, i.e., presumably in cellular homeostasis, is expressed differently in the Spalax brain compared to Rattus brain. Like erythropoietin and myoglobin also neuroglobin is expressed differently in Spalax populations experiencing different oxygen supply (unpublished results). Furthermore, Spalax p53 harbors two amino acid substitutions in its binding domain, which are identical to mutations found in p53 of human cancer cells. These substitutions endow Spalax p53 with several-fold higher activation of cell arrest and DNA repair genes compared to human p53 and favor activation of DNA repair genes over apoptotic genes. The study of specific tumoral variants indicates that such preference of growth arrest over apoptosis possibly results as a response to the hypoxic environmental stress known in tumors. Differences in the structure of other molecules related to homeostasis, namely, hemoglobin, haptoglobin (Nevo, 1999), and cytoglobin (unpublished) were also observed in Spalax.

Stress, adaptation, and speciation in the evolution of the blind mole rat, Spalax, in Israel

Eviatar Nevo
Molecular Phylogenetics and Evolution 66 (2013) 515–525
http://dx.doi.org/10.1016/j.ympev.2012.09.008

Environmental stress played a major role in the evolution of the blind mole rat superspecies Spalax ehrenbergi, affecting its adaptive evolution and ecological speciation underground. Spalax is safeguarded all of its life underground from aboveground climatic fluctuations and predators. However, it encounters multiple stresses in its underground burrows including darkness, energetics, hypoxia, hypercapnia, food scarcity, and pathogenicity. Consequently, it evolved adaptive genomic, proteomic, and phenomic complexes to cope with those stresses. Here I describe some of these adaptive complexes, and their theoretical and applied perspectives. Spalax mosaic molecular and organismal evolution involves reductions or regressions coupled with expansions or progressions caused by evolutionary tinkering and natural genetic engineering. Speciation of Spalax in Israel occurred in the Pleistocene, during the last 2.00–2.35 Mya, generating four species associated intimately with four climatic regimes with increasing aridity stress southwards and eastwards representing an ecological speciational adaptive trend: (Spalax golani, 2n = 54?S. galili, 2n = 52?S. carmeli, 2n = 58?S. judaei, 2n = 60). Darwinian ecological speciation occurred gradually with relatively little genetic change by Robertsonian chromosomal and genic mutations. Spalax genome sequencing has just been completed. It involves multiple adaptive complexes to life underground and is an evolutionary model to a few hundred underground mammals. It involves great promise in the future for medicine, space flight, and deep-sea diving.

Stress is a major driving force of evolution (Parsons, 2005; Nevo, 2011). Parsons defined stress as the ‘‘environmental factor causing potential injurious changes to biological systems with a potential for impacts on evolutionary processes’’. The global climatic transition from the middle Eocene to the early Oligocene (45–35 Ma = Million years ago) led to extensive convergent evolution underground of small subterranean mammals across the planet (Nevo, 1999; Lacey et al., 2000; Bennett and Faulkes, 2000; Begall et al., 2007). The subterranean ecotope provided small mammals with shelter from predators and extreme aboveground climatic stressful fluctuations of temperature and humidity. However, they had to evolve genomic adaptive complexes for the immense underground stresses of darkness, energy for burrowing in solid soil, low productivity and food scarcity, hypoxia, hypercapnia, and high infectivity. These stresses have been described in Nevo (1999, 2011) and Nevo et al. (2001); and Nevo list of Spalax publication at http://evolution.haifa.ac.il with many cited references relevant to these stresses).

blind subterranean mole rat of the Spalax ehrenbergi superspecies

The blind subterranean mole rat of the Spalax ehrenbergi superspecies in Israel. An extreme example of adaptation to life underground

Circadian rhythm and genes

adaptive circadian genes. We identified the circadian rhythm of Spalax
(Nevo et al., 1982) and described, cloned, sequenced, and expressed several circadian genes in Spalax. These include Clock, MOP3, three Period (Per), and cryptochromes (Avivi et al., 2001, 2002, 2003). The Spalax circadian genes are differentially conserved, yet characterized by a significant number of amino acid substitutions. The glutamine-rich area of Clock, which is assumed to function in circadian rhythmicity, is expanded in Spalax compared with that of mice and humans and is different in amino acid composition from that of rats. All three Per genes of Spalax oscillate with a periodicity of 24 h in the suprachaismatic nucleus, eye, and Harderian gland and are expressed in peripheral organs. Per genes are involved in clock resetting. Spalax Per 3 is unique in mammals though its function is still unresolved. The Spalax Per genes contribute to the unique adaptive circadian rhythm to life underground. The cryptochrome (Cry) genes, found in animals and plants, act both as photoreceptors and as ingredients of the negative feedback mechanism of the biological Clock. The CRY 1 protein is significantly closer to the human homolog than to that of mice, as was also shown in parts of the immunogenetic system. Both Cry 1 and Cry 2 mRNAs were found in the SCN, eye, harderian gland, and in peripheral tissues. Remarkably, the distinctly hypertrophied harderian gland is central in Spalax’s unique underground circadian rhythmicity (Pevet et al., 1984).

Spalax eye mosaic evolution
Gene expression in the eye of Spalax
Brain evolution in Spalax to underground stresses
Spalax: four species in Israel

The morphological, physiological, and behavioral Spalax eye patterns are underlain by gene expression representing regressive and progressive associated transcripts. Regressive transcripts involve B-2 microglobulin, transketolase, four keratins, alpha enolase, and different heat shock proteins. Several proteins may be involved in eye degeneration. These include heat shock protein 90alpha (hsp90alpha), found also in the blind fish Astyanax mexicanus, two transcripts of programmed cell death proteins, oculospanin, and peripherin 2, both belonging to the Tetraspanin family, in which 60 different mutations cause eye degeneration in humans. Several progressive transcripts in the Spalax eye are found in the retina of many mammals involving gluthatione, peroxidase 4, B spectrin, and Ankyrin; the last two characterize rod cells in the retina. Some transcripts are involved in metabolic processing of retinal, a vertebrate key component in phototransduction, and a relative of vitamin A.

cross section of the developing eye of the mole rat

Light micrographs showing cross section of the developing eye of the mole rat Spalax ehrenbergi. (A) Optic cup and lens vesicle initially develop normally (x100). (B) Eye at a later embryonic stage. Note appearance of iris-ciliary body rudiment (arrows), and development of the lens nucleus (L). ON, optic nerve (x100). (C) Eye at a still later fetal stage. Note massive growth of the iris-ciliary body complex colobomatous opening (arrow) (x100). (D) Early postnatal stage. The iris-ciliary body complex completely fills the chamber. The lens is vascularized and vacuolated (x100). (E) Adult eye. Eyelids are completely closed and pupil is absent. Note atrophic appearance of the optic disc region (arrow) (x65). (F) Higher magnification of the adult retina. The different retinal layers are retained: PE, pigment epithelium: RE, receptor layer; ON, outer nuclear layer: IN, inner nuclear layer; GC, ganglion cell layer (x500) (from Sanyal et al., 1990, Fig. 1).

The brains of subterranean mammals underwent dramatic evolution in accordance with underground stresses for digging and photoperiodic perception associated with vibrational, tactile, vocal, olfactory, and magnetic communication systems replacing sight, as is seen in Spalax. The brain of Spalax is twice as large as that of the laboratory rat of the same body size. The somatosensory region in the isocortex of Spalax is 1.7 times, the thalamic nuclei 1.3 times, and the motor cortex 3.1 times larger than in the sighted laboratory rat Rattus norvegicus matched to body size.

The ecological stress determinant in Spalax brain evolution is highlighted by the four species of the Spalax ehrenbergi superspecies in Israel. They differentiated chromosomally (by means of Robertsonian mutations and fission), allopatrically, and clinally southwards into four species associated with different climatic regimes, following the gradient of increasing aridity stress and decreasing predictability southwards towards the desert: Spalax galili (2n = 52) ->S. golani (2n = 54)->S. carmeli (2n = 58)->S. judaei (2n = 60), and eastwards S. galili ->S. golani (2n = 52–>54) (Fig. 2). This chromosomal speciation trend southwards is associated with the regional aridity stress southwards (and eastwards) in Israel, budding new species adapted genomically, proteomically, and phenomically (i.e., in morphology, physiology, and behavior) to increasing stresses of higher solar radiation, temperature, and drought southwards (Nevo, 1999; Nevo et al., 2001; Nevo
list of Spalax at http://evolution.haifa.ac.il). A uniquely recent discovery of incipient sympatric ecological speciation at a microscale in Spalax triggered by local stresses occurs within Spalax galili.

retinal input to primary visual structures in Spalax

Relative degree of retinal input to primary visual structures in Spalax, hamster, rat, and Spalacopus cyanus (South American Octodontidae, ‘‘coruro’’). These rodents are of similar body size (120–140 g). B. Relative degree of change in the proportions of retinal input to different primary visual structures in Spalax compared with measures obtained in other rodents. A relative progressive development in Spalax is seen in structures involved in photoperiodic and neuroendocrine functions (SCN, BNST).The main regressive feature is the drastic relative reduction of retinal input to the superior colliculus. The main regressive feature is the drastic reduction of retinal input to the superior colliculus. The relative size of other visual structures in Spalax is modified compared to that of the other species. c. Comparison of the absolute size (volume, mm³ x 10^-4) of visual structures in Spalax and other rodents. The size of the SCN is equivalent in all species. The vLGN and dLGN are reduced by 87–93% in Spalax. The retino-recipient layers of the superior colliculus are reduced by 97%. Abbreviations: SCN: suprachiasmatic nucleus; BNST: bed nucleus of the stria terminalis; dLGN: dorsal lateral geniculate nucleus; SC: superior colliculus [From Cooper et al., 1993 (Fig 3)].

Subterranean life has a high energetic cost if an animal has to burrow in order to obtain its food. For a 150 g Thomomys bottae, burrowing 1 m may be 360–3400 times more expensive energetically than moving the same distance on the surface (Vleck, 1979). Mean rates of oxygen consumption during burrowing at 22 ^oC are from 2.8 to 7.1 times the RMR. Vleck developed a model examining the energetics of foraging by burrowing and found that, in the desert, Thomomys adjusts the burrow segment length to minimize the cost of burrowing. Since burrowing becomes less economic as body size increases, Vleck (1981) predicted that the maximum possible body size that a subterranean mammal can attain depends on a balance between habitat productivity and the cost of burrowing in local soils. Vleck’s cost of burrowing hypothesis has been verified in multiple cases. Heth (1989) demonstrated longer burrows in the rendzina soil and shorter ones in the terra rossa soil, associating lower productivity in the former for Spalax.

Food is a limiting factor for subterranean mammals. The abundance and distribution of food explain some of the ecological, physiological, and behavioral characteristics of subterranean mammals. In a field test of Spalax foraging strategy, we concluded that Spalax was a generalist due to the constraints of the subterranean ecotope. Restricted foraging time primarily during the winter when soil is wet, and the high energetic investment of tunneling to get to food items is significantly reduced than in summertime.
We also identified a decrease in the basic metabolic rate towards the desert, i.e., economizing energetics. The maintenance of adequate O2 transport in a subterranean mammal confronting hypoxia requires adaptation along the O2 transport system, achieved by increasing the flow of O2 in the convection systems (ventilation and perfusion) and by reduction of oxygen pressure (PO2) gradients at the diffusion barriers (lung blood, blood-tissue (Arieli, 1990). The PO2 gradient between blood capillaries and respiring mitochondria capillaries is large, and any adaptation at this level could be significant for O2 transport. Reduction of diffusion distance in a muscle can be achieved, like in Spalax, by increasing the number of capillaries that surround muscle fiber or by reducing fiber areas.

Geographic distribution in Israel of the four chromosomal species belonging to the S. ehrenbergi superspecies

Geographic distribution in Israel of the four chromosomal species belonging to the S. ehrenbergi superspecies that are separated by narrow hybrid zones (2n = 52, 54, 58, and 60, now named as S. galili, S. golani, S. carmeli, and S. judaei, respectively; see Nevo et al., 2001).

Spalacid evolution, based on mtDNA, is driven by climatic oscillations and stresses. The underground ecotope provided subterranean mammals with shelter from extreme climate (temperature and humidity) fluctuations, and predators. However, they had to extensively and intensively adapt to the multiple underground stresses (darkness, energetic, low productivity and
food scarcity, hypoxia, hypercapnia, and high infectivity). All subterranean mammals, including spalacids as an extreme case, share convergent molecular and organismal adaptations to their shared unique underground ecotope. Evolution underground, as exemplified here in spalacids, led to mosaic molecular and organismal evolutionary syndromes to cope with multiple stresses.

Speciation involves all rates – from gradual to rapid. Subterranean mammals, with the spalacid example discussed above, provide uniquely rich evolutionary global tests of speciation and adaptation, convergence, regression, progression, and mosaic evolutionary processes. Adaptation and speciation underground was one of the most dramatic natural experiments verifying Darwinian evolution.

The Spalax genome sequencing has just been completed. It is being analyzed and will soon be published in 2012. This will be a milestone in understanding how numerous mammals across the globe, who found underground shelter from climatic fluctuations and stresses above ground, cope with the new suite of stresses they encountered underground, demanding a new engineering overhaul on all organizational levels, selecting for adaptive complexes to cope with the new underground stresses. The main current and future challenges are to compare and contrast genome sequences and identify the genomic basis of adaptation and speciation.

This global Cenozoic experiment could answer the following open questions: How heterozygous is the whole genome? How prevalent are retrotransposons and what is their functional role? How many genes are involved in the Spalax genome and how are they regulated? What are the genic and regulatory networks resisting the multiple stresses underground? How much of the Spalax genome is conserved and how much is reorganized to cope with the underground stresses? How is the solitary blind mole rat, Spalax, different from the social naked mole rat Heterocephalus? How are the processes of reduction, expansion, and genetic tinkering and engineering reflected across the genome? How effective is copy number variation in regulation? Is there similarity in the transcriptomes of subterranean mammals? How could we harness the rich genome repertoire of Spalax to revolutionize medicine, especially in the realm of hypoxia tolerance and the related major diseases of the western world, e.g., cancer, stroke, and cardiovascular diseases? What is the phylogenetic origin of Spalax? How much of the Spalax genome represents its phylogenetic roots and how much of coding and noncoding genomic regions are shared with other subterranean mammals across the globe in adapting to life underground?

The Atmospheric Environment of the Fossorial Mole Rat (Spalax Ehrenbergi): Effects of Season, Soil Texture, Rain, Temperature and Activity

Arieli
Comp Biochen Physiol. 1978; 63A:569-5151. The fossorial mole rat (Spalax ehrenbergi) may inhabit heavy soil with low gas permeability.
Air composition in burrows in heavy soil deviates from atmospheric air more than that of burrows in light soil.
In winter and spring O2 and CO2 concentrations in breeding mounds were 16.5% O2 and 2.5-3x CO2 and the extreme values measured were 14.0% O2 and 4.8% Cot.
Hypoxia and hypercapnia in the burrow develop shortly after rain and when ambient temperature drops.
Composition of the burrows air is influenced by the solubility of CO2 in soil water and by faster penetration of oxygen than outflowing of CO2.

Hypo-osmotic stress-induced physiological and ion-osmoregulatory responses in European sea bass (Dicentrarchus labrax) are modulated differentially by nutritional status

Amit Kumar Sinha, AF Dasan, R Rasoloniriana, N Pipralia, R Blust, G De Boeck
Comparative Biochemistry and Physiology, Part A 181 (2015) 87–99
http://dx.doi.org/10.1016/j.cbpa.2014.11.024

We investigated the impact of nutritional status on the physiological, metabolic and ion-osmoregulatory performance of European sea bass (Dicentrarchus labrax)when acclimated to seawater (32 ppt), brackishwater (20 and 10 ppt) and hyposaline water (2.5 ppt) for 2 weeks. Following acclimation to different salinities, fish were either fed or fasted (unfed for 14 days). Plasma osmolality, [Na+], [Cl−] and muscle water contentwere severely altered in fasted fish acclimated to 10 and 2.5 ppt in comparison to normal seawater-acclimated fish, suggesting ion regulation and acid–base balance disturbances. In contrast to feed-deprived fish, fed fish were able to avoid osmotic perturbation more effectively. This was accompanied by an increase in Na+/K+-ATPase expression and activity, transitory activation of H+-ATPase (only at 2.5 ppt) and down-regulation of Na+/K+/2Cl− gene expression. Ammonia excretion rate was inhibited to a larger extent in fasted fish acclimated to low salinities while fed fish were able to excrete efficiently. Consequently, the build-up of ammonia in the plasma of fed fish was relatively lower. Energy stores, especially glycogen and lipid, dropped in the fasted fish at low salinities and progression towards the anaerobic metabolic pathway became evident by an increase in plasma lactate level. Overall, the results indicate no osmotic stress in both feeding treatments within the salinity range of 32 to 20 ppt. However, at lower salinities (10–2.5 ppt) feed deprivation tends to reduce physiological, metabolic, ion-osmo-regulatory and molecular compensatory mechanisms and thus limits the fish’s abilities to adapt to a hypo-osmotic environment.

The absence of ion-regulatory suppression in the gills of the aquatic air-breathing fish Trichogaster lalius during oxygen stress

Chun-Yen Huang, Hsueh-Hsi Lin, Cheng-Huang Lin, Hui-Chen Lin
Comparative Biochemistry and Physiology, Part A 179 (2015) 7–16
http://dx.doi.org/10.1016/j.cbpa.2014.08.017

The strategy for most teleost to survive in hypoxic or anoxic conditions is to conserve energy expenditure, which can be achieved by suppressing energy-consuming activities such as ion regulation. However, an air-breathing fish can cope with hypoxic stress using a similar adjustment or by enhancing gas exchange ability, both behaviorally and physiologically. This study examined Trichogaster lalius, an air-breathing fish without apparent gill modification, for their gill ion-regulatory abilities and glycogen utilization under a hypoxic treatment. We recorded air-breathing frequency, branchial morphology, and the expression of ion-regulatory proteins (Na+/K+-ATPase and vacuolar-type H+-ATPase) in the 1st and 4th gills and labyrinth organ (LO), and the expression of glycogen utilization (GP, glycogen phosphorylase protein expression and glycogen content) and other protein responses (catalase, CAT; carbonic anhydrase II, CAII; heat shock protein 70, HSP70; hypoxia-inducible factor-1α, HIF-1α; proliferating cell nuclear antigen, PCNA; superoxidase dismutase, SOD) in the gills of T. lalius after 3 days in hypoxic and restricted conditions. No morphological modification of the 1st and 4th gills was observed. The air breathing behavior of the fish and CAII protein expression both increased under hypoxia. Ion-regulatory abilities were not suppressed in the hypoxic or restricted groups, but glycogen utilization was enhanced within the groups. The expression of HIF-1α, HSP70 and PCNA did not vary among the treatments. Regarding the antioxidant system, decreased CAT enzyme activity was observed among the groups. In conclusion, during hypoxic stress, T. lalius did not significantly reduce energy consumption but enhanced gas exchange ability and glycogen expenditure.

The combined effect of hypoxia and nutritional status on metabolic and ionoregulatory responses of common carp (Cyprinus carpio)

Sofie Moyson, HJ Liew, M Diricx, AK Sinha, R Blusta, G De Boeck
Comparative Biochemistry and Physiology, Part A 179 (2015) 133–143
http://dx.doi.org/10.1016/j.cbpa.2014.09.017

In the present study, the combined effects of hypoxia and nutritional status were examined in common carp (Cyprinus carpio), a relatively hypoxia tolerant cyprinid. Fish were either fed or fasted and were exposed to hypoxia (1.5–1.8mgO2 L−1) at or slightly above their critical oxygen concentration during 1, 3 or 7 days followed by a 7 day recovery period. Ventilation initially increased during hypoxia, but fasted fish had lower ventilation frequencies than fed fish. In fed fish, ventilation returned to control levels during hypoxia, while in fasted fish recovery only occurred after reoxygenation. Due to this, C. carpio managed, at least in part, to maintain aerobic metabolism during hypoxia: muscle and plasma lactate levels remained relatively stable although they tended to be higher in fed fish (despite higher ventilation rates). However, during recovery, compensatory responses differed greatly between both feeding regimes: plasma lactate in fed fish increased with a simultaneous breakdown of liver glycogen indicating increased energy use, while fasted fish seemed to economize energy and recycle decreasing plasma lactate levels into increasing liver glycogen levels. Protein was used under both feeding regimes during hypoxia and subsequent recovery: protein levels reduced mainly in liver for fed fish and in muscle for fasted fish. Overall, nutritional status had a greater impact on energy reserves than the lack of oxygen with a lower hepatosomatic index and lower glycogen stores in fasted fish. Fasted fish transiently increased Na+/K+-ATPase activity under hypoxia, but in general ionoregulatory balance proved to be only slightly disturbed, showing that sufficient energy was left for ion regulation.

The effect of temperature and body size on metabolic scope of activity in juvenile Atlantic cod Gadus morhua L.

Bjørn Tirsgaard, Jane W. Behrens, John F. Steffensen
Comparative Biochemistry and Physiology, Part A 179 (2015) 89–94
http://dx.doi.org/10.1016/j.cbpa.2014.09.033

Changes in ambient temperature affect the physiology and metabolism and thus the distribution of fish. In this study we used intermittent flow respirometry to determine the effect of temperature (2, 5, 10, 15 and 20 °C) and wet body mass (BM) (~30–460 g) on standard metabolic rate (SMR, mg O2 h−1), maximum metabolic rate (MMR, mg O2 h−1) and metabolic scope (MS, mg O2 h−1) of juvenile Atlantic cod. SMR increased with BM irrespectively of temperature, resulting in an average scaling exponent of 0.87 (0.82–0.92). Q10 values were 1.8–2.1 at temperatures between 5 and 15 °C but higher (2.6–4.3) between 2 and 5 °C and lower (1.6–1.4) between 15 and 20 °C in 200 and 450 g cod. MMR increased with temperature in the smallest cod (50 g) but in the larger cod MMR plateaued between 10, 15 and 20 °C. This resulted in a negative correlation between the optimal temperature for MS (T_opt) and BM, T_opt being respectively 14.5, 11.8 and 10.9 °C in a 50, 200 and 450 g cod. Irrespective of BM cold water temperatures resulted in a reduction (30–35%) of MS whereas the reduction of MS at warm temperatures was only evident for larger fish (200 and 450 g), caused by plateauing of MMR at 10 °C and above. Warm temperatures thus seem favorable for smaller (50 g) juvenile cod, but not for larger conspecifics (200 and 450 g).

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Altitude Adaptation

Posted in Amino acids, Biochemical pathways, Biological Networks, Ca2+ triggered activation, Cardiovascular Research, Cell Biology, Curation, Developmental biology, Enzymes and isoenzymes, Fatty acids, Gene Regulation and Evolution, Hematology, Hematopoiesis, Lipid metabolism, Liver & Digestive Diseases Research, Metabolism, Mutant Gene Expression, Myocardial Metabolism, Nitric Oxide in Health and Disease, Proteins, Proteomics, Signaling, Signaling & Cell Circuits, tagged acute high altitude sickness, altitude adaptation, Andeas, cewrebral edema, chronic altitude sickness, cognitive effects, Evolution, hemoglobin gene substitutions, hemoglobins, high elevation plants, Himalayan Plateau, mammals, mountain antelope, Mountain sickness, pulmonary edema, Tibetan goat, Tibetan sheep on February 24, 2015| Leave a Comment »

Altitude Adaptation

Writer and Curator: Larry H. Bernstein, MD, FCAP

Introduction

Land adapted animals depend on respiration for oxygen supply, but have adapted to altitudes that have difference oxygen contents. In this discussion we explore how animals have adapted to oxygen supply in different terrestrial habitats, and also how humans adjust to short term changes in high and extreme altitudes.

High-altitude adaptation is an evolutionary modification in animals, most notably in birds and mammals, by which species are subjected to considerable physiological changes to survive in extremely high mountainous environments. As opposed to short-term adaptation, or more properly acclimatization (which is basically an immediate physiological response to changing environment), the term “high-altitude adaptation” has strictly developed into the description of an irreversible, long-term physiological responses to high-altitude environments, associated with heritable behavioral and genetic changes. Perhaps, the phenomenon is most conspicuous, at least best documented, in human populations such as the Tibetans, the South Americans and the Ethiopians, who live in the otherwise uninhabitable high mountains of the Himalayas, Andes and Ethiopia respectively; and this represents one of the finest examples of natural selection in action.

Oxygen, essential for animal life, is proportionally abundant in the atmosphere with height from the sea level; hence, the highest mountain ranges of the world are considered unsuitable for habitation. Surprisingly, some 140 million people live permanently at high altitudes (>2,500 m) in North, Central and South America, East Africa, and Asia, and flourish very well for millennia in the exceptionally high mountains, without any apparent complications. This has become a recognized instance of the process of Darwinian evolution in humans acting on favorable characters such as enhanced respiratory mechanisms. As a matter of fact, this adaptation is so far the fastest case of evolution in humans that is scientifically documented. Among animals only few mammals (such as yak, ibex, Tibetan gazelle, vicunas, llamas, mountain goats, etc.) and certain birds are known to have completely adapted to high-altitude environments.

These adaptations are an example of convergent evolution, with adaptations occurring simultaneously on three continents. Tibetan humans and Tibetan domestic dogs found the genetic mutation in both species, EPAS1. This mutation has not been seen in Andean humans, showing the effect of a shared environment on evolution.

At elevation higher than 8,000 metres (26,000 ft), which is called the “death zone” in mountaineering, the available oxygen in the air is so low that it is considered insufficient to support life. And higher than 7,600 m is seriously lethal. Yet, there are Tibetans, Ethiopians and Americans who habitually live at places higher than 2,500 m from the sea level. For normal human population, even a brief stay at these places means mountain sickness, which is a syndrome of hypoxia or severe lack of oxygen, with complications such as fatigue, dizziness, breathlessness, headaches, insomnia, malaise, nausea, vomiting, body pain, loss of appetite, ear-ringing, blistering and purpling and of the hands and feet, and dilated veins. Amazingly for the native highlanders, there are no adverse effects; in fact, they are perfectly normal in all respects. Basically, the physiological and genetic adaptations in these people involve massive modification in the oxygen transport system of the blood, especially molecular changes in the structure and functions hemoglobin, a protein for carrying oxygen in the body. This is to compensate for perpetual low oxygen environment. This adaptation is associated with better developmental patterns such as high birth weight, increased lung volumes, increased breathing, and higher resting metabolism.

http://en.wikipedia.org/wiki/High-altitude_adaptation

Acute Mountain Sickness: Pathophysiology, Prevention, and Treatment

Chris Imraya, Alex Wright, Andrew Subudhie,, Robert Roache
Progress in Cardiovascular Diseases 52 (2010) 467–484
http://dx.doi.org:/10.1016/j.pcad.2010.02.003

Barometric pressure falls with increasing altitude and consequently there is a reduction in the partial pressure of oxygen resulting in a hypoxic challenge to any individual ascending to altitude. A spectrum of high altitude illnesses can occur when the hypoxic stress outstrips the subject’s ability to acclimatize. Acute altitude-related problems consist of the common syndrome of acute mountain sickness, which is relatively benign and usually self-limiting, and the rarer, more serious syndromes of high-altitude cerebral edema and high-altitude pulmonary edema. A common feature of acute altitude illness is rapid ascent by otherwise fit individuals to altitudes above 3000 m without sufficient time to acclimatize. The susceptibility of an individual to high altitude syndromes is variable but generally reproducible. Prevention of altitude-related illness by slow ascent is the best approach, but this is not always practical. The immediate management of serious illness requires oxygen (if available) and descent of more than 300 m as soon as possible. In this article, we describe the setting and clinical features of acute mountain sickness and high altitude cerebral edema, including an overview of the known pathophysiology, and explain contemporary practices for both prevention and treatment exploring the comprehensive evidence base for the various interventions.

Acute mountain sickness (AMS) and high-altitude cerebral edema (HACE) strike people who travel too fast to high altitudes that lie beyond their current level of acclimatization. Understanding AMS and HACE is important because AMS can sharply limit recreation and work at high altitude. The syndromes can be identified early and reliably without sophisticated instruments, and when AMS and HACE are recognized early, most cases respond rapidly with complete recovery in a few hours (AMS) to days (HACE).

High-altitude headache (HAH) is the primary symptom of AMS. High-altitude headache in AMS usually occurs with some combination of other symptoms.
These are – insomnia, fatigue (beyond that expected from the day’s activities), dizziness, anorexia, and nausea. The headache often worsens during the night and with exertion. Insomnia is the next most frequent complaint. Poor sleep can occur secondary to periodic breathing, severe headache, dizziness, and shortness of breath, among other causes. Anorexia and nausea are common, with vomiting reported less frequently in trekkers to 4243 m.

AMS is distinguished only by symptoms. The progression of AMS to HACE is marked by altered mental status, including impaired mental capacity, drowsiness, stupor, and ataxia. Coma may develop as soon as 24 hours after the onset of ataxia or change in mental status. The severity of AMS can be scored using the Lake Louise Questionnaire, or the more detailed Environmental Symptoms Questionnaire, or by the use of a simple analogue scale. Today, more than 100 years after the first clear clinical descriptions of AMS and HACE, we have advanced our understanding of the physiology of acclimatization to high altitude, and the pathophysiology of AMS and HACE.

As altitude increases, barometric pressure falls (see Fig ). This fall in barometric pressure causes a corresponding drop in the partial pressure of oxygen (21% of barometric pressure) resulting in hypobaric hypoxia. Hypoxia is the major challenge humans face at high altitude, and the primary cause of AMS and HACE. It follows that oxygen partial pressure is more important than
geographic altitude, as exemplified near the poles where the atmosphere is thinner and, thus, barometric pressure is lower. Lower barometric pressure at the poles can result in oxygen partial pressures that are physiologically equivalent to altitudes 100 to 200 m higher at more moderate latitudes. We define altitude regions as high altitude (1500-3500 m), very high altitude (3500-5500 m), and extreme altitude (>5500 m).

Neurological consequences of increasing altitude

Neurological consequences of increasing altitude: The relation among altitude (classified as high [1500–3500 m], very high [3500-5500 m] and extreme [>5500 m]), the partial pressure of oxygen, and the neurological consequences of acute and gradual exposure to these pressure changes. Neurological consequences will vary greatly from person to person and with rate of ascent. HACE is far more common at higher altitudes, although there are case reports of HACE at 2500 m.

It is important for any discussion of AMS and HACE to have as a starting point an understanding of acclimatization. The process of acclimatization involves a series of adjustments by the body to meet the challenge of hypoxemia. While we have a general understanding of systemic changes associated with acclimatization, the underlying molecular and cellular processes are not yet fully described. Recent findings suggest that the process may be initiated by widespread molecular up-regulation of hypoxia inducible factor-1. Downstream processes ultimately act to offset hypoxemia, including elevated ventilation leading to a rise in arterial oxygen saturation (SaO2), a mild diuresis and contraction of plasma volume such that more oxygen is carried per unit of blood, elevated blood flow and oxygen delivery, and eventually a greater circulating hemoglobin mass. Acclimatization can be viewed as the end-stage process of how humans can best adjust to hypoxia. But optimal acclimatization takes from days to weeks, or perhaps even months.

The initial and immediate strategy to protect the body from hypoxia is to increase ventilation. This compensatory mechanism is triggered by stimulation of the carotid bodies, which sense hypoxemia (low arterial PO2), and increase central respiratory drive. This is a fast response, occurring within minutes of exposure to hypoxia persisting throughout high altitude exposure. This is why one cautions against the use of respiratory depressants such as alcohol and some sleeping medications, which can depress the hypoxic drive to breathe and may thus worsen hypoxemia. Pharmacological simulation of this natural process by acetazolamide, a respiratory stimulant and mild diuretic, largely protects from AMS and HACE by stimulating acclimatization. Circulatory responses are key to improving oxygen delivery, and are likely regulated by marked elevations in sympathetic activity. Field experience suggests that a marked elevation in early morning resting heart rate is a sign of challenges to acclimatization, perhaps secondary to increased hypoxemia, or dehydration. For the pathophysiology of AMS and HACE responses of the cerebral circulation are especially important. Maintenance of cerebral oxygen delivery is a critical factor for survival at high altitude. The balance between hypoxic vasodilation and hypocapnia-induced vasoconstriction determines overall cerebral blood flow (CBF). In a classic study, CBF increased 24% on abrupt ascent to 3810 m, and then returned to normal over 3 to 5 days. Recent studies, largely using regional transcranial Doppler measures of CBF velocity as a proxy for CBF, report discernible individual variation in the CBF response to hypoxia. All advanced brain imaging studies to date have shown both elevations in CBF in hypoxic humans and striking heterogeneity of CBF distribution in the hypoxic brain, with CBF rising up to 33% in the hypothalamus, and 20% in the thalamus with no other significant changes. Also, it is becoming clear that cerebral autoregulation, the process by which cerebral perfusion is maintained as blood pressure varies, is impaired in hypoxia. Thus, hypoxia modulates cerebral autoregulation and raises interesting questions about the importance of this process in AMS and acclimatization, since it appears to be a uniform response in all humans made hypoxemic. Further, hematocrit and hemoglobin concentration are elevated after 12 to 24 hours of hypoxic exposure due to a fall in plasma volume, but after several weeks, plasma volume returns to near sea level values. Normalization of plasma volume coupled with an increase in red cell mass secondary to the hypoxia stimulated erythropoiesis leads to an increase in total blood volume after several weeks of acclimatization. Adequate iron stores are required for adequate hematologic acclimatization to high altitude. Acclimatization, then, is a series of physiological responses to hypoxia that serve to offset hypoxemia, improve systemic oxygen delivery, and avoid AMS and HACE. When acclimatization fails, or the challenge of hypoxia is too great, AMS and HACE can develop.

AMS occurs in susceptible individuals when ascent to high altitude outpaces the ability to acclimatize. For example, most people ascending very rapidly to high altitude will get AMS. The symptoms, although often initially incapacitating, usually resolve in 24 to 48 hrs. The incidence and severity of AMS depend on the rate of ascent and the altitude attained, the length of time at altitude, the degree of physical exertion, and the individual’s physiological susceptibility. The chief significance of AMS is that planned activities may be impossible to complete during the first few days at a new altitude due to symptoms. In addition, in a few individuals, AMS may progress to life-threatening HACE or HAPE. At 4000 m and above, the incidence of AMS ranges from 50% to 65% depending on the rate and mode of ascent, altitude reached, and sleeping altitude. A survey of 3158 travelers visiting resorts in the Rocky Mountains of Colorado revealed that 25% developed AMS, and most decreased their daily activity because of their symptoms.

Singh et al. proposed that the high-altitude syndromes are secondary to the body’s responses to hypobaric hypoxia, not due simply to hypoxemia. They based this conclusion on 2 observations:

there is a delay between the onset of hypoxia and the onset of symptoms after ascent (from hours to days), and
not all symptoms are immediately reversed with oxygen.

On the other hand, scientists have long assumed that AMS and HACE are due solely to hypoxia, based largely on 2 reports:

the pioneering experiments of Paul Bert and
the Glass House experiment of Barcroft.

When these assumptions were tested in a laboratory setting to study symptom responses to hypobaric hypoxia (simulated high altitude), hypoxia alone, and hypobaric normoxia, AMS occurred soonest and with greater severity with simulated altitude, compared with either normobaric hypoxia or normoxic hypobaria. In 2 studies, one in normobaric hypoxia found no MRI signs of vasogenic edema but suggested that AMS was associated with “cytotoxic edema”, whereas a comparable study in hypobaric hypoxia found combined vasogenic and intracellular edema. The conclusions from the 2 studies have very different implications for refining a theory of the pathophysiology of AMS. Although the studies were not designed for a direct comparison between hypobaria and hypoxia, the discrepancy points out an assumption about normobaric hypoxia and the pathophysiology of AMS that may warrant further investigation.

Our central hypothesis regarding the pathophysiology of AMS, and by extension of HACE, is that it is centered on dysfunction within the brain. This is not a new idea, but it is one of current intense interest thanks to advances in brain imaging and neuroscience techniques. Barcroft, writing in 1924, argued that the brain’s response to hypoxia was central to understanding the pathophysiology of mountain sickness.

A low ventilatory response to hypoxia coupled with increased symptoms of AMS led to intensive investigation of a link between the chemical control of ventilation and the pathogenesis of AMS. The results of these investigations suggest that for most people, the ventilatory response to hypoxia has little predictive value for AMS risk. Only if the extremes of ventilator responsiveness are contrasted can accurate predictions be made, where those with extremely low ventilatory drives being more likely to suffer AMS. At the extreme end of the distribution (i.e., for very high responses), the protective role of a brisk hypoxic ventilatory response may be due to increased arterial oxygen content and cerebral oxygen delivery despite mild hypocapnic cerebral vasoconstriction.

Hansen and Evans were the first to publish a comprehensive hypothesis of the pathophysiology of AMS centered on the brain. Their theory posited that compression of the brain, either by increased cerebral venous volume, reduced absorption of cerebral spinal fluid, or increased brain-tissue hydration (edema), initiates the development of the symptoms and signs of AMS and HACE. Ross built on these ideas with his “tight fit hypothesis,” published in 1985, and others have developed these ideas into a series of testable hypotheses congruent with today’s knowledge of AMS and HACE. The tight fit hypothesis states that expanded intracranial volume (due to the reasons put forth by Hansen and Evans, or other causes) plus the volume available for intracranial buffering of that expanded volume would predict who would get AMS. Greater buffering capacity leads to AMS resistance, lower buffering capacity, or a ‘tight fit’ of the brain in the cranial vault, would lead to greater AMS susceptibility. Overall, it is clear that brain volume increases in humans on exposure to hypoxia. It is less certain whether this elevation in brain volume plays a role in AMS.

Hackett’s pioneering MRI study in HACE, with marked white matter edema suggestive of a vasogenic origin, has led to a decade of studies looking for a similar finding in AMS. In moderate to severe AMS, all imaging studies have shown some degree of cerebral edema. But in mild to moderate AMS, admittedly an arbitrary and subjective distinction, brain edema is present in some MRI studies of AMS subjects, but not in all. It seems reasonable to conclude from the available data that the increase in brain volume observed is at least partially due to brain edema, and that earlier studies missed the edema more for technical than physiological reasons. It is less clear whether the brain edema is largely of intracellular or vasogenic origin, and what role if any it plays in the pathophysiology of AMS.

Although we support transcranial doppler for many investigations in integrative physiology, the complex interplay of hypoxia and hypocapnia that is present in acutely hypoxic humans may present a situation where whole brain imaging is a more reliable and accurate tool to discern the role of CBF in the onset of AMS. To date, no brain imaging studies have addressed global cerebral perfusion in AMS.

The management of AMS and HACE is based on our current understanding of the physiological and pathophysiological responses to hypoxia. Hypoxia itself, however, does not immediately lead to AMS as there is a delay of several hours after arrival at high altitude before symptoms develop. Increased knowledge of hypoxic inducible factor and cytokines that alter capillary permeability may lead to the discovery of new drugs for the prevention and alleviation of AMS and HACE.

Much work has focused on the role of vascular endothelial growth factor (VEGF), a potent permeability factor up-regulated by hypoxia. Some studies have found no evidence of an association of changes in plasma concentrations of VEGF and AMS, whereas others support the hypothesis that VEGF contributes to the pathogensis of AMS. Clearly a better understanding of the mechanisms of increased capillary permeability of cerebral capillaries will greatly enhance the management of AMS and HACE.

Flying high: A theoretical analysis of the factors limiting exercise performance in birds at altitude

Graham R. Scott, William K. Milsom
Respiratory Physiology & Neurobiology 154 (2006) 284–301
http://dx.doi.org:/10.1016/j.resp.2006.02.012

The ability of some bird species to fly at extreme altitude has fascinated comparative respiratory physiologists for decades, yet there is still no consensus about what adaptations enable high altitude flight. Using a theoretical model of O2 transport, we performed a sensitivity analysis of the factors that might limit exercise performance in birds. We found that the influence of individual physiological traits on oxygen consumption (˙VO2 ) during exercise differed between sea level, moderate altitude, and extreme altitude. At extreme altitude, hemoglobin (Hb) O2 affinity, total ventilation, and tissue diffusion capacity for O2 (DTO2) had the greatest influences on VO2; increasing these variables should therefore have the greatest adaptive benefit for high altitude flight. There was a beneficial interaction between DTO2 and the P50 of Hb, such that increasing DTO2 had a greater influence on VO2 when P50 was low. Increases in the temperature effect on P50 could also be beneficial for high flying birds, provided that cold inspired air at extreme altitude causes a substantial difference in temperature between blood in the lungs and in the tissues. Changes in lung diffusion capacity for O2, cardiac output, blood Hb concentration, the Bohr coefficient, or the Hill coefficient likely have less adaptive significance at high altitude. Our sensitivity analysis provides theoretical suggestions of the adaptations most likely to promote high altitude flight in birds and provides direction for future in vivo studies.

The bird lung is unique among the lungs of air-breathing vertebrates, with a blood flow that is crosscurrent to gas flow, and a gas flow that occurs unidirectionally through rigid parabronchioles. As such, bird lungs are inherently more efficient than the lungs of other air-breathing vertebrates (Piiper and Scheid, 1972, 1975). While this may partially account for the greater hypoxia tolerance of birds in general when compared to mammals (cf. Scheid, 1990), its presence in all birds excludes the crosscurrent lung as a possible adaptation specific to high altitude fliers. Similarly, an extremely small diffusion distance across the blood–gas interface compared to other air breathers seems to be a characteristic of all bird lungs, and not just those of high fliers (Maina and King, 1982; Powell and Mazzone, 1983; Shams and Scheid, 1989). Partly because of this small diffusion distance, the inherent O2 diffusion capacity across the gas–blood interface (DLO2) is generally high in birds. Interestingly, pulmonary vasoconstriction does not appear to increase during hypoxia in bar-headed geese (Faraci et al., 1984a). This may be a significant advantage during combined exercise and severe hypoxia, and suggests that regulation of lung blood flow could be important in high altitude birds. In addition, the CO2/pH sensitivity of ventilation is commonly assessed by comparing the isocapnic and poikilocapnic hypoxic ventilatory responses; however, the isocapnic ventilatory responses to hypoxia of both low and high altitude birds have not been compared. In this regard, the ventilator response in high altitude birds may also depend on their capacity to maintain intracellular pH during alkalosis, or to buffer changes in extracellular pH due to hyperventilation. It therefore remains to be conclusively determined whether high altitude fliers have a greater capacity to increase ventilation during severe hypoxia.

After diffusing into the blood in the lungs, oxygen is primarily circulated throughout the body bound to hemoglobin. A high cardiac output is therefore important for exercise at high altitude to supply the working muscle with adequate amounts of O2. Indeed, animals selectively bred for exercise performance have higher maximum cardiac outputs, as do species that have evolved for exercise performance. Whether cardiac output limits exercise performance per se, however, is less clear; other factors may limit intense exercise, and in more athletic species (or individuals) cardiac output may be higher simply out of necessity. Excessive cardiac output may even be detrimental if blood transit times in the lungs or tissues are substantially reduced. Unfortunately, very little is known about cardiac performance in high flying birds. Both the high altitude bar-headed goose and the low altitude pekin duck can increase cardiac output at least five-fold during hypoxia at rest (Black and Tenney, 1980), but no comparison of maximum cardiac performance has been made between high and low altitude birds.

Once oxygenated blood is circulated to the tissues, O2 moves to the tissue mitochondria, the site of oxidative phosphorylation and oxygen consumption. Transport of oxygen from the blood to the mitochondria involves several steps. Oxygen must first dissociate from Hb and diffuse through the various compartments of the blood, but in both birds and mammals the conductances of these steps are high, and are unlikely to impose much of a limitation to O2 transport. In contrast, diffusion across the vascular wall and through the extracellular spaces is thought to provide the most sizeable limitation to O2 transport. Consequently, the size of the capillary–muscle fiber interface is an extremely important determinant of a muscle’s aerobic capacity. Finally, oxygen diffuses across the muscle fiber membrane and moves through the cytoplasm until it associates with cytochrome c oxidase, the O2 acceptor in the mitochondrial electron transport chain. Myoglobin probably assists intracellular O2 transport, so diffusion through the muscle likely provides very little resistance to O2 flux.

It is obvious that the ability of some bird species to fly at extreme altitudes is poorly understood. The adaptive benefit of high hemoglobin oxygen affinity is well established, but its relative importance is unknown. Some evidence suggests that traits increasing oxygen diffusion capacity in flight muscle are adaptive in high fliers as well, but the adaptive significance of differences in the respiratory and cardiovascular systems of high altitude fliers is not clear. The remainder of this study assesses these possibilities using theoretical sensitivity analysis, and explores potential adaptations for high altitude flight in birds.

Oxygen transport in birds

Oxygen transport in birds. The crosscurrent parabronchial lung is unidirectionally ventilated by air sacs, and oxygen diffuses into blood capillaries from air capillaries (not shown) all along the length of the parabronchi. Oxygen is then circulated in the blood, and diffuses to mitochondria in the tissues. The rate of oxygen transport at both the lungs and tissues can be calculated using the Fick equation, and the amount of O2 transferred from the lungs into the blood can be calculated using an oxygen conservation equation.

Oxygen tensions in the lung

Oxygen tensions in the lung (A) and tissue (B) capillaries during normoxia. In the crosscurrent avian lung, PO2 varies in two dimensions: PO2 increases along the path of blood flow through the lungs, but does not increase by as much at the end of the parabronchi as at the start (gas PO2 decreases along the length of the parabronchi). In the tissues, blood PO2 decreases continuously along the capillary length as O2 diffuses to tissue mitochondria. To reach a solution, our model iterates between gas transport calculations in the lungs (A) and tissues (B) until a stable result is reached.

varying different biochemical features of hemoglobin (Hb) on oxygen consumption

The effects of varying different biochemical features of hemoglobin (Hb) on oxygen consumption during exercise in normoxia (PIO2 of 150 Torr; red), moderate hypoxia (84 Torr; green dashed), and severe hypoxia (30 Torr; dark blue). (A) P50, the PO2 at 50% Hb saturation; (B and C) Bohr coefficient (φ); and (D and E) Hill coefficient (n) (see Section 2 for a mathematical description of each). In (B)–(E), the effects of each variable were assessed at the P50 of pekin ducks (40 Torr; B and D) as well as the P50 of bar-headed geese (25 Torr; C and E).

Unlike in vivo studies, theoretical sensitivity analyses allow individual physiological variables to be altered independently so their individual effects on oxygen consumption can be assessed. By applying this analysis to hypoxia in birds, we feel we can predict which factors most likely limit oxygen consumption and exercise performance. As a consequence, our analysis identifies which steps in the oxygen cascade can provide the basis for adaptive change in birds that evolved for high altitude flight, namely ventilation and tissue diffusion capacity.

Since our interest was in the factors limiting exercise performance at altitude, the starting data for our model were obtained from previous studies on pekin ducks near maximal oxygen consumption. These ducks were exercising on a treadmill, however, and were not flying. Unfortunately, to the best of our knowledge only one previous study has made all the required measurements for this analysis during flight, and this was only done in normoxia (in pigeons, Butler et al., 1977). Pekin ducks are the only species for which we could find all the required measurements for our analysis during exercise in both normoxia and hypoxia. Only the lung and tissue diffusion capacities remained to be calculated in our analysis, but previous experimental determinations of DLO2 in pekin ducks were similar to the values calculated in this study (Scheid et al., 1977). Similar values for DTO2 are not available.

The physiological variables limiting exercise performance in birds during moderate hypoxia are similar to those limiting performance in normoxia. DTO2 continues to pose the greatest limitation, and limitations imposed by the circulation (˙Q and CHb) are still greater at a lower P50. Unlike normoxia, however, ˙VO2 in moderate hypoxia appears to be limited less by the circulation and more by respiratory variables, as is also the case in humans (Wagner, 1996). The most substantial difference between severe hypoxia and normoxia/moderate hypoxia is in the effects of altering ventilation. Ventilation appears to become a major limitation to exercise performance at extreme altitude. DTO2 also appears to limit ˙VO2 in severe hypoxia, but only at lower P50 values. This is not entirely unsurprising: in severe hypoxia the venous blood of pekin ducks (a species which has a higher P50) is almost completely deoxygenated in vivo, so there are no possible benefits of increasing DTO2 . At the lower P50, there is a substantially higher arterial oxygen content, so more oxygen can be removed, and increasing DTO2 can have a greater influence. In humans during severe hypoxia, DTO2, DLO2, and ˙V have the greatest influence on exercise performance.

Tissue diffusion capacity should also be adaptive in high altitude birds with a high hemoglobin O2 affinity. In the present study, a simultaneous decrease in P50 (from 40 to 25 Torr) and increase in DTO2 (twofold) increased ˙VO2 by 51%. Thus, in high flying birds that are known to have a low P50, such as the barheaded goose and Ruppell’s griffon (Gyps rueppellii), increases in flight muscle diffusion capacity should be of extreme importance. This suggestion is supported by research demonstrating greater muscle capillarization in bar-headed geese than in low altitude fliers, as the size of the capillary–muscle fiber interface is known to be the primary structural determinant of O2 flux into the muscle.

Our analysis suggests that an enhanced capacity to increase ventilation should also benefit birds significantly in severe hypoxia, and could therefore be an important source of adaptation for high altitude flight. This is likely true regardless of P50; although there is a small amount of interaction between P50 and ventilation, increasing ˙V always had a substantial effect on oxygen consumption. Data from the literature addressing this possibility have unfortunately been inconclusive. Both bar-headed geese and pekin ducks can effectively increase ventilation, thus reducing the inspired-arterial O2 difference, during severe poikilocapnic hypoxia at rest, as well as during moderate poikilocapnic hypoxia and running exercise.

oxyhemoglobin dissociation curve

In contrast to the Bohr effect and Hill coefficient, the temperature effect on Hb-O2 binding affinity may have a substantial effect on oxygen consumption, and may therefore be a source of adaptive change for high altitude flight. An effect of temperature on ˙VO2 may arise if hyperventilation during flight at extreme altitude cools the pulmonary blood. This would reduce the P50 of Hb in the lungs, and thus facilitate oxygen uptake. When this blood enters the exercising muscles it would then be rewarmed to body temperature, and oxygen would be released from Hb. Our modelling suggests that a temperature effect on Hb could significantly enhance ˙VO2 . The greater the difference in temperature between blood in the lungs and in the muscles, and the greater the temperature effect on Hb-O2 binding, the greater the increase in ˙VO2 . At normal levels of temperature sensitivity, the increase in ˙VO2 was approximately 5% for every 1 ◦C difference. It could be adaptive at high altitude to alter the magnitude of the temperature effect on Hb while allowing lung temperature to fall. At present, however, it is unknown whether the Hb of high altitude birds has a heightened sensitivity to temperature, or whether pulmonary blood is actually cooled during high altitude flight.

Using a theoretical sensitivity analysis that allows individual physiological variables to be altered independently, we have identified the factors most likely to limit oxygen consumption and exercise performance in birds, and by extension, the physiological changes that are likely adaptive for high altitude flight. The adaptive benefits of some of these changes, in particular hemoglobin oxygen affinity, are already well established for high flying birds. For other traits, such as an enhanced hypoxic ventilatory response or O2 diffusion capacity of flight muscle, adaptive differences have not been conclusively recognized in studies in vivo. Furthermore, the beneficial interaction between increasing DTO2 and decreasing hemoglobin P50 has not yet been demonstrated in vivo. Our theoretical analysis suggests that changes in these respiratory processes could also adapt birds to environmental extremes, and future studies should explore these findings.

Adaptation and Convergent Evolution within the Jamesonia-Eriosorus Complex in High-Elevation Biodiverse Andean Hotspots

Patricia Sanchez-Baracaldo, Gavin H. Thomas
PLoS ONE 9(10): e110618. http://dx.doi.org:/10.1371/journal.pone.0110618

The recent uplift of the tropical Andes (since the late Pliocene or early Pleistocene) provided extensive ecological opportunity for evolutionary radiations. We test for phylogenetic and morphological evidence of adaptive radiation and convergent evolution to novel habitats (exposed, high-altitude paramo habitats) in the Andean fern genera Jamesonia and Eriosorus. We construct time-calibrated phylogenies for the Jamesonia-Eriosorus clade. We then use recent phylogenetic comparative methods to test for evolutionary transitions among habitats, associations between habitat and leaf morphology, and ecologically driven variation in the rate of morphological evolution. Paramo species (Jamesonia) display morphological adaptations consistent with convergent evolution in response to the demands of a highly exposed environment but these adaptations are associated with microhabitat use rather than the paramo per se. Species that are associated with exposed microhabitats (including Jamesonia and Eriorsorus) are characterized by many but short pinnae per frond whereas species occupying sheltered microhabitats (primarily Eriosorus) have few but long pinnae per frond. Pinnae length declines more rapidly with altitude in sheltered species. Rates of speciation are significantly higher among paramo than non-paramo lineages supporting the hypothesis of adaptation and divergence in the unique Pa´ramo biodiversity hotspot.

AltitudeOmics: Rapid Hemoglobin Mass Alterations with Early Acclimatization to and De-Acclimatization from 5,260 m in Healthy Humans

Benjamin J. Ryan, NB Wachsmuth, WF Schmidt, WC Byrnes, et al.
PLoS ONE 9(10): e108788. http://dx.doi.org:/10.1371/journal.pone.0108788

It is classically thought that increases in hemoglobin mass (Hb mass) take several weeks to develop upon ascent to high altitude and are lost gradually following descent. However, the early time course of these erythropoietic adaptations has not been thoroughly investigated and data are lacking at elevations greater than 5,000 m, where the hypoxic stimulus is dramatically increased. As part of the AltitudeOmics project, we examined Hb mass in healthy men and women at sea level (SL) and 5,260 m following 1, 7, and 16 days of high altitude exposure (ALT1/ALT7/ALT16). Subjects were also studied upon return to 5,260 m following descent to 1,525 m for either 7 or 21 days. Compared to SL, absolute Hb mass was not different at ALT1 but increased by 3.7-5.8% (mean 6 SD; n = 20; p<0.01) at ALT7 and 7.6-6.6% (n = 21; p=0.001) at ALT16. Following descent to 1,525 m, Hb mass was reduced compared to ALT16 (-6.0+3.7%; n = 20; p = 0.001) and not different compared to SL, with no difference in the loss in Hb mass between groups that descended for 7 (-6.3+3.0%; n = 13) versus 21 days (-5.7+5.0; n = 7). The loss in Hb mass following 7 days at 1,525 m was correlated with an increase in serum ferritin
(r =20.64; n = 13; p,0.05), suggesting increased red blood cell destruction. Our novel findings demonstrate that Hb mass increases within 7 days of ascent to 5,260 m but that the altitude-induced Hb mass adaptation is lost within 7 days of descent to 1,525 m. The rapid time course of these adaptations contrasts with the classical dogma, suggesting the need to further examine mechanisms responsible for Hb mass adaptations in response to severe hypoxia.

Cardiovascular adjustments for life at high altitude

Roger Hainsworth, Mark J. Drinkhill
Respiratory Physiology & Neurobiology 158 (2007) 204–211
http://dx.doi.org:/10.1016/j.resp.2007.05.006

The effects of hypobaric hypoxia in visitors depend not only on the actual elevation but also on the rate of ascent. There are increases in sympathetic activity resulting in increases in systemic vascular resistance, blood pressure and heart rate. Pulmonary vasoconstriction leads to pulmonary hypertension, particularly during exercise. The sympathetic excitation results from hypoxia, partly through chemoreceptor reflexes and partly through altered baroreceptor function. Systemic vasoconstriction may also occur as a reflex response to the high pulmonary arterial pressures. Many communities live permanently at high altitude and most dwellers show excellent adaptation although there are differences between populations in the extent of the ventilatory drive and the erythropoiesis. Despite living all their lives at altitude, some dwellers, particularly Andeans, may develop a maladaptation syndrome known as chronic mountain sickness. The most prominent characteristic of this is excessive polycythemia, the cause of which has been attributed to peripheral chemoreceptor dysfunction. The hyperviscous blood leads to pulmonary hypertension, symptoms of cerebral hypoperfusion, and eventually right heart failure and death.

High altitude places are not only destinations of adventurous travelers, many people are born, live their lives and die in these cold and hypoxic regions. According to WHO, in 1996 there were approximately 140 million people living at altitudes over 2,500m and there are several areas of permanent habitation at over 4,000 m. These are in three main regions of the world: the Andes of South America, the highlands of Eastern Africa, and the Himalayas of South-Central Asia. This review is concerned with the effects of exposure to high altitude on the cardiovascular system and its autonomic control, in visitors, and the means by which the permanent high altitude dwellers have adapted to their environment.

For visitors the period of initial adaptation, i.e. the first days and weeks following arrival at attitude, is a critical time since it is during this period that acute mountain sickness and/or pulmonary edema may occur. The processes of adaptation occurring during this initial period may well determine the individual’s ability to continue to function normally. Recent studies in animals and man have highlighted the role of the autonomic nervous system in adaptation and in particular the importance of sympathetic activation of the cardiovascular system following high altitude exposure.

An increase in resting heart rate in response to acute hypoxia has been
described in several species including man. Vogel and Harris (1967)
investigated the effects of simulated exposure to high altitude in man
at pressures equivalent to 600, 3,400 and 4,600m using a hypobaric
chamber. Each level of chamber pressure was developed over a 30 min
period andwas maintained for 48 h in an attempt to simulate expedition
conditions. After 10 h at the equivalent of 3,400 m resting
heart rate was significantly increased and by 40 h it had increased by
16% from the resting value at 600 m. At 4,600 m it increased by 34%.
Similar findings, an increase in heart rate of 18%, were shown following
ascent to 4,300 m for periods up to 5 weeks. However, this study also
demonstrated that the rate of ascent also influenced the magnitude of
the heart rate increase. A gradual increase in altitude over a period
of 2 weeks resulted in the resting heart rate increasing by 25%
compared with an abrupt ascent which resulted in an increase of
only 9%. As subjects acclimatize at altitudes up to about 4,500 m
much of the increase in heart rate is lost and resting heart rates
return towards their sea level values. Acute hypoxia also causes
increases in cardiac output both at rest and for given levels of
exercise compared with values during normoxia.

The effect of hypoxia on the pulmonary circulation is dramatic
resulting in pulmonary hypertension caused by an increase in
pulmonary vascular resistance. The onset has been shown in man
to be very rapid, reaching a maximum within 5 min. Zhao et al.
(2001) demonstrated that breathing 11% oxygen for 30 min
increased mean pulmonary artery pressure by 56%, from 16 to
25 mmHg. The effect of hypoxia on the pulmonary circulation is
even more pronounced during exercise, as demonstrated in studies
carried out on subjects of Operation Everest II. Resting pulmonary
artery pressure increased from 15 mmHg at sea level to 34 mmHg
at the equivalent of 8,840 m. During near maximal exercise at
8,840 m it increased from the sea level value of 33–54 mm Hg.
In the short term the mechanism of this pulmonary artery vaso-
constriction has been shown to involve inhibition of O2 sensitive
K+ channels leading to depolarization of pulmonary artery smooth
muscle cells and activation of voltage gated Ca2+ channels. This
causes Ca2+ influx and vasocon-striction. This process is
immediately reversed by breathing oxygen.

Healthy high altitude residents show excellent adaptation to their
environment. These adaptations are likely to be associated with
altered gene expression as the expression of genes associated with
vascular control and reactions to hypoxia have been found to be high
in altitude dwellers. Different communities, however, seem to adopt
different adaptation strategies. For example Andeans hyperventilate
to decrease end-tidal and arterial CO2 levels to as low as 25 mmHg
and have hemoglobin levels well above those in sea-level people.
Tibetans Hyperventilate but have normal hemoglobin levels below
4,000 m. Ethiopian highlanders, on the other hand, have CO2 and
hemoglobin levels similar to those of sea-level dwellers.

Blood volumes are larger in high altitude dwellers. In Andeans this
is due to large packed cell volumes whereas in Ethiopians it was the
plasma volumes that were large. Probably as the result of the large
blood volumes, tolerance to orthostatic stress was greater than that
in sea-level residents.

CMS is a condition frequently found in long term residents of high
altitudes, particularly in the Andes where it is a major public health
problem. It also occurs in residents on the Tibetan plateau, although
not in Ethiopians. Patients with CMS develop excessive polycythemia
and various clinical features including dyspnea, palpitations, insomnia,
dizziness, headaches, confusion, loss of appetite, lack of mental
concentration and memory alterations. Patients may also complain
of decreased exercise tolerance, bone pains, acral paresthesia and
occasionally hemoptysis. The impairment of mental function may
be reversed by phlebotomy. Physical examination reveals cyanosis,
due to the combination of polycythemia and low oxygen saturation,
and a marked pigmentation of the skin exposed to the sun.
Hyperemia of conjunctivae is characteristic and the retinal vessels
are also dilated and engorged. The second heart sound is frequently
accentuated and there is an increased cardiac size, mainly due to
right ventricular hypertrophy. As the condition progresses, overt
congestive heart failure becomes evident, characterized by dyspnea
at rest and during mild effort, peripheral edema, distension of
superficial veins, and progressive cardiac dilation.

The major mechanism for the control of blood pressure is through
regulation of peripheral vascular resistance, but most studies have
examined only the control of heart rate. We have recently studied
the responses of forearm vascular resistance to carotid baroreceptor
stimulation in high altitude residents with and without CMS, both at
their resident altitude and shortly after descent to sea level. Results
showed that baroreflex “set point” was higher in CMS, but only at
altitude. At sea level, values were similar.

The chronic hypoxia at high altitude stresses many of the body’s
homeostatic mechanisms. There have been many investigations
which have examined the effects on respiration. However, cardio-
vascular effects are no less important and it is largely through effects
on the cardiovascular system that both acute and chronic mountain
sickness are caused. The hypoxia exerts both direct and reflex effects.
In the lung it causes vasoconstriction and pulmonary hypertension.
The sympathetic nervous system is excited partly through a central
effect of the hypoxia, through stimulation of chemoreceptors and
possibly pulmonary arterial baroreceptors and altered systemic
baroreceptor function. In some individuals the excessive hemopoiesis
causes increased blood viscosity and tissue hypoperfusion leading
to the syndrome of chronic mountain sickness.

New Insights in the Pathogenesis of High-Altitude Pulmonary Edema

Urs Scherrer, Emrush Rexhaj, Pierre-Yves Jayet, et al.
Progress in Cardiovascular Diseases 52 (2010) 485–492
http://dx.doi.org:/10.1016/j.pcad.2010.02.004

High-altitude pulmonary edema is a life-threatening condition occurring in predisposed but otherwise healthy individuals. It therefore permits the study of underlying mechanisms of pulmonary edema in the absence of confounding factors such as coexisting cardiovascular or pulmonary disease, and/or drug therapy. There is evidence that some degree of asymptomatic alveolar fluid accumulation may represent a normal phenomenon in healthy humans shortly after arrival at high altitude. Two fundamental mechanisms then determine whether this fluid accumulation is cleared or whether it progresses to HAPE: the quantity of liquid escaping from the pulmonary vasculature and the rate of its clearance by the alveolar respiratory epithelium. The former is directly related to the degree of hypoxia induced pulmonary hypertension, whereas the latter is determined by the alveolar epithelial sodium transport. Here, we will review evidence that, in HAPE-prone subjects, impaired pulmonary endothelial and epithelial NO synthesis and/or bioavailability may represent a central underlying defect predisposing to exaggerated hypoxic pulmonary vasoconstriction and, in turn, capillary stress failure and alveolar fluid flooding. We will then demonstrate that exaggerated pulmonary hypertension, although possibly a condition sine qua non, may not always be sufficient to induce HAPE and how defective alveolar fluid clearance may represent a second important pathogenic mechanism.

Cerebral Blood Flow at High Altitude

Philip N. Ainslie and Andrew W. Subudhi
High Altitude Medicine & Biology 2014; 15(2): 133–140
http://dx.doi.org:/10.1089/ham.2013.1138

This brief review traces the last 50 years of research related to cerebral blood flow (CBF) in humans exposed to high altitude. The increase in CBF within the first 12 hours at high altitude and its return to near sea level values after 3–5 days of acclimatization was first documented with use of the Kety-Schmidt technique in 1964. The degree of change in CBF at high altitude is influenced by many variables, including arterial oxygen and carbon dioxide tensions, oxygen content, cerebral spinal fluid pH, and hematocrit, but can be collectively summarized in terms of the relative strengths of four key integrated reflexes:

hypoxic cerebral vasodilatation;
2) hypocapnic cerebral vasoconstriction;
3) hypoxic ventilatory response; and
4) hypercapnic ventilatory response.

Understanding the mechanisms underlying these reflexes and their interactions with one another is critical to advance our understanding of global and regional CBF regulation. Whether high altitude populations exhibit cerebrovascular adaptations to chronic levels of hypoxia or if changes in CBF are related to the development of acute mountain sickness are currently unknown; yet overall, the integrated CBF response to high altitude appears to be sufficient to meet the brain’s large and consistent demand for oxygen.

Relative to its size, the brain is the most oxygen dependent organ in the body, but many pathophysiological and environmental processes may either cause or result in an interruption to its oxygen supply. As such, studying the brain at high altitude is an appropriate model to investigate both acute and chronic effects of hypoxemia on cerebrovascular function. The cerebrovascular responses to high altitude are complex, involving mechanistic interactions of physiological, metabolic, and biochemical processes.

This short review is organized as follows: An historical overview of the earliest CBF measurements collected at high altitude introduces a summary of reported CBF changes at altitude over the last 50 years in both lowlanders and high-altitude natives. The most tenable candidate mechanism(s) regulating CBF at altitude are summarized with a focus on available data in humans, and a role for these mechanisms in the pathophysiology of AMS is considered. Finally, suggestions for future directions are provided.

Angelo Mosso (1846–1910) is undoubtedly the forefather of high altitude cerebrovascular physiology. In order to pursue his principal curiosity of the physiological effects of hypobaria, Mosso built barometric chambers and was reported to expose himself pressures as low as 192 mmHg (equivalent to > 10,000 m). He was also responsible for the building of the Capanna Margherita laboratory on Monta Rosa at 4,559 m. In both settings, Mosso utilized his hydrosphygmomanometer to measure changes in ‘‘brain pulsations’’ in patients that had suffered removal of skull sections, due to illness or trauma. Indicative of changes in CBF, these recordings preceded the next estimates of CBF in humans by some 50 years.

At sea level, Kety and Schmidt (1945) were the first to quantify human CBF using an inert tracer (nitrous oxide, N2O) combined with arterial and jugular venous sampling. This method for the measurement of global CBF is based on the Fick principle, whereby the integrated difference of multiple arterial and venous blood samples during the first 10 or more minutes after the sudden introduction into the lung of a soluble gas tracer is inversely proportional to cerebral blood flow. In 1948, they showed that breathing 10% oxygen increased CBF by 35%; however, it was not until 1964 that the first measurements of CBF were made in humans at high altitude. The motivation for these high altitude experiments was stimulated, in part, from the earlier discovery of the brain’s ventral medullary cerebrospinal fluid (CSF) pH sensors in animals. Following the location of these central chemoreceptors, Severinghaus and colleagues examined in humans the role of CSF pH and bicarbonate in acclimatization to high altitude (3,810 m) at the White Mountain (California, USA) laboratories (Severinghaus et al., 1963). A year later, at the same location, John Severinghaus performed his seminal study of CBF at high altitude. He was joined by Tom Hornbein—shortly after his first ascent of Everest by the West Ridge—who was part of the research team and also volunteered for the study (Fig.). The results showed clear time dependent changes in CBF during acclimatization to high altitude (HA).

the Kety-Schmidt nitrous oxide method of measuring CBF

From left to right, Larry Saidman (administering the gas), Tom Hornbien (volunteer), Ed Munson (drawing jugular venous blood samples), and John Severinghaus. Here (1964) the Kety-Schmidt nitrous oxide method of measuring CBF is used. The subject breathed about 15% N2O for 15 min while arterial and jugular venous blood was frequently sampled. (B) Results from Severinghaus et al. (1966). Graphs shows that CBF as estimated by cerebral A-VO2 differences from sea level controls increased about 24% within hours of arrival at 3810 m, and fell over 4 days to about 13% above control. CBF by the N2O method was increased by 40% on day 1, and returned to 6% above control on day 4. However, the N2O method data had greater variance. Acute normoxia on day 1 and day 4 returned CBF to sea level values within 15 min. Photograph courtesy of Dr. John W Severinghaus.

Native Tibetan (or Himalayan) and Andean populations arrived approximately 25,000 and 11,000 years ago, suggesting that these populations either carried traits that allowed them to thrive at high altitude or were able to adapt to the environment. The physiological and genetic traits associated with native high-altitude populations have been elegantly reviewed (Beall, 2007; Erzurum et al., 2007; Frisancho, 2013). As such, this topic is briefly summarized here with the focus on CBF at altitude in context of Andean and Tibetan high-altitude residents.

In general, native Andeans have lower CBF values compared to sea level natives. The first evidence suggesting lower flow was reported in 8 Peruvian natives living at 4300m altitude in Cerro de Pasco (Milledge and Sørensen, 1972). The authors found the mean arterial–venous oxygen content difference across the brain was 7.9 – 1 vol%, about 20% higher than the published sea level mean of 6.5 vol%. They suggested that CBF probably was proportionately about 20% below sea level normal values, assuming that brain metabolic rate was normal, and postulated that the mechanism might be high blood viscosity given the high hematocrit (58 – 6%) in these subjects. However, since the cerebral metabolic rate for oxygen (CMRO2) is constant even in severe hypoxia (Kety and Schmidt 1948b; Ainslie et al. 2013), the inverse linear relationship between CBF and arterial–venous oxygen content differences could also explain the reduction in CBF, as less flow would be needed to match the oxygen demand of the brain when arterial content is elevated. A similar study (Sørensen et al., 1974), using arterio-venous differences combined (in a subgroup) with a modified version of Kety–Schmidt method (krypton instead of N2O,) conducted in high-altitude residents in La Paz in Bolivia at 3800 m, also reported a 15%–20% reduction in CBF (with a reported average hematocrit of 50%) compared to a sea level control group.

Percent changes in cerebral blood flow

Percent changes in cerebral blood flow (D%CBF, graph A), arterial oxygen content (Cao2, graph B), and cerebral oxygen delivery (CDO2, graph C) with time at high-altitude from seven studies at various altitudes and durations. Severinghaus et al. (1966) studied CBF using the Kety-Schmidt technique in five subjects brought rapidly by car to 3810 m. Using the Xe133 method, Jensen et al. (1990) measured CBF in 12 subjects at 3475 m. Huang et al. (1987) measured ICA and VA blood velocities as a metric of CBF on Pikes Peak (4300 m). Baumgartner et al. (1994) studied 24 subjects who rapidly ascended to 3200m by cable car, slept one night at 3600 m, and ascended by foot to 4559m the next day. Cerebral blood flow was estimated by transcranial Doppler ultrasound. About two-thirds of the subjects developed symptoms of AMS, data included are the mean of all subjects. Lucas et al. (2011) employed an 8–9 day ascent to 5050m and estimated changes in CBF by transcranial Doppler ultrasound of the middle cerebral artery. Willie et al. (2013) following the same ascent measured flow (Duplex ultrasound; and TCCD) in the ICA and VA and estimated global flow from: 2*ICA + 2* VA. The same methodological approach was used time Subudhi upon rapid ascent via car and oxygen breathing to 5240 m (Subudhi et al. 2013). Cao2 was calculated from: (1.39 · [Hb] · SaO2) + Pao2 *0.003. In some studies [Hb] data were not available, and typical data from previous studies over comparable time at related elevation were used. In other studies, Pao2 was not always reported; therefore, Sao2 was used to estimate Pao2 via (Severinghaus, 1979).

Only two studies have measured serial changes in CBF during progressive ascent to high altitude, but the findings may help explain small discrepancies between studies. In 2011, Wilson et al. (2011) measured diameter and velocity in the MCA (using transcranial color-coded Duplex-ultrasound, TCCD) following partial acclimation to 5300m (n = 24), 6400 m (n = 14), and 7950m (n = 5). Remarkable elevations (200%) in flow in the MCA occurred at 7950 m. Notably, the authors estimated *24% dilation of the MCA occurred at 6400 m. Dilation of the MCA further increased to *90% at 7950m (Fig.) and was rapidly reversed with oxygen supplementation (Fig.). Cerebral oxygen delivery and oxygenation were maintained by commensurate elevations of CBF even at these extreme altitudes. In another recent study, CBF and MCA diameter were measured at 1338 m, 3440 m, 4371 m, and over time at 5050 m (Willie et al., 2013). Dilation of the MCA was observed upon arrival at 5050 m with subsequent normalization of CBF and MCA diameter by days 10–12. Such findings are consistent with unchanged diameter following 17 days at 5400m (Wilson et al., 2011). It is important to note that according to Poiseuille’s Law, flow is proportional to radius raised to the fourth power. Therefore, consistent with previous concerns about TCD (Giller, 2003), that the MCA dilates at such levels of hypoxemia indicates that previous studies using TCD at altitude may have underestimated flow (see previous Fig.) and thus may explain differences between studies. These findings are particularly important because they suggest regional regulation of CBF occurs in both large and small cerebral arteries.

Changes in blood flow in the middle cerebral artery (MCA) upon progressive ascent to 7950 m

Changes in blood flow in the middle cerebral artery (MCA) upon progressive ascent to 7950 m. Data were collected following partial acclimation to 5300 m (n = 24), at 6400 m (n = 14), and at 7950 m (n = 5). Remarkable elevations (200%) in flow in the MCA occurred at 7950 m following removal of breathing supplementary oxygen and breathing air for 20 min. Dilation (*24%) of the MCA occurred at 6400 m, which was further increased to 90% at 7950 m. Oxygen supplementation at this highest altitude rapidly reversed the observed MCA vessel dilation (denoted by blue triangle). Elevations in CBF via cerebral vasodilation were adequate to maintain oxygen delivery, even at these extreme altitudes. Modified from Wilson et al. (2011).

Summary of the major factors acting to increase ( plus) and decrease (minus) CBF during exposure to hypoxia

Summary of the major factors acting to increase ( plus) and decrease (minus) CBF during exposure to hypoxia. Cao2, arterial oxygen content; CBV, cerebral blood volume; EDHF, endothelium-derived hyperpolarizing factor; ET-1, endothelin-1; HCT, hematocrit; NO, nitric oxide; O2-, superoxide; PGE, prostaglandins; SNA, sympathetic nerve activity; VAH, ventilatory acclimatization to hypoxia/altitude. Modified from Ainslie and Ogoh (2010); Ainslie et al. (2014).

It is clear that many aspects of CBF regulation and brain function at high altitude warrant further investigation. Indeed, several questions remain. For example, over the period of ventilatory acclimatization (weeks to months), how do interactions between the hypoxic ventilatory response, hypercapnic ventilatoy response, hypoxic cerebral vasodilatation, and hypocapnic cerebral vasoconstriction interact to alter CBF? Furthermore, what is the role of NO and/or adenosine in mediating cerebral vasodilation at high altitude? And last, what is the time-course of recovery in CBF following descent to sea level?

Cognitive Impairments at High Altitudes and Adaptation

Xiaodan Yan
High Alt Med Biol. 15:141–145, 2014
http://dx.doi.org:/10.1089/ham.2014.1009

High altitude hypoxia has been shown to have significant impact on cognitive performance. This article reviews the aspects in which, and the conditions under which, decreased cognitive performance has been observed at high altitudes. Neural changes related to high altitude hypoxia are also reviewed with respect to their possible contributions to cognitive impairments. In addition, potential adaptation mechanisms are reviewed among indigenous high altitude residents and long-term immigrant residents, with discussions about methodological concerns related to these studies.

The amount of cognitive impairments at high altitudes is related to the chronicity of exposure. Acute exposure usually refers to a duration of several weeks, whereas chronic exposure usually refer to ‘‘extended permanence’’ in the high altitude environment (Virue´s-Ortega and others, 2004). The altitude of ascending or residence is another factor affecting the severity of impairments. This review will first summarize the cognitive impairments in acute exposure, then talk about impairments in chronic exposure, with discussions about the effect of altitudes in corresponding sections.

High altitude-related neurocognitive impairments with ascending altitudes

High altitude-related neurocognitive impairments with ascending altitudes in acute high altitude exposure (Wilson and others, 2009).

human brain consumes about 20% of the total oxygen intake

The human brain consumes about 20% of the total oxygen intake, which is disproportional to its size (about 2% of the total body weight). In this figure, oxygen consumption is reflected from glucose consumption in positron emission tomography (PET) (Alavi and Reivich, 2002).

The possibility of adaptation to high altitude hypoxia has always been an intriguing issue. In the acute cases, the human body does have some capacity for acclimatization, which varies significantly for different individuals. The question is, in chronic cases, for example, does growing up at high altitude regions guarantee sufficient adaption to occur to compensate for the risk of cognitive impairments? Existing research tends to suggest that, although some level of adaptation does occur, neural and cognitive impairments are still observed in these populations who are native or long-term residents at high altitude.

Although multiple studies have suggested that growing up at high altitudes is associated with cognitive impairments, it is not to say that adaptation does not happen with prolonged chronic exposure to high altitudes. One study has revealed that as a function of the length of low altitude residence (across the range of 1–5 years), some neuroimaging parameters of original highlanders who grew up at high altitude regions had shown the trend of converging towards the patterns of original low altitude residents, although such changes were not accompanied by statistically significant changes in cognitive performance (Yan and others, 2010). It is possible that, given sufficiently long time for normoxia adaptation, the neural and cognitive impairments associated with high altitude hypoxia may be alleviated to a certain extent.

In summary, various cognitive impairments associated with high altitude hypoxia have been reported from existing studies, which are accompanied by findings about neural impairments, suggesting that these cognitive impairments have legitimate neural basis. The specific relationships between physiological symptoms and cognitive impairments appear to be complicated and require further elucidation. There are cognitive impairments associated with both acute and chronic exposure to high altitudes; however, particular caution should be taken when interpreting the findings about cognitive impairments among native high altitude residents because of the differences
in cultural and socioeconomic factors. Existing studies have suggested that there can be some level of adaptation to high altitudes, in spite of the fact that some neuronal impairment may be irreversible.

Exercise Capacity and Selected Physiological Factors by Ancestry and Residential Altitude: Cross-Sectional Studies of 9–10-Year-Old Children in Tibet

Bianba, Sveinung Berntsen, Lars Bo Andersen, Hein Stigum, et al.
High Alt Med Biol. 2014; 15:162–169
http://dx.doi.org:/10.1089/ham.2013.1084

Aim: Several physiological compensatory mechanisms have enabled Tibetans to live and work at high altitude, including increased ventilation and pulmonary diffusion capacity, both of which serve to increase oxygen transport in the blood. The aim of the present study was to compare exercise capacity (maximal power output) and selected physiological factors (arterial oxygen saturation and heart rate at rest and during maximal exercise, resting hemoglobin concentration, and forced vital capacity) in groups of native Tibetan children living at different residential altitudes (3700 vs. 4300 m above sea level) and across ancestry (native Tibetan vs. Han Chinese children living at the same altitude of 3700 m). Methods: A total of 430 9–10-year-old native Tibetan children from Tingri (4300 m) and 406 native Tibetan and 406 Han Chinese immigrants (77% lowland-born and 33% highland-born) from Lhasa (3700 m) participated in two cross-sectional studies. The maximal power output (Wmax) was assessed using an ergometer cycle. Results: Lhasa Tibetan children had a 20% higher maximal power output (watts/kg) than Tingri Tibetan and 4% higher than Lhasa Han Chinese. Maximal heart rate, arterial oxygen saturation at rest, lung volume, and arterial oxygen saturation were significantly associated with exercise capacity at a given altitude, but could not fully account for the differences in exercise capacity observed between ancestry groups or altitudes. Conclusions: The superior exercise capacity in native Tibetans vs. Han Chinese may reflect a better adaptation to life at high altitude. Tibetans at the lower residential altitude of 3700 m demonstrated a better exercise capacity than residents at a higher altitude of 4300m when measured at their respective residential altitudes. Such altitude- or ancestry-related difference could not be fully attributed to the physiological factors measured.

Group size effects on foraging and vigilance in migratory Tibetan antelope

Xinming Lian, Tongzuo Zhang, Yifan Cao, Jianping Su, Simon Thirgood
Behavioural Processes 76 (2007) 192–197
http://dx.doi.org:/10.1016/j.beproc.2007.05.001

Large group sizes have been hypothesized to decrease predation risk and increase food competition. We investigated group size effects on vigilance and foraging behavior during the migratory period in female Tibetan antelope Pantholops hodgsoni, in the Kekexili Nature Reserve of Qinghai Province, China. During June to August, adult female antelope and yearling females gather in large migratory groups and cross the Qinghai–Tibet highway to calving grounds within the Nature Reserve and return to Qumalai county after calving. Large groups of antelope aggregate in the migratory corridor where they compete for limited food resources and attract the attention of mammalian and avian predators and scavengers. We restricted our sampling to groups of less than 30 antelopes and thus limit our inference accordingly. Focal-animal sampling was used to record the behavior of the free-ranging antelope except for those with lambs. Tibetan antelope spent more time foraging in larger groups but frequency of foraging bouts was not affected by group size. Conversely, the time spent vigilant and frequency of vigilance bouts decreased with increased group size. We suggest that these results are best explained by competition for food and risk of predation.

High altitude exposure alters gene expression levels of DNA repair enzymes, and modulates fatty acid metabolism by SIRT4 induction in human skeletal muscle

Zoltan Acsa, Zoltan Boria, Masaki Takedaa, Peter Osvatha, et al.
Respiratory Physiology & Neurobiology 196 (2014) 33–37
http://dx.doi.org/10.1016/j.resp.2014.02.006

We hypothesized that high altitude exposure and physical activity associated with the attack to Mt Everest could alter mRNA levels of DNA repair and metabolic enzymes and cause oxidative stress-related challenges in human skeletal muscle. Therefore, we have tested eight male mountaineers (25–40 years old) before and after five weeks of exposure to high altitude, which included attacks to peaks above 8000 m. Data gained from biopsy samples from vastus lateralis revealed increased mRNA levels of both cytosolic and mitochondrial superoxide dismutase. On the other hand 8-oxoguanine DNA glycosylase(OGG1) mRNA levels tended to decrease while Ku70 mRNA levels and SIRT6 decreased with altitude exposure. The levels of SIRT1 and SIRT3 mRNA did not change significantly. But SIRT4 mRNA level increased significantly, which could indicate decreases in fatty acid metabolism, since SIRT4 is one of the important regulators of this process. Within the limitations of this human study, data suggest that combined effects of high altitude exposure and physical activity climbing to Mt. Everest, could jeopardize the integrity of the particular chromosome.

High-altitude adaptations in vertebrate hemoglobins

Roy E. Weber
Respiratory Physiology & Neurobiology 158 (2007) 132–142
http://dx.doi.org:/10.1016/j.resp.2007.05.001

Vertebrates at high altitude are subjected to hypoxic conditions that challenge aerobic metabolism. O2 transport from the respiratory surfaces to tissues requires matching between theO2 loading and unloading tensions and theO2-affinity of blood, which is an integrated function of hemoglobin’s intrinsic O2-affinity and its allosteric interaction with cellular effectors (organic phosphates, protons and chloride). Whereas short-term altitudinal adaptations predominantly involve adjustments in allosteric interactions, long-term, genetically-coded adaptations typically involve changes in the structure of the hemoglobin molecules. The latter commonly comprise substitutions of amino acid residues at the effector binding sites, the heme protein contacts, or at inter-subunit contacts that stabilize either the low-affinity (‘Tense’) or the high-affinity (‘Relaxed’) structures of the molecules. Molecular heterogeneity (multiple iso-Hbs with differentiated oxygenation properties) can further broaden the range of physico-chemical conditions where Hb functions under altitudinal hypoxia. This treatise reviews the molecular and cellular mechanisms that adapt hemoglobin-oxygen affinities in mammals, birds and ectothermic vertebrates at high altitude.

Vertebrate animals display remarkable ability to tolerate high altitudes and cope with the concomitant decreases in O2 tension that potentially constrain aerobic life (Monge and Leon-Velarde, 1991;Weber, 1995; Samaja et al., 2003). Compared to an ambient PO2 of approximately 160 mm Hg at sea level, inspired tension approximates only 95 mm Hg for llamas and frogs from Andean habitats above 4000 m, 45 mm Hg for bar-headed geese that fly across the Himalayas, and 33 mm Hg for Ruppell’s griffon that soars at 11,300 m over Africa’s Ivory Coast. Apart from the distinct adaptations manifest in blood’s O2-transporting properties, tolerance to decreased O2 availability may entail reconfigurations at the organ and cellular levels that include a switch to partial anaerobiosis. Driven by needs to reduce aerobic metabolic rate and maintain functional integrity (Ramirez et al., 2007), these pertain to a core triad of adaptations:

metabolic suppression,
tolerance to metabolite (e.g. lactate) accumulation, and
defenses against increased free radicals associated with return to high O2 tensions (Bickler and Buck, 2007).

The response to oxygen lack comprises two phases

defense, which includes metabolic arrest (a suppression of ATP-demand and ATP-supply) and channel arrest (decreases cell membrane permeability), and
rescue, which commonly involves preferential expression of proteins that are implicated in extending metabolic down-regulation (Hochachka et al., 1996).

These responses vary greatly in different species and different tissues. Thus, although mixed-venous lactate concentrations increase strongly in sea-level as well as high-altitude acclimated pigeons that are exposed to altitude (from 1–2 mM at sea level to 5–7 mM at 9000 m) (Weinstein et al., 1985), and humans performing submaximal work at high altitude show a transient ‘lactate paradox’ (lower peak lactate levels that humans living at sea level (Lundby et al., 2000)), many species do not exhibit altitude-related changes in anaerobic metabolism.

Organismic adaptations to survive and perform physical exercise at extreme altitudinal hypoxia are diverse. In birds the undisputed high-altitude champions, where flapping flight may raise the energy demand 10–20-fold compared to resting levels (Scott et al., 2006), a highly efficient “cross-current” ventilation perfusion arrangement in the lungs may increase arterial O2 tensions above the tensions in expired air (Scheid, 1979) and drastically reduce the difference between inhalant and arterial O2 tensions (to 1 mm Hg in bar-headed geese subjected to simulated altitude of 11580 m) (Black and Tenney, 1980). The Andean frog Telmatobius culeus has a highly ‘oversized’ (folded) and vascularized skin that is ventilated by ‘bobbing’ behavior to support water(=skin) breathing. Manifold organismic adaptations moreover include combinations of increased muscle Mb concentrations (Reynafarje and Morrison, 1962) increased muscle capillarization (manifest in mammals and birds (cf. Monge et al., 1991)) and decreased red cell size (seen in amphibians but not high-altitude reptiles (Ruiz et al., 1989; Ruiz et al., 1993)). Amphibians exhibit an interspecific correlation between erythrocyte count and the degree of vascularization of respiratory surfaces and muscle tissues (Hutchison and Szarski, 1965), that reflect differences in their ability to tolerate altitudinal hypoxia.

A sensitivity analysis of the factors that may limit exercise performance identifies high Hb-O2 affinity, together with high total ventilation and high tissue diffusion capacity as the physiological traits that have greatest adaptive benefit for bird flight at extreme high altitude (Scott and Milsom, 2006). Blood O2 affinity is a combination of the intrinsic O2 affinity of the ‘stripped’ (purified) Hb molecules and the interaction of allosteric effectors (like organic phosphates, protons and chloride ions) that decrease Hb-O2 affinity inside the rbcs (Weber and Fago, 2004). Short-term adaptations in O2 affinity are commonly mediated by changes in erythrocytic effectors such as organic phosphates (2,3-diphosphoglycerate, DPG, in mammals, inositol pentaphosphate, IPP, in birds, ATP in reptiles, and ATP and DPG in amphibians), whereas long-term adaptations (that include interspecific ones that are genetically determined) commonly involve changes in Hb structure (amino acid exchanges) that alter Hb’s intrinsic O2 affinity or its sensitivity to allosteric effectors.

Vertebrate Hbs are tetrameric molecules composed of two α (or α-like) chains and two β (or β-like) chains, which in humans consist of 141 and 146 amino acid residues, respectively. Each subunit exhibits a highly characteristic “globin fold” comprised of seven or eight α-helices (labelled A, B, C, etc.) linked by nonhelical (EF, FG) segments, and N- and C-terminal extensions termed NA and HC, respectively. Individual amino acid residues are identified by their sequential positions in chain or/and the helix; thus α1131(H14)-Ser refers to Serine that is the 131^st residue of α1 chain and the 14th of the H. During (de-) oxygenation Hb switches between two major structural states:

the high affinity oxygenated R (relaxed) state that prevails at the respiratory surfaces, and
the low affinity, deoxygenated T (tense) state that occurs predominantly in the tissues and is constrained by additional hydrogen bonds and salt bridges.

The Hbs exhibit cooperative homotropic interactions between the O2 binding heme groups (that cause the S-shaped O2 equilibrium curves and increase O2 loading and unloading for a given change in O2 tension) as well as inhibitory, heterotropic interactions between the hemes and the binding sites of effectors that decrease O2 affinity (increase the half-saturation O2 loading tension, P50) and facilitate O2 unloading.

A comparison of Hbs from different species (cf. Perutz, 1983) reveals that variation in the sensitivities to effectors correlates generally with exchanges of very few of the approximately 287 amino acid residues that comprise each αβ dimer. Thus in adult human Hb (HbA) at physiological pH, the majority of the Bohr effect (pH dependence of Hb-O2 affinity that facilitates O2 release in relatively acid working muscles) results from proton binding at the C-terminal residues of the β-chains (β146-His) (cf. Lukin and Ho, 2004). Correspondingly DPG binds to only four β-chain residues (β1-Val, β2-His, β82-Lys and β143-His), CO2 binding (carbamate formation) occurs at the uncharged amino-termini of both chains (α1-Val and β1-Val), and monovalent anions like chloride are considered to bind at one α-chain site (between α1-Val and α131–Ser) and one β-chain site (between β82-Lys and β1-Val) (cf. Riggs, 1988).

The small number of sites that primarily determine Hb-O2 affinity and its sensitivity to effectors aligns with the neutral theory of molecular evolution (Kimura, 1979), which holds that the majority of amino acid substitutions are non-adaptive and harmless—and facilitates identification of key molecular mechanisms implicated in adaptations at altitude.

The role of effectors in altitude adaptation is aptly illustrated in humans where Hb structure (intrinsic O2 affinity) remains unchanged. Newcomers and permanent residents at moderate altitude (e.g. 2000 m) show increased DPG levels, resulting in a decreased O2 affinity that positions arterial and mixed venous O2 tensions on the steep part of the O2 equilibrium curve, increasing O2 capacitance ([1]bO2) and O2 transport, without materially compromising O2 loading (Turek et al., 1973; Mairbaurl, 1994). The increased DPG correlates with erythropoietin-mediated formation of new rbcs that have higher glycolytic rates and higher DPG and ATP levels than old rbcs. However, faster increases in P50 than in DPG level indicate contributions from other factors, such as chloride and ATP, and Mg ions that neutralize the anionic effectors (Mairbaurl et al., 1993). At higher altitudes (4559 m) increased hyperventilation that drives off CO2 causes respiratory alkalosis (Mairbaurl, 1994). The higher pH increases O2 affinity via the Bohr effect and, offsetting the effect of increased DPG, leads to a similar O2 affinity and arterio-venous O2 saturation difference as at sea level (Fig.). O2 unloading in the tissues is moreover enhanced by metabolic acidification of capillary blood (Fig.).

Obviously right-shifted curves (that favor O2 unloading) becomes counterproductive at extreme altitudes where O2 loading becomes compromised, predicting that decreased O2 affinity becomes maladaptive under severe hypoxic stress. This is consistent with the observation that a carbamylation-induced increase in blood O2 affinity of rats (that lowers P50 from 27 to 15 mm Hg), increases survival under hypobaric hypoxia equivalent to 9200 meters’ altitude (Eaton et al., 1974). The altitude limit where increased affinity rather than a decreased affinity optimizes tissue O2 supply < 5000 m in man (Samaja et al., 2003)] depends on organismic adaptations (e.g. efficiency of gas exchange) and thus will vary between species. Mammals that permanently inhabit high altitudes and show high blood O2 affinities include the Andean rodent Chinchilla brevicaudata living at 3000–5000 m (blood P50 = 23 mm Hg compared to 38 mm Hg in the rat) (Ostojic et al., 2002). The deer mouse, Peromyscus maniculatus that occurs continuously from sea level to altitudes above 4300 m shows a strong correlation between blood O2 affinity and native altitude (Snyder et al., 1988). That genetically based differences in cofactor levels may contribute to this relationship follows from lower DPG/Hb ratios found in specimens resident, and native to, high altitude than in those from low altitude, after long-term acclimation of both groups to low altitude (Snyder, 1982).

O2 equilibrium curves of human blood illustrating the effects of increases in red cell DPG and pH at high-altitude

O2 equilibrium curves of human blood illustrating the effects of increases in red cell DPG and pH at high-altitude (4559 m). Solid curves refer to arterial blood (P50 = 26 mm,upper section) and cubical venous blood (P50 = 27.5 mm Hg, lower section); their displacement reflects the Bohr effect. The broken curves depict effects of increased DPG levels (↑DPG) at unchanged pH, increased pH (↑pH) at unchanged DPG, and of decreased tissue pH (↓pH) resulting from higher degrees of metabolic acidification in the tissues. Open and shaded vertical columns indicate O2 unloaded at sea level and 4559 m, respectively, for venous O2 tensions (PvO2) of 25 and 15 mm Hg,respectively [Modified after (Mairbaurl, 1994)].

Camelids. The high blood-O2 affinities in Andean camelids (llama, vicunia, alpaca and guanaco) whose natural habitats exceed 3000 m (Bartels et al., 1963) compared to those of similarly-sized lowland mammals are well-established. In the camelids a β2His→Asn substitution deletes two of the seven DPG contacts in the tetrameric Hb, which increases blood O2 affinity by reducing the DPG effect. Although the intrinsic Hb-O2 affinity is lower in llama than in the related, lowland camel (Bauer et al., 1980), llama blood has a higher O2 affinity due to a three-fold lower DPG-binding than in camel Hb that has the same DPG binding sites as humans (Bauer et al., 1980). In vicunia, a higher O2 affinity than in llama (that has identical β-chains), correlates with the α130Ala→Thr substitution, which introduces a hydroxyl polar group that predictably reduces the chloride binding at adjacent α131Asn residue .

Sheep and goats commonly express two isoforms, HbA and HbB. The heterogeneity is controlled by two autosomal alleles with codominant expression. Whereas individuals expressing HbA have higher blood-O2 affinity than those that express HbB, heterozygotes that express both forms at equimolar concentrations in the same erythrocytes show intermediate affinity. Anemic blood loss induces switching from HbA to HbC that has a similarly high affinity. Hbs A, B and C have identical α-chains but different β[1]-chains. It appears unknown whether altitudinal exposure (which like anemia, induces tissue hypoxia) modulates Hb heterogeneity via selective expression of specific β-chains.

Compared to most mammals that possess one major adult and one major fetal Hb, yak, Poephagus (=Bos) grunniens, a native to altitudes of 3000–6000 m in Tibet, Nepal and Bhutan, has two or four major adult Hbs and two major fetal Hbs. These Hbs exhibit higher intrinsic affinities than closely-related bovine Hb, marked DPG sensitivities and, exceptional amongst mammals, differentiated O2 affinities that indicates an extended range of ambient O2 tensions (and altitudes) in which the composite Hb functions.

(Not shown). Representation of interchain contacts considered to underly differentiated O2 affinities in Rueppell’s griffon isoHbs A, A , D and D that have identical β- chains but different α- chains. Accordingly the van der Waal’s contact between β134Ile and β₁125-Asp in Hbs A , D and D stabilizes the low-affinity, T-state less strongly than the H-bond between Thr 134 and β₁125-Asp and thus increases O2 affinity in Hbs A, D and D. Analogously, the hydrogen bonds between α₁38-β₂97/99 that stabilize the high-affinity oxystructure (raising O2 affinity in isoHbs D and D) cannot form in HbA and HbA that have Pro at α₁38.

Ostriches, the largest extant birds, exhibit a β2His→Gln exchange (that reduces phosphate interaction). They moreover ‘use’ ITP (inositol phosphate) that carries fewer negative charges, and predictably has lesser allosteric effect, than IPP (Isaacks et al., 1977), predicting a high blood O2 affinity that is compatible with ‘scaling’ and (as in elephants) increases high altitude tolerance.

Whereas some adult birds express one major iso-Hb (HbA), the majority of species, reportedly all that fly at high altitudes (Hiebl et al., 1987), also express a less abundant HbD. HbD has the same β-chains as HbA but different α-chains (α^D) and exhibits higher O2 affinities (Huisman et al., 1964). There is no consistent evidence for hypoxia-induced changes in HbD expression.

An example of how “molecular anatomy is just as key to understanding molecular adaptation as phylogeny and physiological ecology” (Golding and Dean, 1998) is Hb of the high-altitude tolerant bar-headed goose that has a sharply higher blood O2 affinity than that of the closely related graylag goose that is restricted to lower altitudes (P50 = 29.7 and 39.5mmHg at 37 ◦C and pH 7.4). The Hbs differ by only four (greylag→bar-headed) amino acid exchanges: α18Gly→Ser, α63Ala→Val, β125Glu→Asp and α119Pro→Ala. The last mentioned exchange that is unique in birds, predictably increases O2 affinity, by deleting a contact between α₁119 and β₁55 that destabilizes the T-structure (Perutz, 1983). Moreover, Andean ‘goose’ Hb that also has high blood O2 affinity shows β55 Leu→Ser that deletes the same contact. Significantly, two human Hb mutants (α₁19Pro–Ala and β₁55Met→Ser) engineered by site-directed mutagenesis to mimic the mutations found in bar-headed and Andean geese possess markedly higher O2 affinities than native HbA.

Although “the study of molecular adaptation has long been fraught with difficulties not the least of which is identifying out the hundreds of amino acid replacements, those few directly responsible for major adaptations” Hb’s adaptations to high altitude are a prime example of how “an amino acid replacement of modest effect at the molecular level causes a dramatic expansion in an ecological niche” [quotations from (Golding et al., 1998)].

However, the pathway of molecular O2 from the respiratory medium to the cellular combustion sites via the Hb molecules is regulated by a symphony of supplementary adaptations that span different levels of biological organization, each of which (according to the principle of symmorphosis) may become maximally recruited in extreme cases (as in birds actively flying above 10,000 m). Apart from hyperventilation, that appears to occur ubiquitously (and increases blood O2 affinity via increased pH), different species subjected to less extreme hypoxic stress utilize different adaptations among the arsenal of organismic, cellular and molecular strategies that favor efficient aerobic utilization of the scarce O2 available at high altitude. No clear correlations exist between the adaptive strategies recruited by different animals on the one hand, and their phylogenetic position, mode of life or ecological niches on the other. An overall limitation is that short-term adaptive adjustments in O2 affinity (that may occur within individual animals) necessarily involves rapid adaptive responses, such as changes in the levels of erythrocytic effectors, whereas the long-term acclimations that have accumulated in permanent high-altitude dwellers during evolutionary development.

Genetic Diversity of Microsatellite DNA Loci of Tibetan Antelope (Chiru, Pantholops hodgsonii) in Hoh Xil National Nature Reserve, Qinghai, China

Hui Zhou, Diqiang Li, Yuguang Zhang, Tao Yang, Yi Liu
J Genetics and Genomics (Formerly Acta Genetica Sinica) 2007; 34(7): 600-607

The Tibetan antelope (Pantholops hodgsonii), indigenous to China, became an endangered species because of considerable reduction both in number and distribution during the 20th century. Presently, it is listed as an AppendixⅠspecies by CITES and as CategoryⅠ by the Key Protected Wildlife List of China. Understanding the genetic diversity and population structure of the Tibetan antelope is significant for the development of effective conservation plans that will ensure the recovery and future persistence of this species. Twenty-five microsatellites were selected to obtain loci with sufficient levels of polymorphism that can provide in-formation for the analysis of population structure. Among the 25 loci that were examined, nine of them showed high levels of genetic diversity. The nine variable loci (MCM38, MNS64, IOBT395, MCMAI, TGLA68, BM1329, BMS1341, BM3501, and MB066) were used to examine the genetic diversity of the Tibetan antelope (n = 75) in Hoh Xil National Nature Reserve(HXNNR), Qinghai, China. The results obtained by estimating the number of population suggested that all the 75 Tibetan antelope samples were from the same population. The mean number of alleles per locus was 9.4 ± 0.5300 (range, 7–12) and the mean effective number of alleles was 6.519 ± 0.5271 (range, 4.676–9.169). The observed mean and expected heterozygosity were 0.844 ± 0.0133 (range, 0.791–0.897) and 0.838 ± 0.0132 (range, 0.786–0.891), respectively. Mean Polymorphism Information Content (PIC) was 0.818 ± 0.0158 (range, 0.753–0.881). The value of Fixation index (Fis) ranged from −0.269 to −0.097 with the mean of −0.163 ± 0.0197. Mean Shannon’s information index was 1.990 ± 0.0719 among nine loci (range, 1.660–2.315). These results provide baseline data for the evaluation of the level of genetic variation in Tibetan antelope, which will be important for the development of conservation strategies in future.

Expression profiling of abundant genes in pulmonary and cardiac muscle tissues of Tibetan Antelope (Pantholops hodgsonii)

Xiaomei Tong, Yingzhong Yang, Weiwei Wang, Zenzhong Bai, et al.
Gene 523 (2013) 187–191
http://dx.doi.org/10.1016/j.gene.2013.03.011

The Tibetan Antelope (TA), which has lived at high altitude for millions of years, was selected as the model species of high hypoxia-tolerant adaptation. Here we constructed two cDNA libraries from lung and cardiac muscle tissues, obtained EST sequences from the libraries, and acquired extensive expression data related energy metabolism genes. Comparative analyses of synonymous (Ks) and nonsynonymous (Ka) substitution rates of nucleus-encoded mitochondrial unigenes among different species revealed that many antelope genes have undergone rapid evolution. Surfactant-associated protein A (SP-A) and surfactant-associated protein B (SP-B) genes in the AT lineage experienced accelerated evolution compared to goat and sheep, and these two genes are highly expressed in the lung tissue. This study suggests that many specific genes of lung and cardiac muscle tissues showed unique expression profiles and may undergo fast adaptive evolution in TA. These data provide useful information for studying on molecular adaptation to high-altitude in humans as well as other mammals.

Exogenous Sphingosine-1-Phosphate Boosts Acclimatization in Rats Exposed to Acute Hypobaric Hypoxia: Assessment of Haematological and Metabolic Effects

Sonam Chawla, Babita Rahar, Mrinalini Singh, Anju Bansal, et al.
PLoS ONE 9(6): e98025. http://dx.doi.org:/10.1371/journal.pone.0098025

Background: The physiological challenges posed by hypobaric hypoxia warrant exploration of pharmacological entities to improve acclimatization to hypoxia. The present study investigates the preclinical efficacy of sphingosine-1-phosphate (S1P) to improve acclimatization to simulated hypobaric hypoxia. Experimental Approach: Efficacy of intravenously administered S1P in improving hematological and metabolic acclimatization was evaluated in rats exposed to simulated acute hypobaric hypoxia (7620 m for 6 hours) following S1P pre-treatment for three days. Major Findings: Altitude exposure of the control rats caused systemic hypoxia, hypocapnia (plausible sign of hyperventilation) and respiratory alkalosis due to suboptimal renal compensation indicated by an overt alkaline pH of the mixed venous blood. This was associated with pronounced energy deficit in the hepatic tissue along with systemic oxidative stress and inflammation. S1P pre-treatment improved blood oxygen-carrying-capacity by increasing hemoglobin, hematocrit, and RBC count, probably as an outcome of hypoxia inducible factor-1a mediated erythropoiesis and renal S1P receptor 1 mediated hemoconcentation. The improved partial pressure of oxygen in the blood could further restore aerobic respiration and increase ATP content in the hepatic tissue of S1P treated animals. S1P could also protect the animals from hypoxia mediated oxidative stress and inflammation. Conclusion: The study findings highlight S1P’s merits as a preconditioning agent for improving acclimatization to acute hypobaric hypoxia exposure. The results may have long term clinical application for improving physiological acclimatization of subjects venturing into high altitude for occupational or recreational purposes.

S1P Stabilizes HIF-1a and Boosts HIF-1a Mediated Hypoxia Adaptive Responses

S1P pre-conditioning led to 1.9 fold higher HIF-1a level in the kidney tissue (p<0.001) and 1.3 fold higher HIF-1a level in the liver tissue (p<0.001) in 1 mg/kg b.w. S1P group than in hypoxia control group. However, the hypoxia control group also had 1.3 folds higher HIF-1a levels in both liver and kidney tissues than in normoxia control groups, indicating a non-hypoxic boost of HIF-1a in S1P treated animals (Figure 1a and b). Further, plasma Epo levels were also observed to be significantly higher following S1P pre-treatment compared to the hypoxia control groups (p=0.05) (Figure 1a). Epo being primarily secreted by the kidneys and its expression being under regulation of HIF-1a, the raised plasma Epo level could be attributed to higher HIF-1a level in the kidney.

Figure 1. (not shown) Effect of S1P treatment on HIF-1a accumulation and downstream gene expression. a) Renal HIF-1a accumulation and Epo accumulation in plasma. HIF-1a accumulation in the renal tissue homogenate and build-up of erythropoietin in plasma was quantified. b) Hepatic HIF-1a accumulation. c) Effect S1P pre-treatment on circulatory VEGF. Vascular endothelial growth factor (VEGF) was quantified in plasma of experimental animals. These estimations were carried out using sandwich ELISA, and were carried out in triplicates for each experimental animal. Values are representative of mean 6 SD (n = 6). Statistical significance was calculated using ANOVA/post hoc Bonferroni. NC: Normoxia control, HC: Hypoxia control, 1: 1 mg S1P/kg b.w., 10: 10 mg S1P/kg b.w., 100: 100 mg S1P/kg b.w., p<0.05 compared with the normoxic control, p<0.01 compared with the normoxic control, p<0.001 compared with the normoxic control, p<0.05 compared with the hypoxic control, p<0.01 compared with the hypoxic control, p<0.001 compared with the hypoxic control. http://dx.doi.org:/10.1371/journal.pone.0098025.g001

Figure 2.(not shown) Effect of S1P treatment on S1P1 expression in renal tissue. Representative immune-blot of S1P1. Densitometric analysis of blot normalized against the loading control (α-tubulin). Values are representative of mean 6 SD (n = 6). Statistical significance was calculated using ANOVA/post hoc Bonferroni. NC: Normoxia control, HC: Hypoxia control, 1: 1 mg S1P/kg b.w., 10: 10 mg S1P/kg b.w., 100: 100 mg S1P/kg b.w., p<0.05 compared with the normoxic control, p<0.01 compared with the normoxic control, p<0.001 compared with the normoxic control, p< 0.05 compared with the hypoxic control, p<0.01 compared with the hypoxic control, p<0.001 compared with the hypoxic control. http://dx.doi.org:/10.1371/journal.pone.0098025.g002

Cloning of hypoxia-inducible factor 1α cDNA from a high hypoxia tolerant mammal—plateau pika (Ochotona curzoniae)

T.B. Zhao, H.X. Ning, S.S. Zhu, P. Sun, S.X. Xu, Z.J. Chang, and X.Q. Zhao
Biochemical and Biophysical Research Communications 316 (2004) 565–572
http://dx.doi.org:/10.1016/j.bbrc.2004.02.087

Hypoxia-inducible factor 1 is a transcription factor composed of HIF-1α and HIF-1β. It plays an important role in the signal transduction of cell response to hypoxia. Plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant and cold adaptation species living only at 3000–5000m above sea level on the Qinghai-Tibet Plateau. In this study, HIF-1α cDNA of plateau pika was cloned and its expression in various tissues was studied. The results indicated that plateau pika HIF-1α cDNA was highly identical to those of the human (82%), bovine (89%), mouse (82%), and Norway rat (77%). The deduced amino acid sequence (822 bp) showed 90%, 92%, 86%, and 86% identities with those of the human, bovine, house mouse, and Norway rat, respectively. Northern blot analyses detected two isoforms named pLHIF-1α and pSHIF-1α. The HIF-1α mRNA was highly expressed in the brain and kidney, and much less in the heart, lung, liver, muscle, and spleen, which was quite different from the expression pattern of mouse mRNA. Meanwhile, a new variant of plateau pika HIF-1α mRNA was identified by RT-PCR and characterized. The deduced protein, composed of 536 amino acids, lacks a part of the oxygen-dependent degradation domain (ODD), both transactivation domains (TADs), and the nuclear localization signal motif (NLS). Our results suggest that HIF-1α may play an important role in the pika’s adaptation to hypoxia, especially in brain and kidney, and pika HIF-1α function pattern may be different from that of mouse HIF-1α. Furthermore, for the high ratio of HIF-1α homology among the animals, the HIF-1α gene may be a good phylogenetic performer in recovering the true phylogenetic relationships among taxa.

Comparative Proteomics Analyses of Kobresia pygmaea Adaptation to Environment along an Elevational Gradient on the Central Tibetan Plateau

Xiong Li, Yunqiang Yang, Lan Ma, Xudong Sun, et al.
PLoS ONE 9(6): e98410. http://dx.doi.org:/10.1371/journal.pone.0098410

Variations in elevation limit the growth and distribution of alpine plants because multiple environmental stresses impact plant growth, including sharp temperature shifts, strong ultraviolet radiation exposure, low oxygen content, etc. Alpine plants have developed special strategies to help survive the harsh environments of high mountains, but the internal mechanisms remain undefined. Kobresia pygmaea, the dominant species of alpine meadows, is widely distributed in the Southeastern Tibet Plateau, Tibet Autonomous Region, China. In this study, we mainly used comparative proteomics analyses to investigate the dynamic protein patterns for K. pygmaea located at four different elevations (4600, 4800, 4950 and 5100 m). A total of 58 differentially expressed proteins were successfully detected and functionally characterized. The proteins were divided into various functional categories, including material and energy metabolism, protein synthesis and degradation, redox process, defense response, photosynthesis, and protein kinase. Our study confirmed that increasing levels of antioxidant and heat shock proteins and the accumulation of primary metabolites, such as proline and abscisic acid, conferred K. pygmaea with tolerance to the alpine environment. In addition, the various methods K. pygmaea used to regulate material and energy metabolism played important roles in the development of tolerance to environmental stress. Our results also showed that the way in which K. pygmaea mediated stomatal characteristics and photosynthetic pigments constitutes an enhanced adaptation to alpine environmental stress. According to these findings, we concluded that K. pygmaea adapted to the high-elevation environment on the Tibetan Plateau by aggressively accumulating abiotic stress related metabolites and proteins and by the various life events mediated by proteins. Based on the species flexible physiological and biochemical processes, we surmised that environment change has only a slight impact on K. pygmaea except for possible impacts to populations on vulnerable edges of the species’ range
Altered mitochondrial biogenesis and its fusion gene expression is involved in the high-altitude adaptation of rat lung

Loganathan Chitra, Rathanam Boopathy
Respiratory Physiology & Neurobiology 192 (2014) 74– 84
http://dx.doi.org/10.1016/j.resp.2013.12.007

Intermittent hypobaric hypoxia-induced preconditioning (IHH-PC) of rat favored the adaption of lungs to severe HH conditions, possibly through stabilization of mitochondrial function. This is based on the data generated on regulatory coordination of nuclear DNA-encoded mitochondrial biogenesis; dynamics,and mitochondrial DNA (mtDNA)-encoded oxidative phosphorylation (mt-OXPHOS) genes expression. At16th day after start of IHH-PC (equivalent to 5,000 m, 6 h/d, 2 w of treatment), rats were exposed to severe HH stimulation at 9142 m for 6 h. The IHH-PC significantly counteracted the HH-induced effect of increased lung: water content; tissue damage; and oxidant injury. Further, IHH-PC significantly increased the mitochondrial number, mtDNA content and mt- OXPHOS complex activity in the lung tissues. This observation is due to an increased expression of genes involved in mitochondrial biogenesis (PGC-1α,ERRα, NRF1, NRF2 and TFAM), fusion (Mfn1 and Mfn2) and mt OXPHOS. Thus, the regulatory pathway formed by PGC-1α/ERRα/Mfn2 axes is required for the mitochondrial adaptation provoked by IHH-PC regimen to counteract subsequent HH stress.

Molecular characteristics of Tibetan antelope (Pantholops hodgsonii) mitochondrial DNA control region and phylogenetic inferences with related species

Feng, B. Fan, K. Li, Q.D. Zhang, et al.
Small Ruminant Research 75 (2008) 236–242
http://dx.doi.org:/10.1016/j.smallrumres.2007.06.011

Although Tibetan antelope (Pantholops hodgsonii) is a distinctive wild species inhabiting the Tibet-Qinghai Plateau, its taxonomic classification within the Bovidae is still unclear and little molecular information has been reported to date. In this study of Tibetan antelope, the complete control regions of mtDNA were sequenced and compared to those of Tibetan sheep (Ovis aries) and goat (Capra hircus). The length of the control region in Tibetan antelope, sheep and goat is 1067, 1181/1106 and 1121 bp, respectively. A 75-bp repeat sequence was found near the 5’ end of the control region of Tibetan antelope and sheep, the repeat numbers of which were two in Tibetan antelope and three or four in sheep. Three major domain regions, including HVI, HVII and central domain, in Tibetan antelope, sheep and goat were outlined, as well as other less conserved blocks, such as CSB-1, CSB-2, ETAS-1 and ETAS-2. NJ cluster analysis of the three species revealed that Tibetan antelope was more closely related to Tibetan sheep than Tibetan goat. These results were further confirmed by phylogenetic analysis using the partial control region sequences of these and 13 other antelope species. Tibetan antelope is better assigned to the Caprinae rather than the Antilopinae subfamily of the Bovidae.

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Neonatal Pathophysiology

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Neonatal Pathophysiology

Writer and Curator: Larry H. Bernstein, MD, FCAP

Introduction

This curation deals with a large and specialized branch of medicine that grew since the mid 20th century in concert with the developments in genetics and as a result of a growing population, with large urban populations, increasing problems of premature deliveries. The problems of prematurity grew very preterm to very low birth weight babies with special problems. While there were nurseries, the need for intensive care nurseries became evident in the 1960s, and the need for perinatal care of pregnant mothers also grew as a result of metabolic problems of the mother, intrauterine positioning of the fetus, and increasing numbers of teen age pregnancies as well as nutritional problems of the mother. There was also a period when the manufacturers of nutritional products displaced the customary use of breast feeding, which was consequential. This discussion is quite comprehensive, as it involves a consideration of the heart, the lungs, the brain, and the liver, to a large extent, and also the kidneys and skeletal development.

It is possible to outline, with a proportionate emphasis based on frequency and severity, this as follows:

Genetic and metabolic diseases
Nervous system
Cardiovascular
Pulmonary
Skeletal – bone and muscle
Hematological
Liver
Esophagus, stomach, and intestines
Kidneys
Immune system

Fetal Development

Gestation is the period of time between conception and birth when a baby grows and develops inside the mother’s womb. Because it’s impossible to know exactly when conception occurs, gestational age is measured from the first day of the mother’s last menstrual cycle to the current date. It is measured in weeks. A normal gestation lasts anywhere from 37 to 41 weeks.

Week 5 is the start of the “embryonic period.” This is when all the baby’s major systems and structures develop. The embryo’s cells multiply and start to take on specific functions. This is called differentiation. Blood cells, kidney cells, and nerve cells all develop. The embryo grows rapidly, and the baby’s external features begin to form.

Week 6-9: Brain forms into five different areas. Some cranial nerves are visible. Eyes and ears begin to form. Tissue grows that will the baby’s spine and other bones. Baby’s heart continues to grow and now beats at a regular rhythm. Blood pumps through the main vessels. Your baby’s brain continues to grow. The lungs start to form. Limbs look like paddles. Essential organs begin to grow.

Weeks 11-18: Limbs extended. Baby makes sucking motion. Movement of limbs. Liver and pancreas produce secretions. Muscle and bones developing.

Week 19-21: Baby can hear. Mom feels baby – and quickening.

http://www.nlm.nih.gov/medlineplus/ency/article/002398.htm

fetal-development

https://polination.files.wordpress.com/2014/02/abortion-new-research-into-fetal-development.jpg

Inherited Metabolic Disorders

The original cause of most genetic metabolic disorders is a gene mutation that occurred many, many generations ago. The gene mutation is passed along through the generations, ensuring its preservation.

Each inherited metabolic disorder is quite rare in the general population. Considered all together, inherited metabolic disorders may affect about 1 in 1,000 to 2,500 newborns. In certain ethnic populations, such as Ashkenazi Jews (Jews of central and eastern European ancestry), the rate of inherited metabolic disorders is higher.

Hundreds of inherited metabolic disorders have been identified, and new ones continue to be discovered. Some of the more common and important genetic metabolic disorders include:

Lysosomal storage disorders : Lysosomes are spaces inside cells that break down waste products of metabolism. Various enzyme deficiencies inside lysosomes can result in buildup of toxic substances, causing metabolic disorders including:

Hurler syndrome (abnormal bone structure and developmental delay)
Niemann-Pick disease (babies develop liver enlargement, difficulty feeding, and nerve damage)
Tay-Sachs disease (progressive weakness in a months-old child, progressing to severe nerve damage; the child usually lives only until age 4 or 5)
Gauchers disease and others

Galactosemia: Impaired breakdown of the sugar galactose leads to jaundice, vomiting, and liver enlargement after breast or formula feeding by a newborn.

Maple syrup urine disease: Deficiency of an enzyme called BCKD causes buildup of amino acids in the body. Nerve damage results, and the urine smells like syrup.

Phenylketonuria (PKU): Deficiency of the enzyme PAH results in high levels of phenylalanine in the blood. Mental retardation results if the condition is not recognized.

Glycogen storage diseases: Problems with sugar storage lead to low blood sugar levels, muscle pain, and weakness.

Metal metabolism disorders: Levels of trace metals in the blood are controlled by special proteins. Inherited metabolic disorders can result in protein malfunction and toxic accumulation of metal in the body:

Wilson disease (toxic copper levels accumulate in the liver, brain, and other organs)

Hemochromatosis (the intestines absorb excessive iron, which builds up in the liver, pancreas, joints, and heart, causing damage)

Organic acidemias: methylmalonic acidemia and propionic acidemia.

Urea cycle disorders: ornithine transcarbamylase deficiency and citrullinemia

Hemoglobinopathies – thalassemias, sickle cell disease

Red cell enzyme disorders – glucose-6-phosphate dehydrogenase, pyruvate kinase

This list is by no means complete.

http://www.webmd.com/a-to-z-guides/inherited-metabolic-disorder-types-and-treatments

New variations in the galactose-1-phosphate uridyltransferase (GALT) gene

Clinical and molecular spectra in galactosemic patients from neonatal screening in northeastern Italy: Structural and functional characterization of new variations in the galactose-1-phosphate uridyltransferase (GALT) gene

E Viggiano, A Marabotti, AP Burlina, C Cazzorla, MR D’Apice, et al.
Gene 559 (2015) 112–118
http://dx.doi.org/10.1016/j.gene.2015.01.013
Galactosemia (OMIM 230400) is a rare autosomal recessive inherited disorder caused by deficiency of galactose-1-phosphate uridyltransferase (GALT; OMIM 606999) activity. The incidence of galactosemia is 1 in 30,000–60,000, with a prevalence of 1 in 47,000 in the white population. Neonates with galactosemia can present acute symptoms, such as severe hepatic and renal failure, cataract and sepsis after milk introduction. Dietary restriction of galactose determines the clinical improvement in these patients. However, despite early diagnosis by neonatal screening and dietary treatment, a high percentage of patients develop long-term complications such as cognitive disability, speech problems, neurological and/or movement disorders and, in females, ovarian dysfunction.

With the benefit of early diagnosis by neonatal screening and early therapy, the acute presentation of classical galactosemia can be prevented. The objectives of the current study were to report our experience with a group of galactosemic patients identified through the neonatal screening programs in northeastern Italy during the last 30 years.

No neonatal deaths due to galactosemia complications occurred after the introduction of the neonatal screening program. However, despite the early diagnosis and dietary treatment, the patients with classical galactosemia showed one or more long-term complications.

A total of 18 different variations in the GALT gene were found in the patient cohort: 12 missense, 2 frameshift, 1 nonsense, 1 deletion, 1 silent variation, and 1 intronic. Six (p.R33P, p.G83V, p.P244S, p.L267R, p.L267V, p.E271D) were new variations. The most common variation was p.Q188R (12 alleles, 31.5%), followed by p.K285N (6 alleles, 15.7%) and p.N314D (6 alleles, 15.7%). The other variations comprised 1 or 2 alleles. In the patients carrying a new mutation, the biochemical analysis of GALT activity in erythrocytes showed an activity of < 1%. In silico analysis (SIFT, PolyPhen-2 and the computational analysis on the static protein structure) showed potentially damaging effects of the six new variations on the GALT protein, thus expanding the genetic spectrum of GALT variations in Italy. The study emphasizes the difficulty in establishing a genotype–phenotype correlation in classical galactosemia and underlines the importance of molecular diagnostic testing prior to making any treatment.

Diagnosis and Management of Hereditary Hemochromatosis

Reena J. Salgia, Kimberly Brown
Clin Liver Dis 19 (2015) 187–198
http://dx.doi.org/10.1016/j.cld.2014.09.011

Hereditary hemochromatosis (HH) is a diagnosis most commonly made in patients with elevated iron indices (transferrin saturation and ferritin), and HFE genetic mutation testing showing C282Y homozygosity.

The HFE mutation is believed to result in clinical iron overload through altering hepcidin levels resulting in increased iron absorption.

The most common clinical complications of HH include cirrhosis, diabetes, nonischemic cardiomyopathy, and hepatocellular carcinoma.

Liver biopsy should be performed in patients with HH if the liver enzymes are elevated or serum ferritin is greater than 1000 mg/L. This is useful to determine the degree of iron overload and stage the fibrosis.

Treatment of HH with clinical iron overload involves a combination of phlebotomy and/or chelation therapy. Liver transplantation should be considered for patients with HH-related decompensated cirrhosis.

Health economic evaluation of plasma oxysterol screening in the diagnosis of Niemann–Pick Type C disease among intellectually disabled using discrete event simulation

CDM van Karnebeek, Tima Mohammadi, Nicole Tsaod, Graham Sinclair, et al.
Molecular Genetics and Metabolism 114 (2015) 226–232
http://dx.doi.org/10.1016/j.ymgme.2014.07.004

Background: Recently a less invasive method of screening and diagnosing Niemann–Pick C (NP-C) disease has emerged. This approach involves the use of a metabolic screening test (oxysterol assay) instead of the current practice of clinical assessment of patients suspected of NP-C (review of medical history, family history and clinical examination for the signs and symptoms). Our objective is to compare costs and outcomes of plasma oxysterol screening versus current practice in diagnosis of NP-C disease among intellectually disabled (ID) patients using decision-analytic methods.
Methods: A discrete event simulation model was conducted to follow ID patients through the diagnosis and treatment of NP-C, forecast the costs and effectiveness for a cohort of ID patients and compare the outcomes and costs in two different arms of the model: plasma oxysterol screening and routine diagnosis procedure (anno 2013) over 5 years of follow up. Data from published sources and clinical trials were used in simulation model. Unit costs and quality-adjusted life-years (QALYs) were discounted at a 3% annual rate in the base case analysis. Deterministic and probabilistic sensitivity analyses were conducted.
Results: The outcomes of the base case model showed that using plasma oxysterol screening for diagnosis of NP-C disease among ID patients is a dominant strategy. It would result in lower total cost and would slightly improve patients’ quality of life. The average amount of cost saving was $3642 CAD and the incremental QALYs per each individual ID patient in oxysterol screening arm versus current practice of diagnosis NP-C was 0.0022 QALYs. Results of sensitivity analysis demonstrated robustness of the outcomes over the wide range of changes in model inputs.
Conclusion: Whilst acknowledging the limitations of this study, we conclude that screening ID children and adolescents with oxysterol tests compared to current practice for the diagnosis of NP-C is a dominant strategy with clinical and economic benefits. The less costly, more sensitive and specific oxysterol test has potential to save costs to the healthcare system while improving patients’ quality of life and may be considered as a routine tool in the NP-C diagnosis armamentarium for ID. Further research is needed to elucidate its effectiveness in patients presenting characteristics other than ID in childhood and adolescence.

Neurological and Behavioral Disorders

Estrogen receptor signaling during vertebrate development

Maria Bondesson, Ruixin Hao, Chin-Yo Lin, Cecilia Williams, Jan-Åke Gustafsson
Biochimica et Biophysica Acta 1849 (2015) 142–151
http://dx.doi.org/10.1016/j.bbagrm.2014.06.005

Estrogen receptors are expressed and their cognate ligands produced in all vertebrates, indicative of important and conserved functions. Through evolution estrogen has been involved in controlling reproduction, affectingboth the development of reproductive organs and reproductive behavior. This review broadly describes the synthesis of estrogens and the expression patterns of aromatase and the estrogen receptors, in relation to estrogen functions in the developing fetus and child. We focus on the role of estrogens for the development of reproductive tissues, as well as non-reproductive effects on the developing brain. We collate data from human, rodent, bird and fish studies and highlight common and species-specific effects of estrogen signaling on fetal development. Morphological malformations originating from perturbed estrogen signaling in estrogen receptor and aromatase knockout mice are discussed, as well as the clinical manifestations of rare estrogen receptor alpha and aromatase gene mutations in humans. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Memory function and hippocampal volumes in preterm born very-low-birth-weight (VLBW) young adults

Synne Aanes, Knut Jørgen Bjuland, Jon Skranes, Gro C.C. Løhaugen
NeuroImage 105 (2015) 76–83
http://dx.doi.org/10.1016/j.neuroimage.2014.10.023

The hippocampi are regarded as core structures for learning and memory functions, which is important for daily functioning and educational achievements. Previous studies have linked reduction in hippocampal volume to working memory problems in very low birth weight (VLBW; ≤1500 g) children and reduced general cognitive ability in VLBW adolescents. However, the relationship between memory function and hippocampal volume has not been described in VLBW subjects reaching adulthood. The aim of the study was to investigate memory function and hippocampal volume in VLBW young adults, both in relation to perinatal risk factors and compared to term born controls, and to look for structure–function relationships. Using Wechsler Memory Scale-III and MRI, we included 42 non-disabled VLBW and 61 control individuals at age 19–20 years, and related our findings to perinatal risk factors in the VLBW-group. The VLBW young adults achieved lower scores on several subtests of the Wechsler Memory Scale-III, resulting in lower results in the immediate memory indices (visual and auditory), the working memory index, and in the visual delayed and general memory delayed indices, but not in the auditory delayed and auditory recognition delayed indices. The VLBW group had smaller absolute and relative hippocampal volumes than the controls. In the VLBW group inferior memory function, especially for the working memory index, was related to smaller hippocampal volume, and both correlated with lower birth weight and more days in the neonatal intensive care unit (NICU). Our results may indicate a structural–functional relationship in the VLBW group due to aberrant hippocampal development and functioning after preterm birth.

The relation of infant attachment to attachment and cognitive and behavioural outcomes in early childhood

Yan-hua Ding, Xiu Xua, Zheng-yan Wang, Hui-rong Li, Wei-ping Wang
Early Human Development 90 (2014) 459–464
http://dx.doi.org/10.1016/j.earlhumdev.2014.06.004

Background: In China, research on the relation of mother–infant attachment to children’s development is scarce.
Aims: This study sought to investigate the relation of mother–infant attachment to attachment, cognitive and behavioral development in young children. Study design: This study used a longitudinal study design.
Subjects: The subjects included healthy infants (n=160) aged 12 to 18 months.
Outcome measures: Ainsworth’s “Strange Situation Procedure” was used to evaluate mother–infant attachment types. The attachment Q-set (AQS) was used to evaluate the attachment between young children and their mothers. The Bayley scale of infant development-second edition (BSID-II) was used to evaluate cognitive developmental level in early childhood. Achenbach’s child behavior checklist (CBCL) for 2- to 3-year-oldswas used to investigate behavioral problems.
Results: In total, 118 young children (73.8%) completed the follow-up; 89.7% of infants with secure attachment and 85.0% of infants with insecure attachment still demonstrated this type of attachment in early childhood (κ = 0.738, p b 0.05). Infants with insecure attachment collectively exhibited a significantly lower mental development index (MDI) in early childhood than did infants with secure attachment, especially the resistant type. In addition, resistant infants were reported to have greater social withdrawal, sleep problems and aggressive behavior in early childhood.
Conclusion: There is a high consistency in attachment development from infancy to early childhood. Secure mother–infant attachment predicts a better cognitive and behavioral outcome; whereas insecure attachment, especially the resistant attachment, may lead to a lower cognitive level and greater behavioral problems in early childhood.

representations of the HPA axis

representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus

Fetal programming of schizophrenia: Select mechanisms

Monojit Debnatha, Ganesan Venkatasubramanian, Michael Berk
Neuroscience and Biobehavioral Reviews 49 (2015) 90–104
http://dx.doi.org/10.1016/j.neubiorev.2014.12.003

Mounting evidence indicates that schizophrenia is associated with adverse intrauterine experiences. An adverse or suboptimal fetal environment can cause irreversible changes in brain that can subsequently exert long-lasting effects through resetting a diverse array of biological systems including endocrine, immune and nervous. It is evident from animal and imaging studies that subtle variations in the intrauterine environment can cause recognizable differences in brain structure and cognitive functions in the offspring. A wide variety of environmental factors may play a role in precipitating the emergent developmental dysregulation and the consequent evolution of psychiatric traits in early adulthood by inducing inflammatory, oxidative and nitrosative stress (IO&NS) pathways, mitochondrial dysfunction, apoptosis, and epigenetic dysregulation. However, the precise mechanisms behind such relationships and the specificity of the risk factors for schizophrenia remain exploratory. Considering the paucity of knowledge on fetal programming of schizophrenia, it is timely to consolidate the recent advances in the field and put forward an integrated overview of the mechanisms associated with fetal origin of schizophrenia.

NMDA receptor dysfunction in autism spectrum disorders

Eun-Jae Lee, Su Yeon Choi and Eunjoon Kim
Current Opinion in Pharmacology 2015, 20:8–13
http://dx.doi.org/10.1016/j.coph.2014.10.007

Autism spectrum disorders (ASDs) represent neurodevelopmental disorders characterized by two core symptoms;

(1) impaired social interaction and communication, and
(2) restricted and repetitive behaviors, interests, and activities.

ASDs affect ~ 1% of the population, and are considered to be highly genetic in nature. A large number (~600) of ASD-related genetic variations have been identified (sfari.org), and target gene functions are apparently quite diverse. However, some fall onto common pathways, including synaptic function and chromosome remodeling, suggesting that core mechanisms may exist.

Abnormalities and imbalances in neuronal excitatory and inhibitory synapses have been implicated in diverse neuropsychiatric disorders including autism spectrum disorders (ASDs). Increasing evidence indicates that dysfunction of NMDA receptors (NMDARs) at excitatory synapses is associated with ASDs. In support of this, human ASD-associated genetic variations are found in genes encoding NMDAR subunits. Pharmacological enhancement or suppression of NMDAR function ameliorates ASD symptoms in humans. Animal models of ASD display bidirectional NMDAR dysfunction, and correcting this deficit rescues ASD-like behaviors. These findings suggest that deviation of NMDAR function in either direction contributes to the development of ASDs, and that correcting NMDAR dysfunction has therapeutic potential for ASDs.

Among known synaptic proteins implicated in ASD are metabotropic glutamate receptors (mGluRs). Functional enhancement and suppression of mGluR5 are associated with fragile X syndrome and tuberous sclerosis, respectively, which share autism as a common phenotype. More recently, ionotropic glutamate receptors, namely NMDA receptors (NMDARs) and AMPA receptors (AMPARs), have also been implicated in ASDs. In this review, we will focus on NMDA receptors and summarize evidence supporting the hypothesis that NMDAR dysfunction contributes to ASDs, and, by extension, that correcting NMDAR dysfunction has therapeutic potential for ASDs. ASD-related human NMDAR genetic variants.

Chemokines roles within the hippocampus

IL-1 mediates stress-induced activation of the HPA axis

A systemic model of the beneficial role of immune processes in behavioral and neural plasticity

Three Classes of Glutamate Receptors

Clinical studies on ASDs have identified genetic variants of NMDAR subunit genes. Specifically, de novo mutations have been identified in the GRIN2B gene, encoding the GluN2B subunit. In addition, SNP analyses have linked both GRIN2A (GluN2A subunit) and GRIN2B with ASDs. Because assembled NMDARs contain four subunits, each with distinct properties, ASD-related GRIN2A/ GRIN2B variants likely alter the functional properties of NMDARs and/or NMDAR-dependent plasticity.

Pharmacological modulation of NMDAR function can improve ASD symptoms. D-cycloserine (DCS), an NMDAR agonist, significantly ameliorates social withdrawal and repetitive behavior in individuals with ASD. These results suggest that reduced NMDAR function may contribute to the development of ASDs in humans.

We can divide animal studies into two groups. The first group consists of animals in which NMDAR modulators were shown to normalize both NMDAR dysfunction and ASD-like behaviors, establishing strong association between NMDARs and ASD phenotypes (Fig.). In the second group, NMDAR modulators were shown to rescue ASD-like behaviors, but NMDAR dysfunction and its correction have not been demonstrated.

ASD models with data showing rescue of both NMDAR dysfunction and ASD like behaviors Mice lacking neuroligin-1, an excitatory postsynaptic adhesion molecule, show reduced NMDAR function in the hippocampus and striatum, as evidenced by a decrease in NMDA/AMPA ratio and long-term potentiation (LTP). Neuroligin-1 is thought to enhance synaptic NMDAR function, by directly interacting with and promoting synaptic localization of NMDARs.

Fig not shown.

Bidirectional NMDAR dysfunction in animal models of ASD. Animal models of ASD with bidirectional NMDAR dysfunction can be positioned on either side of an NMDAR function curve. Model animals were divided into two groups.

Group 1: NMDAR modulators normalize both NMDAR dysfunction and ASD-like behaviors (green).

Group 2: NMDAR modulators rescue ASD-like behaviors, but NMDAR dysfunction and its rescue have not been demonstrated (orange). Note that Group 2 animals are tentatively placed on the left-hand side of the slope based on the observed DCS rescue of their ASD-like phenotypes, but the directions of their NMDAR dysfunctions remain to be experimentally determined.

ASD models with data showing rescue of ASD-like behaviors but no demonstrated NMDAR dysfunction

Tbr1 is a transcriptional regulator, one of whose targets is the gene encoding the GluN2B subunit of NMDARs. Mice haploinsufficient for Tbr1 (Tbr1+/-) show structural abnormalities in the amygdala and limited GluN2B induction upon behavioral stimulation. Both systemic injection and local amygdalar infusion of DCS rescue social deficits and impaired associative memory in Tbr1+/- mice. However, reduced NMDAR function and its DCS-dependent correction have not been demonstrated.

Spatial working memory and attention skills are predicted by maternal stress during pregnancy

André Plamondon, Emis Akbari, Leslie Atkinson, Meir Steiner
Early Human Development 91 (2015) 23–29
http://dx.doi.org/10.1016/j.earlhumdev.2014.11.004

Introduction: Experimental evidence in rodents shows that maternal stress during pregnancy (MSDP) negatively impacts spatial learning and memory in the offspring. We aim to investigate the association between MSDP (i.e., life events) and spatial working memory, as well as attention skills (attention shifting and attention focusing), in humans. The moderating roles of child sex, maternal anxiety during pregnancy and postnatal care are also investigated. Methods: Participants were 236mother–child dyads that were followed from the second trimester of pregnancy until 4 years postpartum. Measurements included questionnaires and independent observations.
Results: MSDP was negatively associated with attention shifting at 18monthswhen concurrent maternal anxiety was low. MSDP was associated with poorer spatial working memory at 4 years of age, but only for boys who experienced poorer postnatal care.
Conclusion: Consistent with results observed in rodents, MSDP was found to be associated with spatial working memory and attention skills. These results point to postnatal care and maternal anxiety during pregnancy as potential targets for interventions that aim to buffer children from the detrimental effects of MSDP.

Acute and massive bleeding from placenta previa and infants’ brain damage

Ken Furuta, Shuichi Tokunaga, Seishi Furukawa, Hiroshi Sameshima
Early Human Development 90 (2014) 455–458
http://dx.doi.org/10.1016/j.earlhumdev.2014.06.002

Background: Among the causes of third trimester bleeding, the impact of placenta previa on cerebral palsy is not well known.
Aims: To clarify the effect ofmaternal bleeding fromplacenta previa on cerebral palsy, and in particular when and how it occurs.
Study design: A descriptive study.
Subjects: Sixty infants born to mothers with placenta previa in our regional population-based study of 160,000 deliveries from 1998 to 2012. Premature deliveries occurring atb26 weeks of gestation and placenta accrete were excluded.
Outcome measures: Prevalence of cystic periventricular leukomalacia (PVL) and cerebral palsy (CP).
Results: Five infants had PVL and 4 of these infants developed CP (1/40,000 deliveries). Acute and massive bleeding (>500 g) within 8 h) occurred at around 30–31 weeks of gestation, and was severe enough to deliver the fetus. None of the 5 infants with PVL underwent antenatal corticosteroid treatment, and 1 infant had mild neonatal hypocapnia with a PaCO2 < 25 mm Hg. However, none of the 5 PVL infants showed umbilical arterial academia with pH < 7.2, an abnormal fetal heart rate monitoring pattern, or neonatal hypotension.
Conclusions: Our descriptive study showed that acute and massive bleeding from placenta previa at around 30 weeks of gestation may be a risk factor for CP, and requires careful neonatal follow-up. The underlying process connecting massive placental bleeding and PVL requires further investigation.

Impact of bilirubin-induced neurologic dysfunction on neurodevelopmental outcomes

Courtney J. Wusthoff, Irene M. Loe
Seminars in Fetal & Neonatal Medicine 20 (2015) 52e57
http://dx.doi.org/10.1016/j.siny.2014.12.003

Extreme neonatal hyperbilirubinemia has long been known to cause the clinical syndrome of kernicterus, or chronic bilirubin encephalopathy (CBE). Kernicterus most usually is characterized by choreoathetoid cerebral palsy (CP), impaired upward gaze, and sensorineural hearing loss, whereas cognition is relatively spared. The chronic condition of kernicterus may be, but is not always, preceded in the acute stage by acute bilirubin encephalopathy (ABE). This acute neonatal condition is also due to hyperbilirubinemia, and is characterized by lethargy and abnormal behavior, evolving to frank neonatal encephalopathy, opisthotonus, and seizures. Less completely defined is the syndrome of bilirubin-induced neurologic dysfunction (BIND).

Bilirubin-induced neurologic dysfunction (BIND) is the constellation of neurologic sequelae following milder degrees of neonatal hyperbilirubinemia than are associated with kernicterus. Clinically, BIND may manifest after the neonatal period as developmental delay, cognitive impairment, disordered executive function, and behavioral and psychiatric disorders. However, there is controversy regarding the relative contribution of neonatal hyperbilirubinemia versus other risk factors to the development of later neurodevelopmental disorders in children with BIND. In this review, we focus on the empiric data from the past 25 years regarding neurodevelopmental outcomes and BIND, including specific effects on developmental delay, cognition, speech and language development, executive function, and the neurobehavioral disorders, such as attention deficit/hyperactivity disorder and autism.

As noted in a technical report by the American Academy of Pediatrics Subcommittee on Hyperbilirubinemia, “it is apparent that the use of a single total serum bilirubin level to predict long-term outcomes is inadequate and will lead to conflicting results”. As described above, this has certainly been the case in research to date. To clarify how hyperbilirubinemia influences neurodevelopmental outcome, more sophisticated consideration is needed both of how to assess bilirubin exposure leading to neurotoxicity, and of those comorbid conditions which may lower the threshold for brain injury.

For example, premature infants are known to be especially susceptible to bilirubin neurotoxicity, with kernicterus reported following TB levels far lower than the threshold expected in term neonates. Similarly, among extremely preterm neonates, BBC is proportional to gestational age, meaning that the most premature infants have the highest UB, even for similar TB levels. Thus, future studies must be adequately powered to examine preterm infants separately from term infants, and should consider not just peak TB, but also BBC, as independent variables in neonates with hyperbilirubinemia. Similarly, an analysis by the NICHD NRN found that, among ELBW infants, higher UB levels were associated with a higher risk of death or NDI. However, increased TB levels were only associated with death or NDI in unstable infants. Again, UB or BBC appeared to be more useful than TB.

Are the neuromotor disabilities of bilirubin-induced neurologic dysfunction disorders related to the cerebellum and its connections?

Jon F. Watchko, Michael J. Painter, Ashok Panigrahy
Seminars in Fetal & Neonatal Medicine 20 (2015) 47e51
http://dx.doi.org/10.1016/j.siny.2014.12.004

Investigators have hypothesized a range of subcortical neuropathology in the genesis of bilirubin induced neurologic dysfunction (BIND). The current review builds on this speculation with a specific focus on the cerebellum and its connections in the development of the subtle neuromotor disabilities of BIND. The focus on the cerebellum derives from the following observations:
(i) the cerebellum is vulnerable to bilirubin-induced injury; perhaps the most vulnerable region within the central nervous system;
(ii) infants with cerebellar injury exhibit a neuromotor phenotype similar to BIND; and (iii) the cerebellum has extensive bidirectional circuitry projections to motor and non-motor regions of the brain-stem and cerebral cortex that impact a variety of neurobehaviors.
Future study using advanced magnetic resonance neuroimaging techniques have the potential to shed new insights into bilirubin’s effect on neural network topology via both structural and functional brain connectivity measurements.

Bilirubin-induced neurologic damage is most often thought of in terms of severe adverse neuromotor (dystonia with or without athetosis) and auditory (hearing impairment or deafness) sequelae. Observed together, they comprise the classic neurodevelopmental phenotype of chronic bilirubin encephalopathy or kernicterus, and may also be seen individually as motor or auditory predominant subtypes. These injuries reflect both a predilection of bilirubin toxicity for neurons (relative to glial cells) and the regional topography of bilirubin-induced neuronal damage characterized by prominent involvement of the globus pallidus, subthalamic nucleus, VIII cranial nerve, and cochlear nucleus.

It is also asserted that bilirubin neurotoxicity may be associated with other less severe neurodevelopmental disabilities, a condition termed “subtle kernicterus” or “bilirubin-induced neurologic dysfunction” (BIND). BIND is defined by a constellation of “subtle neurodevelopmental disabilities without the classical findings of kernicterus that, after careful evaluation and exclusion of other possible etiologies, appear to be due to bilirubin neurotoxicity”. These purportedly include:

(i) mild-to-moderate disorders of movement (e.g., incoordination, clumsiness, gait abnormalities, disturbances in static and dynamic balance, impaired fine motor skills, and ataxia); (ii) disturbances in muscle tone; and
(iii) altered sensorimotor integration. Isolated disturbances of central auditory processing are also included in the spectrum of BIND.

Cerebellar vulnerability to bilirubin-induced injury
Cerebellar injury phenotypes and BIND
Cerebellar projections

Transverse section of cerebellum and brainstem

Transverse section of cerebellum and brain-stem from a 34 gestational-week premature kernicteric infant formalin-fixed for two weeks. Yellow staining is evident in the cerebellar dentate nuclei (upper arrow) and vestibular nuclei at the pontomedullary junction (lower arrowhead). Photo is courtesy of Mahmdouha Ahdab-Barmada and reprinted with permission from Taylor-Francis Group (Ahdab Barmada M. The neuropathology of kernicterus: definitions and debate. In: Maisel MJ, Watchko JF editors. Neonatal jaundice. Amsterdam: Harwood Academic Publishers; 2000. p. 75e88

Whether cerebellar injury is primal or an integral part of disturbed neural circuitry in bilirubin-induced CNS damage is unclear. Movement disorders, however, are increasingly recognized to arise from abnormalities of neuronal circuitry rather than localized, circumscribed lesions. The cerebellum has extensive bidirectional circuitry projections to an array of brainstem nuclei and the cerebral cortex that modulate and refine motor activities. In this regard, the cerebellum is characteristically subdivided into three lobes based on neuroanatomic and phylogenetic criteria as well as by their primary afferent and efferent connections. They include:
(i) flocculonodular lobe (archicerebellum);
(ii) anterior lobe (paleocerebellum); and
(iii) posterior lobe (neocerebellum).

The archicerebellum, the oldest division phylogenically, receives extensive input from the vestibular system and is therefore also known as the vestibulocerebellum and is important for equilibrium control. The paleocerebellum, also a primitive region, receives extensive somatosensory input from the spinal cord, including the anterior and posterior spinocerebellar pathways that convey unconscious proprioception, and is therefore also known as the spinocerebellum. The neocerebellum is the most recently evolved region, receives most of the input from the cerebral cortex, and is thus termed the cerebrocerebellum. This area has greatly expanded in association with the extensive development of the cerebral cortex in mammals and especially primates. To cause serious longstanding dysfunction, cerebellar injury must typically involve the deep cerebellar nuclei and their projections.

Schematic of the bidirectional connectivity between the cerebellum and other

Schematic of the bidirectional connectivity between the cerebellum and other brain regions including the cerebral cortex. Most cerebro-cerebellar afferent projections pass through the basal (anterior or ventral) pontine nuclei and intermediate cerebellar peduncle, whereas most cerebello-cerebral efferent projections pass through the dentate and ventrolateral thalamic nuclei. DCN, deep cerebellar nuclei; RN, red nucleus; ATN, anterior thalamic nucleus; PFC, prefrontal cortex; MC, motor cortex; PC, parietal cortex; TC, temporal cortex; STN, subthalamic nucleus; APN, anterior pontine nuclei. Reprinted under the terms of the Creative Commons Attribution License from D’Angelo E, Casali S. Seeking a unified framework for cerebellar function and dysfunction: from circuit to cognition. Front Neural Circuits 2013; 6:116.

Given the vulnerability of the cerebellum to bilirubin-induced injury, cerebellar involvement should also be evident in classic kernicterus, contributing to neuromotor deficits observed therein. It is of interest, therefore, that cerebellar damage may play a role in the genesis of bilirubin-induced dystonia, a prominent neuromotor feature of chronic bilirubin encephalopathy in preterm and term neonates alike. This complex movement disorder is characterized by involuntary sustained muscle contractions that result in abnormal position and posture. Moreover, dystonia that is brief in duration results in chorea, and, if brief and repetitive, leads to athetosis ‒ conditions also classically observed in kernicterus. Recent evidence suggests that dystonic movements may depend on disruption of both basal ganglia and cerebellar neuronal networks, rather than isolated dysfunction of only one motor system.

Dystonia is also a prominent feature in Gunn rat pups and neonatal Ugt1‒/‒-deficient mice both robust models of kernicterus. The former is used as an experimental model of dystonia. Although these models show basal ganglia injury, the sine qua non of bilirubin-induced murine neuropathology is cerebellar damage and resultant cerebellar hypoplasia.

Studies are needed to define more precisely the motor network abnormalities in kernicterus and BIND. Magnetic resonance imaging (MRI) has been widely used in evaluating infants at risk for bilirubin-induced brain injury using conventional structural T1-and T2-weighted imaging. Infants with chronic bilirubin encephalopathy often demonstrate abnormal bilateral, symmetric, high-signal intensity on T2-weighted MRI of the globus pallidus and subthalamic nucleus, consistent with the neuropathology of kernicterus. Early postnatal MRI of at-risk infants, although frequently showing increased T1-signal in these regions, may give false-positive findings due to the presence of myelin in these structures.

Diffusion tensor imaging and tractography could be used to delineate long-term changes involving specific white matter pathways, further elucidating the neural basis of long-term disability in infants and children with chronic bilirubin encephalopathy and BIND. It will be equally valuable to use blood oxygen level-dependent (BOLD) “resting state” functional MRI to study intrinsic connectivity in order to identify vulnerable brain networks in neonates with kernicterus and BIND. Structural networks of the CNS (connectome) and functional network topology can be characterized in infants with kernicterus and BIND to determine disease-related pattern(s) with respect to both long- and short-range connectivity. These findings have the potential to shed novel insights into the pathogenesis of these disorders and their impact on complex anatomical connections and resultant functional deficits.

Audiologic impairment associated with bilirubin-induced neurologic damage

Cristen Olds, John S. Oghalai
Seminars in Fetal & Neonatal Medicine 20 (2015) 42e46
http://dx.doi.org/10.1016/j.siny.2014.12.006

Hyperbilirubinemia affects up to 84% of term and late preterm infants in the first week of life. The elevation of total serum/plasma bilirubin (TB) levels is generally mild, transitory, and, for most children, inconsequential. However, a subset of infants experiences lifelong neurological sequelae. Although the prevalence of classic kernicterus has fallen steadily in the USA in recent years, the incidence of jaundice in term and premature infants has increased, and kernicterus remains a significant problem in the global arena. Bilirubin-induced neurologic dysfunction (BIND) is a spectrum of neurological injury due to acute or sustained exposure of the central nervous system(CNS) to bilirubin. The BIND spectrum includes kernicterus, acute bilirubin encephalopathy, and isolated neural pathway dysfunction.

Animal studies have shown that unconjugated bilirubin passively diffuses across cell membranes and the blood‒brain barrier (BBB), and bilirubin not removed by organic anion efflux pumps accumulates within the cytoplasm and becomes toxic. Exposure of neurons to bilirubin results in increased oxidative stress and decreased neuronal proliferation and presynaptic neuro-degeneration at central glutaminergic synapses. Furthermore, bilirubin administration results in smaller spiral ganglion cell bodies, with decreased cellular density and selective loss of large cranial nerve VIII myelinated fibers. When exposed to bilirubin, neuronal supporting cells have been found to secrete inflammatory markers, which contribute to increased BBB permeability and bilirubin loading.

The jaundiced Gunn rat is the classic animal model of bilirubin toxicity. It is homozygous for a premature stop codon within the gene for UDP-glucuronosyltransferase family 1 (UGT1). The resultant gene product has reduced bilirubin-conjugating activity, leading to a state of hyperbilirubinemia. Studies with this rat model have led to the concept that impaired calcium homeostasis is an important mechanism of neuronal toxicity, with reduced expression of calcium-binding proteins in affected cells being a sensitive index of bilirubin-induced neurotoxicity. Similarly, application of bilirubin to cultured auditory neurons from brainstem cochlear nuclei results in hyperexcitability and excitotoxicity.

The auditory pathway and normal auditory brainstem response (ABR).

The auditory pathway and normal auditory brain-stem response (ABR). The ipsilateral (green) and contralateral (blue) auditory pathways are shown, with structures that are known to be affected by hyperbilirubinemia highlighted in red. Roman numerals in parentheses indicate corresponding waves in the normal human ABR (inset). Illustration adapted from the “Ear Anatomy” series by Robert Jackler and Christine Gralapp, with permission.

Bilirubin-induced neurologic dysfunction (BIND)

Vinod K. Bhutani, Ronald Wong
Seminars in Fetal & Neonatal Medicine 20 (2015) 1
http://dx.doi.org/10.1016/j.siny.2014.12.010

Beyond the traditional recognized areas of fulminant injury to the globus pallidus as seen in infants with kernicterus, other vulnerable areas include the cerebellum, hippocampus, and subthalamic nuclear bodies as well as certain cranial nerves. The hippocampus is a brain region that is particularly affected by age related morphological changes. It is generally assumed that a loss in hippocampal volume results in functional deficits that contribute to age-related cognitive deficits. Lower grey matter volumes within the limbic-striato-thalamic circuitry are common to other etiological mechanisms of subtle neurologic injury. Lower grey matter volumes in the amygdala, caudate, frontal and medial gyrus are found in schizophrenia and in the putamen in autism. Thus, in terms of brain volumetrics, schizophrenia and autism spectrum disorders have a clear degree of overlap that may reflect shared etiological mechanisms. Overlap with injuries observed in infants with BIND raises the question about how these lesions are arrived at in the context of the impact of common etiologies.

Stress-induced perinatal and transgenerational epigenetic programming of brain development and mental health

Olena Babenko, Igor Kovalchuk, Gerlinde A.S. Metz
Neuroscience and Biobehavioral Reviews 48 (2015) 70–91
http://dx.doi.org/10.1016/j.neubiorev.2014.11.013

Research efforts during the past decades have provided intriguing evidence suggesting that stressful experiences during pregnancy exert long-term consequences on the future mental wellbeing of both the mother and her baby. Recent human epidemiological and animal studies indicate that stressful experiences in utero or during early life may increase the risk of neurological and psychiatric disorders, arguably via altered epigenetic regulation. Epigenetic mechanisms, such as miRNA expression, DNA methylation, and histone modifications are prone to changes in response to stressful experiences and hostile environmental factors. Altered epigenetic regulation may potentially influence fetal endocrine programming and brain development across several generations. Only recently, however, more attention has been paid to possible transgenerational effects of stress. In this review we discuss the evidence of transgenerational epigenetic inheritance of stress exposure in human studies and animal models. We highlight the complex interplay between prenatal stress exposure, associated changes in miRNA expression and DNA methylation in placenta and brain and possible links to greater risks of schizophrenia, attention deficit hyperactivity disorder, autism, anxiety- or depression-related disorders later in life. Based on existing evidence, we propose that prenatal stress, through the generation of epigenetic alterations, becomes one of the most powerful influences on mental health in later life. The consideration of ancestral and prenatal stress effects on lifetime health trajectories is critical for improving strategies that support healthy development and successful aging.

Sensitive time-windows for susceptibility in neurodevelopmental disorders

Rhiannon M. Meredith, Julia Dawitz and Ioannis Kramvis
Trends in Neurosciences, June 2012; 35(6): 335-344
http://dx.doi.org:/10.1016/j.tins.2012.03.005

Many neurodevelopmental disorders (NDDs) are characterized by age-dependent symptom onset and regression, particularly during early postnatal periods of life. The neurobiological mechanisms preceding and underlying these developmental cognitive and behavioral impairments are, however, not clearly understood. Recent evidence using animal models for monogenic NDDs demonstrates the existence of time-regulated windows of neuronal and synaptic impairments. We propose that these developmentally-dependent impairments can be unified into a key concept: namely, time-restricted windows for impaired synaptic phenotypes exist in NDDs, akin to critical periods during normal sensory development in the brain. Existence of sensitive time-windows has significant implications for our understanding of early brain development underlying NDDs and may indicate vulnerable periods when the brain is more susceptible to current therapeutic treatments.

Fig (not shown)

Misregulated mechanisms underlying spine morphology in NDDs. Several proteins implicated in monogenic NDDs (highlighted in red) are linked to the regulation of the synaptic cytoskeleton via F-actin through different Rho-mediated signaling pathways (highlighted in green). Mutations in OPHN1, TSC1/2, FMRP, p21-activated kinase (PAK) are directly linked to human NDDs of intellectual disability. For instance, point mutations in OPHN1 and a PAK isoform are linked to non-syndromic mental retardation, whereas mutations or altered expression of TSC1/2 and FMRP are linked to TSC and FXS, respectively. Cytoplasmic interacting protein (CYFIP) and LIM-domain kinase 1 (LIMK1) are known to interact with FMRP and PAK, respectively [105]. LIMK1 is one of many dysregulated proteins contributing to the NDD Williams syndrome. Mouse models are available for all highlighted (red) proteins and reveal specific synaptic and behavioral deficits. Local protein synthesis in synapses, dendrites and glia is also regulated by proteins such as TSC1/2 and the FMRP/CYFIP complex. Abbreviations: 4EBP, 4E binding protein; eIF4E, eukaryotic translation initiation factor 4E.

Fig (not shown)

Sensitive time-windows, synaptic phenotypes and NDD gene targets. Sensitive time-windows exist in neural circuits, during which gene targets implicated in NDDs are normally expressed. Misregulation of these genes can affect multiple synaptic phenotypes during a restricted developmental period. The effect upon synaptic phenotypes is dependent upon the temporal expression of these NDD genes and their targets. (a) Expression outside a critical period of development will have no effect upon synaptic phenotypes. (b,c) A temporal expression pattern that overlaps with the onset (b) or closure (c) of a known critical period can alter the synaptic phenotype during that developmental time-window.

Outstanding questions

(1) Can treatment at early presymptomatic stages in animal models for NDDs prevent or ease the later synaptic, neuronal, and behavioral impairments?

(2) Are all sensory critical periods equally misregulated in mouse models for a specific NDD? Are there different susceptibilities for auditory, visual and somatosensory neurocircuits that reflect the degree of impairments observed in patients?

(3) If one critical period is missed or delayed during formation of a layer-specific connection in a network, does the network overcome this misregulated connectivity or plasticity window?

(4) In monogenic NDDs, does the severity of misregulating one particular time-window for synaptic establishment during development correlate with the importance of that gene for that synaptic circuit?

(5) Why do critical periods close in brain development?

(6) What underlies the regression of some altered synaptic phenotypes in Fmr1-KO mice?

(7) Can the concept of susceptible time-windows be applied to other NDDs, including schizophrenia and Tourette’s syndrome?

Cardiovascular

Cardiac output monitoring in newborns

Willem-Pieter de Boode
Early Human Development 86 (2010) 143–148
http://dx.doi.org:/10.1016/j.earlhumdev.2010.01.032

There is an increased interest in methods of objective cardiac output measurement in critically ill patients. Several techniques are available for measurement of cardiac output in children, although this remains very complex in newborns. Cardiac output monitoring could provide essential information to guide hemodynamic management. An overview is given of various methods of cardiac output monitoring with advantages and major limitations of each technology together with a short explanation of the basic principles.

Fick principle

According to the Fick principle the volume of blood flow in a given period equals the amount of substance entering the blood stream in the same period divided by the difference in concentrations of the substrate upstream respectively downstream to the point of entry in the circulation. This substance can be oxygen (O2-Fick) or carbon dioxide (CO2-FICK), so cardiac output can be calculated by dividing measured pulmonary oxygen uptake by the arteriovenous oxygen concentration difference. The direct O2-Fick method is regarded as gold standard in cardiac output monitoring in a research setting, despite its limitations. When the Fick principle is applied for carbon dioxide (CO2 Fick), the pulmonary carbon dioxide exchange is divided by the venoarterial CO2 concentration difference to calculate cardiac output.

In the modified CO2 Fick method pulmonary CO2 exchange is measured at the endotracheal tube. Measurement of total CO2 concentration in blood is more complex and simultaneous sampling of arterial and central venous blood is required. However, frequent blood sampling will result in an unacceptable blood loss in the neonatal population.

Blood flow can be calculated if the change in concentration of a known quantity of injected indicator is measured in time distal to the point of injection, so an indicator dilution curve can be obtained. Cardiac output can then be calculated with the use of the Stewart–Hamilton equation. Several indicators are used, such as indocyanine green, Evans blue and brilliant red in dye dilution, cold solutions in thermodilution, lithium in lithium dilution, and isotonic saline in ultrasound dilution.

Cardiovascular adaptation to extra uterine life

Alice Lawford, Robert MR Tulloh
Paediatrics And Child Health 2014; 25(1): 1-6.

The adaptation to extra uterine life is of interest because of its complexity and the ability to cause significant health concerns. In this article we describe the normal changes that occur and the commoner abnormalities that are due to failure of normal development and the effect of congenital cardiac disease. Abnormal development may occur as a result of problems with the mother, or with the fetus before birth. After birth it is essential to determine whether there is an underlying abnormality of the fetal pulmonary or cardiac development and to determine the best course of management of pulmonary hypertension or congenital cardiac disease. Causes of underdevelopment, maldevelopment and maladaptation are described as are the causes of critical congenital heart disease. The methods of diagnosis and management are described to allow the neonatologist to successfully manage such newborns.

Fetal vascular structures that exist to direct blood flow

Fetal structure	Function
Arterial duct	Connects pulmonary artery to the aorta and shunts blood right to left; diverting flow away from fetal lungs
Foramen ovale	Opening between the two atria thatdirects blood flow returning to right atrium through the septal wall into the left atrium bypassing lungs
Ductus venosus	Receives oxygenated blood fromumbilical vein and directs it to the inferior vena cava and right atrium
Umbilical arteries	Carrying deoxygenated blood fromthe fetus to the placenta
Umbilical vein	Carrying oxygenated blood from theplacenta to the fetus

Maternal causes of congenital heart disease

Maternal disorders	rubella, SLE, diabetes mellitus
Maternal drug use	Warfarin, alcohol
Chromosomal abnormality	Down, Edward, Patau, Turner, William, Noonan

Fetal and Neonatal Circulation The fetal circulation is specifically adapted to efficiently exchange gases, nutrients, and wastes through placental circulation. Upon birth, the shunts (foramen ovale, ductus arteriosus, and ductus venosus) close and the placental circulation is disrupted, producing the series circulation of blood through the lungs, left atrium, left ventricle, systemic circulation, right heart, and back to the lungs.

Clinical monitoring of systemic hemodynamics in critically ill newborns

Willem-Pieter de Boode
Early Human Development 86 (2010) 137–141
http://dx.doi.org:/10.1016/j.earlhumdev.2010.01.031

Circulatory failure is a major cause of mortality and morbidity in critically ill newborn infants. Since objective measurement of systemic blood flow remains very challenging, neonatal hemodynamics is usually assessed by the interpretation of various clinical and biochemical parameters. An overview is given about the predictive value of the most used indicators of circulatory failure, which are blood pressure, heart rate, urine output, capillary refill time, serum lactate concentration, central–peripheral temperature difference, pH, standard base excess, central venous oxygen saturation and color.

Key guidelines

➢ The clinical assessment of cardiac output by the interpretation of indirect parameters of systemic blood flow is inaccurate, irrespective of the level of experience of the clinician

➢ Using blood pressure to diagnose low systemic blood flow will consequently mean that too many patients will potentially be undertreated or overtreated, both with substantial risk of adverse effects and iatrogenic damage.

➢ Combining different clinical hemodynamic parameters enhances the predictive value in the detection of circulatory failure, although accuracy is still limited.

➢ Variation in time (trend monitoring) might possibly be more informative than individual, static values of clinical and biochemical parameters to evaluate the adequacy of neonatal circulation.

Monitoring oxygen saturation and heart rate in the early neonatal period

J.A. Dawson, C.J. Morley
Seminars in Fetal & Neonatal Medicine 15 (2010) 203e207
http://dx.doi.org:/10.1016/j.siny.2010.03.004

Pulse oximetry is commonly used to assist clinicians in assessment and management of newly born infants in the delivery room (DR). In many DRs, pulse oximetry is now the standard of care for managing high risk infants, enabling immediate and dynamic assessment of oxygenation and heart rate. However, there is little evidence that using pulse oximetry in the DR improves short and long term outcomes. We review the current literature on using pulse oximetry to measure oxygen saturation and heart rate and how to apply current evidence to management in the DR.

Practice points

Understand how SpO2 changes in the first minutes after birth.
Apply a sensor to an infant’s right wrist as soon as possible after birth.
Attach sensor to infant then to oximeter cable.
Use two second averaging and maximum sensitivity.

Using pulse oximetry assists clinicians:

Assess changes in HR in real time during transition.
Assess oxygenation and titrate the administration of oxygen to maintain oxygenation within the appropriate range for SpO2 during the first minutes after birth.

Research directions

What are the appropriate centiles to target during the minutes after birth to prevent hypoxia and hyperoxia: 25th to 75th, or 10th to 90th, or just the 50th (median)?
Can the inspired oxygen be titrated against the SpO2 to keep the SpO2 in the ‘normal range’?
Does the use of centile charts in the DR for HR and oxygen saturation reduce the rate of hyperoxia when infants are treated with oxygen.
Does the use of pulse oximetry immediately after birth improve short term outcomes, e.g. efficacy of immediate respiratory support, intubation rates in the DR, percentage of inspired oxygen, rate of use of adrenalin or chest compressions, duration of hypoxia/hyperoxia and bradycardia.
Does the use of pulse oximetry in the DR improve short term respiratory and long term neurodevelopmental outcomes for preterm infants, e.g. rate of intubation, use of surfactant, and duration of ventilation, continuous positive airway pressure, or supplemental oxygen?
Can all modern pulse oximeters be used effectively in the DR or do some have a longer delay before giving an accurate signal and more movement artefact?
Would a longer averaging time result in more stable data?

Peripheral haemodynamics in newborns: Best practice guidelines

Michael Weindling, Fauzia Paize
Early Human Development 86 (2010) 159–165
http://dx.doi.org:/10.1016/j.earlhumdev.2010.01.033

Peripheral hemodynamics refers to blood flow, which determines oxygen and nutrient delivery to the tissues. Peripheral blood flow is affected by vascular resistance and blood pressure, which in turn varies with cardiac function. Arterial oxygen content depends on the blood hemoglobin concentration (Hb) and arterial pO2; tissue oxygen delivery depends on the position of the oxygen-dissociation curve, which is determined by temperature and the amount of adult or fetal hemoglobin. Methods available to study tissue perfusion include near-infrared spectroscopy, Doppler flowmetry, orthogonal polarization spectral imaging and the peripheral perfusion index. Cardiac function, blood gases, Hb, and peripheral temperature all affect blood flow and oxygen extraction. Blood pressure appears to be less important. Other factors likely to play a role are the administration of vasoactive medications and ventilation strategies, which affect blood gases and cardiac output by changing the intrathoracic pressure.

graphic

NIRS with partial venous occlusion to measure venous oxygen saturation

NIRS with partial venous occlusion to measure venous oxygen saturation. Taken from Yoxall and Weindling

Schematic representation of the biphasic relationship between oxygen delivery and oxygen consumption in tissue

graphic

Schematic representation of the biphasic relationship between oxygen delivery and oxygen consumption in tissue. (a) oxygen delivery (DO2). (b) As DO2 decreases, VO2 is dependent on DO2. The slope of the line indicates the FOE, which in this case is about 0.50. (c) The slope of the line indicates the FOE in the normal situation where oxygenation is DO2 independent, usually < 0.35

The oxygen-dissociation curve

graphic

The oxygen-dissociation curve

Considerable information about the response of the peripheral circulation has been obtained using NIRS with venous occlusion. Although these measurements were validated against blood co-oximetry in human adults and infants, they can only be made intermittently by a trained operator and are thus not appropriate for general clinical use. Further research is needed to find other better measures of peripheral perfusion and oxygenation which may be easily and continuously monitored, and which could be useful in a clinical setting.

Peripheral oxygenation and management in the perinatal period

Michael Weindling
Seminars in Fetal & Neonatal Medicine 15 (2010) 208e215
http://dx.doi.org:/10.1016/j.siny.2010.03.005

The mechanisms for the adequate provision of oxygen to the peripheral tissues are complex. They involve control of the microcirculation and peripheral blood flow, the position of the oxygen dissociation curve including the proportion of fetal and adult hemoglobin, blood gases and viscosity. Systemic blood pressure appears to have little effect, at least in the non-shocked state. The adequate delivery of oxygen (DO2) depends on consumption (VO2), which is variable. The balance between VO2 and DO2 is given by fractional oxygen extraction (FOE ¼ VO2/DO2). FOE varies from organ to organ and with levels of activity. Measurements of FOE for the whole body produce a range of about 0.15-0.33, i.e. the body consumes 15-33% of oxygen transported.

Fig (not shown)

Biphasic relationship between oxygen delivery (DO2) and oxygen consumption (VO2) in tissue. Dotted lines show fractional oxygen extraction (FOE). ‘A’ indicates the normal situation when VO2 is independent ofDO2 and FOE is about 0.30. AsDO2 decreases in the direction of the arrow, VO2 remains independent of DO2 until the critical point is reached at ‘B’; in this illustration, FOE is about 0.50. The slope of the dotted line indicates the FOE (¼ VO2/DO2), which increases progressively as DO2 decreases.

$Relationship between haemoglobin F fraction (HbF) and peripheral fractional oxygen extraction$

Relationship between haemoglobin F fraction (HbF) and peripheral fractional oxygen extraction

Graphic
(A)Relationship between haemoglobin F fraction (HbF) and peripheral fractional oxygen extraction in anaemic and control infants. (From Wardle et al.) (B) HbF synthesis and concentration. (From Bard and Widness.) (C) Oxygen dissociation curve.

$Peripheral fractional oxygen extraction in babies$

Peripheral fractional oxygen extraction in babies

graphic

Peripheral fractional oxygen extraction in babies with asymptomatic or symptomatic anemia compared to controls. Bars represent the median for each group. (From Wardle et al.)

Practice points

Peripheral tissue DO2 is complex: cardiac function, blood gases, Hb concentration and the proportion of HbF, and peripheral temperature all play a part in determining blood flow and oxygen extraction in the sick, preterm infant. Blood pressure appears to be less important.
Other factors likely to play a role are the administration of vasoactive medications and ventilation strategies, which affect blood gases and cardiac output by changing intrathoracic pressure.
Central blood pressure is a poor surrogate measurement for the adequacy of DO2 to the periphery. Direct measurement, using NIRS, laser Doppler flowmetry or other means, may give more useful information.
Reasons for total hemoglobin concentration (Hb) being a relatively poor indicator of the adequacy of the provision of oxygen to the tissues:

Hb is only indirectly related to red blood cell volume, which may be a better indicator of the body’s oxygen delivering capacity.
Hb-dependent oxygen availability depends on the position of the oxygen-hemoglobin dissociation curve.
An individual’s oxygen requirements vary with time and from organ to organ. This means that DO2 also needs to vary.
It is possible to compensate for a low Hb by increasing cardiac output and ventilation, and so the ability to compensate for anemia depends on an individual’s cardio-respiratory reserve as well as Hb.
The normal decrease of Hb during the first few weeks of life in both full-term and preterm babies usually occurs without symptoms or signs of anemia or clinical consequences.

The relationship between VO2 and DO2 is complex and various factors need to be taken into account, including the position of the oxygen dissociation curve, determined by the proportion of HbA and HbF, temperature and pH. Furthermore, diffusion of oxygen from capillaries to the cell depends on the oxygen tension gradient between erythrocytes and the mitochondria, which depends on microcirculatory conditions, e.g. capillary PO2, distance of the cell from the capillary (characterized by intercapillary distances) and the surface area of open capillaries. The latter can change rapidly, for example, in septic shock where arteriovenous shunting occurs associated with tissue hypoxia in spite of high DO2 and a low FOE.

Changes in local temperature deserve particular consideration. When the blood pressure is low, there may be peripheral vasoconstriction with decreased local perfusion and DO2. However, the fall in local tissue temperature would also be expected to be associated with a decreased metabolic rate and a consequent decrease in VO2. Thus a decreased DO2 may still be appropriate for tissue needs.

Pulmonary

Accurate Measurements of Oxygen Saturation in Neonates: Paired Arterial and Venous Blood Analyses

Shyang-Yun Pamela K. Shiao
Newborn and Infant Nurs Rev, 2005; 5(4): 170–178
http://dx.doi.org:/10.1053/j.nainr.2005.09.001

Oxygen saturation (So2) measurements (functional measurement, So2; and fractional measurement, oxyhemoglobin [Hbo2]) and monitoring are commonly investigated as a method of assessing oxygenation in neonates. Differences exist between the So2 and Hbo2 when blood tests are performed, and clinical monitors indicate So2 values. Oxyhemoglobin will decrease with the increased levels of carbon monoxide hemoglobin (Hbco) and methemo-globin (MetHb), and it is the most accurate measurements of oxygen (O2) association of hemoglobin (Hb). Pulse oximeter (for pulse oximetry saturation [Spo2] measurement) is commonly used in neonates. However, it will not detect the changes of Hb variations in the blood for accurate So2 measurements. Thus, the measurements from clinical oximeters should be used with caution. In neonates, fetal hemoglobin (HbF) accounts for most of the circulating Hb in their blood. Fetal hemoglobin has a high O2 affinity, thus releases less O2 to the body tissues, presenting a left-shifted Hbo2 dissociation curve.5,6 To date, however, limited data are available with HbF correction, for accurate arterial and venous (AV) So2 measurements (arterial oxygen saturation [Sao2] and venous oxygen saturation [Svo2]) in neonates, using paired AV blood samples.

In a study of critically ill adult patients, increased pulmonary CO production and elevation in arterial Hbco but not venous Hbco were documented by inflammatory stimuli inducing pulmonary heme oxygenase–1. In normal adults, venous Hbco level might be slightly higher than or equal to arterial Hbco because of production of CO by enzyme heme oxygenase–2, which is predominantly produced in the liver and spleen. However, hypoxia or pulmonary inflammation could induce heme oxygenase–1 to increase endogenous CO, thus elevating pulmonary arterial and systemic arterial Hbco levels in adults. Both endogenous and exogenous CO can suppress proliferation of pulmonary smooth muscles, a significant consideration for the prevention of chronic lung diseases in newborns. Despite these considerations, a later study in healthy adults indicated that the AV differences in Hbco were from technical artifacts and perhaps from inadequate control of different instruments. Thus, further studies are needed to provide more definitive answers for the AV differences of Hbco for adults and neonates with acute and chronic lung diseases.

Methemoglobin is an indicator of Hb oxidation and is essential for accurate measurement of Hbo2, So2, and oxygenation status. No evidence exists to show the AV MetHb difference, although this difference was elucidated with the potential changes of MetHb with different O2 levels. Methemoglobin can be increased with nitric oxide (NO) therapy, used in respiratory distress syndrome (RDS) to reduce pulmonary hypertension and during heart surgery. Nitric oxide, in vitro, is an oxidant of Hb, with increased O2 during ischemia reperfusion. In hypoxemic conditions in vivo, nitrohemoglobin is a product generated by vessel responsiveness to nitrovasodilators. Nitro-hemoglobin can be spontaneously reversible in vivo, requiring no chemical agents or reductase. However, when O2 levels were increased experimentally in vitro following acidic conditions (pH 6.5) to simulate reperfusion conditions, MetHb levels were increased for the hemolysates (broken red cells). Nitrite-induced oxidation of Hb was associated with an increase in red blood cell membrane rigidity, thus contributing to Hb breakdown. A newer in vitro study of whole blood cells, however, concluded that MetHb formation is not dependent on increased O2 levels. Additional studies are needed to examine in vivo reperfusion of O2 and MetHb effects.

Purpose: The aim of this study was to examine the accuracy of arterial oxygen saturation (Sao2) and venous oxygen saturation (Svo2) with paired arterial and venous (AV) blood in relation to pulse oximetry saturation (Spo2) and oxyhemoglobin (Hbo2) with fetal hemoglobin determination, and their Hbo2 dissociation curves. Method: Twelve preterm neonates with gestational ages ranging from 27 to 34 weeks at birth, who had umbilical AV lines inserted, were investigated. Analyses were performed with 37 pairs of AV blood samples by using a blood volume safety protocol. Results: The mean differences between Sao2 and Svo2, and AV Hbo2 were both 6 percent (F6.9 and F6.7 percent, respectively), with higher Svo2 than those reported for adults. Biases were 2.1 – 0.49 for Sao2, 2.0 – 0.44 for Svo2, and 3.1 – 0.45 for Spo2, compared against Hbo2. With left-shifted Hbo2 dissociation curves in neonates, for the critical values of oxygen tension values between 50 and 75 millimeters of mercury, Hbo2 ranged from 92 to 93.4 percent; Sao2 ranged from 94.5 to 95.7 percent; and Spo2 ranged from 93.7 to 96.3 percent (compared to 85–94 percent in healthy adults). Conclusions: In neonates, both left-shifted Hbo2 dissociation curve and lower AV differences of oxygen saturation measurements indicated low flow of oxygen to the body tissues. These findings demonstrate the importance of accurate assessment of oxygenation statues in neonates.

In these neonates, the mean AV blood differences for both So2 and Hbo2 were about 6 percent, which was much lower than those reported for healthy adults (23 percent) for O2 supply and demand. In addition, with very high levels of HbF releasing less O2 to the body tissue, the results of blood analyses are worrisome for these critically ill neonates for low systemic oxygen states. O’Connor and Hall determined AV So2 in neonates without HbF determination. Much of the AV So2 difference is dependent on Svo2 measurement. The ranges of Svo2 spanned for 35 percent, and the ranges of Sao2 spanned 6 percent in these neonates. The greater intervals for Svo2 measurements contribute to greater sensitivity for the measurements (than Sao2 measurements) in responding to nursing care and changes of O2 demand. Thus, Svo2 measurement is essential for better assessment of oxygenation status in neonates.

The findings of this study on AV differences of So2 were limited with very small number of paired AV blood samples. However, critically ill neonates need accurate assessment of oxygenation status because of HbF, which releases less O2 to the tissues. Decreased differences of AV So2 measurements added further possibilities of lower flow of O2 to the body tissues and demonstrated the greater need to accurately assess the proper oxygenation in the neonates. The findings of this study continued to clarify the accuracy of So2 measurements for neonates. Additional studies are needed to examine So2 levels in neonates to further validate these findings by using larger sample sizes.

Neonatal ventilation strategies and long-term respiratory outcomes

Sandeep Shetty, Anne Greenough
Early Human Development 90 (2014) 735–739
http://dx.doi.org/10.1016/j.earlhumdev.2014.08.020

Long-term respiratory morbidity is common, particularly in those born very prematurely and who have developed bronchopulmonary dysplasia (BPD), but it does occur in those without BPD and in infants born at term. A variety of neonatal strategies have been developed, all with short-term advantages, but meta-analyses of randomized controlled trials (RCTs) have demonstrated that only volume-targeted ventilation and prophylactic high-frequency oscillatory ventilation (HFOV) may reduce BPD. Few RCTs have incorporated long-term follow-up, but one has demonstrated that prophylactic HFOV improves respiratory and functional outcomes at school age, despite not reducing BPD. Results from other neonatal interventions have demonstrated that any impact on BPD may not translate into changes in long-term outcomes. All future neonatal ventilation RCTs should have long-term outcomes rather than BPD as their primary outcome if they are to impact on clinical practice.

A Model Analysis of Arterial Oxygen Desaturation during Apnea in Preterm Infants

Scott A. Sands, BA Edwards, VJ Kelly, MR Davidson, MH Wilkinson, PJ Berger
PLoS Comput Biol 5(12): e1000588
http://dx.doi.org:/10.1371/journal.pcbi.1000588

Rapid arterial O2 desaturation during apnea in the preterm infant has obvious clinical implications but to date no adequate explanation for why it exists. Understanding the factors influencing the rate of arterial O2 desaturation during apnea (_SSaO2 ) is complicated by the non-linear O2 dissociation curve, falling pulmonary O2 uptake, and by the fact that O2 desaturation is biphasic, exhibiting a rapid phase (stage 1) followed by a slower phase when severe desaturation develops (stage 2). Using a mathematical model incorporating pulmonary uptake dynamics, we found that elevated metabolic O2 consumption accelerates _SSaO2 throughout the entire desaturation process. By contrast, the remaining factors have a restricted temporal influence: low pre-apneic alveolar PO2 causes an early onset of desaturation, but thereafter has little impact; reduced lung volume, hemoglobin content or cardiac output, accelerates _SSaO2 during stage 1, and finally, total blood O2 capacity (blood volume and hemoglobin content) alone determines _SSaO2 during stage 2. Preterm infants with elevated metabolic rate, respiratory depression, low lung volume, impaired cardiac reserve, anemia, or hypovolemia, are at risk for rapid and profound apneic hypoxemia. Our insights provide a basic physiological framework that may guide clinical interpretation and design of interventions for preventing sudden apneic hypoxemia.

A novel approach to study oxidative stress in neonatal respiratory distress syndrome

Reena Negi, D Pande, K Karki, A Kumar, RS Khanna, HD Khanna
BBA Clinical 3 (2015) 65–69
http://dx.doi.org/10.1016/j.bbacli.2014.12.001

Oxidative stress is an imbalance between the systemic manifestation of reactive oxygen species and a biological system’s ability to readily detoxify the reactive intermediates or to repair the resulting damage. It is a physiological event in the fetal-to-neonatal transition, which is actually a great stress to the fetus. These physiological changes and processes greatly increase the production of free radicals, which must be controlled by the antioxidant defense system, the maturation of which follows the course of the gestation. This could lead to several functional alterations with important repercussions for the infants. Adequately mature and healthy infants are able to tolerate this drastic change in the oxygen concentration. A problem occurs when the intrauterine development is incomplete or abnormal. Preterm or intrauterine growth retarded (IUGR) and low birth weight neonates are typically of this kind. An oxidant/antioxidant imbalance in infants is implicated in the pathogenesis of the major complications of prematurity including respiratory distress syndrome (RDS), necrotizing enterocolitis (NEC), chronic lung disease, retinopathy of prematurity and intraventricular hemorrhage (IVH).

Background: Respiratory distress syndrome of the neonate (neonatal RDS) is still an important problem in treatment of preterm infants. It is accompanied by inflammatory processes with free radical generation and oxidative stress. The aim of study was to determine the role of oxidative stress in the development of neonatal RDS. Methods: Markers of oxidative stress and antioxidant activity in umbilical cord blood were studied in infants with neonatal respiratory distress syndrome with reference to healthy newborns. Results: Status of markers of oxidative stress (malondialdehyde, protein carbonyl and 8-hydroxy-2-deoxy guanosine) showed a significant increase with depleted levels of total antioxidant capacity in neonatal RDS when compared to healthy newborns. Conclusion: The study provides convincing evidence of oxidative damage and diminished antioxidant defenses in newborns with RDS. Neonatal RDS is characterized by damage of lipid, protein and DNA, which indicates the augmentation of oxidative stress. General significance: The identification of the potential biomarker of oxidative stress consists of a promising strategy to study the pathophysiology of neonatal RDS.

Neonatal respiratory distress syndrome represents the major lung complications of newborn babies. Preterm neonates suffer from respiratory distress syndrome (RDS) due to immature lungs and require assisted ventilation with high concentrations of oxygen. The pathogenesis of this disorder is based on the rapid formation of the oxygen reactive species, which surpasses the detoxification capacity of antioxidative defense system. The high chemical reactivity of free radical leads to damage to a variety of cellular macro molecules including proteins, lipids and nucleic acid. This results in cell injury and may induce respiratory cell death.

Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acids peroxidation. The present study showed increased concentration of MDA in neonates with respiratory disorders than that of control in consonance with the reported study.

Anemia, Apnea of Prematurity, and Blood Transfusions

Kelley Zagol, Douglas E. Lake, Brooke Vergales, Marion E. Moorman, et al
J Pediatr 2012;161:417-21
http://dx.doi.org:/10.1016/j.jpeds.2012.02.044

The etiology of apnea of prematurity is multifactorial; however, decreased oxygen carrying capacity may play a role. The respiratory neuronal network in neonates is immature, particularly in those born preterm, as demonstrated by their paradoxical response to hypoxemia. Although adults increase the minute ventilation in response to hypoxemia, newborns have a brief increase in ventilation followed by periodic breathing, respiratory depression, and occasionally cessation of respiratory effort. This phenomenon may be exacerbated by anemia in preterm newborns, where a decreased oxygen carrying capacity may result in decreased oxygen delivery to the central nervous system, a decreased efferent output of the respiratory neuronal network, and an increase in apnea.

Objective Compare the frequency and severity of apneic events in very low birth weight (VLBW) infants before and after blood transfusions using continuous electronic waveform analysis. Study design We continuously collected waveform, heart rate, and oxygen saturation data from patients in all 45 neonatal intensive care unit beds at the University of Virginia for 120 weeks. Central apneas were detected using continuous computer processing of chest impedance, electrocardiographic, and oximetry signals. Apnea was defined as respiratory pauses of >10, >20, and >30 seconds when accompanied by bradycardia (<100 beats per minute) and hypoxemia (<80% oxyhemoglobin saturation as detected by pulse oximetry). Times of packed red blood cell transfusions were determined from bedside charts. Two cohorts were analyzed. In the transfusion cohort, waveforms were analyzed for 3 days before and after the transfusion for all VLBW infants who received a blood transfusion while also breathing spontaneously. Mean apnea rates for the previous 12 hours were quantified and differences for 12 hours before and after transfusion were compared. In the hematocrit cohort, 1453 hematocrit values from all VLBW infants admitted and breathing spontaneously during the time period were retrieved, and the association of hematocrit and apnea in the next 12 hours was tested using logistic regression. Results Sixty-seven infants had 110 blood transfusions during times when complete monitoring data were available. Transfusion was associated with fewer computer-detected apneic events (P < .01). Probability of future apnea occurring within 12 hours increased with decreasing hematocrit values (P < .001). Conclusions Blood transfusions are associated with decreased apnea in VLBW infants, and apneas are less frequent at higher hematocrits.

Bronchopulmonary dysplasia: The earliest and perhaps the longest lasting obstructive lung disease in humans

Silvia Carraro, M Filippone, L Da Dalt, V Ferraro, M Maretti, S Bressan, et al.
Early Human Development 89 (2013) S3–S5
http://dx.doi.org/10.1016/j.earlhumdev.2013.07.015

Bronchopulmonary dysplasia (BPD) is one of the most important sequelae of premature birth and the most common form of chronic lung disease of infancy, an umbrella term for a number of different diseases that evolve as a consequence of a neonatal respiratory disorder. BPD is defined as the need for supplemental oxygen for at least 28 days after birth, and its severity is graded according to the respiratory support required at 36 post-menstrual weeks.

BPD was initially described as a chronic respiratory disease occurring in premature infants exposed to mechanical ventilation and oxygen supplementation. This respiratory disease (later named “old BPD”) occurred in relatively large premature newborn and, from a pathological standpoint, it was characterized by intense airway inflammation, disruption of normal pulmonary structures and lung fibrosis.

Bronchopulmonary dysplasia (BPD) is one of the most important sequelae of premature birth and the most common form of chronic lung disease of infancy. From a clinical standpoint BPD subjects are characterized by recurrent respiratory symptoms, which are very frequent during the first years of life and, although becoming less severe as children grow up, they remain more common than in term-born controls throughout childhood, adolescence and into adulthood. From a functional point of view BPD subjects show a significant airflow limitation that persists during adolescence and adulthood and they may experience an earlier and steeper decline in lung function during adulthood. Interestingly, patients born prematurely but not developing BPD usually fare better, but they too have airflow limitations during childhood and later on, suggesting that also prematurity per se has life-long detrimental effects on pulmonary function. For the time being, little is known about the presence and nature of pathological mechanisms underlying the clinical and functional picture presented by BPD survivors. Nonetheless, recent data suggest the presence of persistent neutrophilic airway inflammation and oxidative stress and it has been suggested that BPD may be sustained in the long term by inflammatory pathogenic mechanisms similar to those underlying COPD. This hypothesis is intriguing but more pathological data are needed. A better understanding of these pathogenetic mechanisms, in fact, may be able to orient the development of novel targeted therapies or prevention strategies to improve the overall respiratory health of BPD patients.

We have a limited understanding of the presence and nature of pathological mechanisms in the lung of BPD survivors. The possible role of asthma-like inflammation has been investigated because BPD subjects often present with recurrent wheezing and other symptoms resembling asthma during their childhood and adolescence. But BPD subjects have normal or lower than normal exhaled nitric oxide levels and exhaled air temperatures, whereas they are higher than normal in asthmatic patients.

Of all obstructive lung diseases in humans, BPD has the earliest onset and is possibly the longest lasting. Given its frequent association with other conditions related to preterm birth (e.g. growth retardation, pulmonary hypertension, neurodevelopmental delay, hearing defects, and retinopathy of prematurity), it often warrants a multidisciplinary management.

Effects of Sustained Lung Inflation, a lung recruitment maneuver in primary acute respiratory distress syndrome, in respiratory and cerebral outcomes in preterm infants

Chiara Grasso, Pietro Sciacca, Valentina Giacchi, Caterina Carpinato, et al.
Early Human Development 91 (2015) 71–75
http://dx.doi.org/10.1016/j.earlhumdev.2014.12.002

Background: Sustained Lung Inflation (SLI) is a maneuver of lung recruitment in preterm newborns at birth that can facilitate the achieving of larger inflation volumes, leading to the clearance of lung fluid and formation of functional residual capacity (FRC). Aim: To investigate if Sustained Lung Inflation (SLI) reduces the need of invasive procedures and iatrogenic risks. Study design: 78 newborns (gestational age ≤ 34 weeks, weighing ≤ 2000 g) who didn’t breathe adequately at birth and needed to receive SLI in addition to other resuscitation maneuvers (2010 guidelines). Subjects: 78 preterm infants born one after the other in our department of Neonatology of Catania University from 2010 to 2012. Outcome measures: The need of intubation and surfactant, the ventilation required, radiological signs, the incidence of intraventricular hemorrhage (IVH), periventricular leukomalacia, retinopathy in prematurity from III to IV plus grades, bronchopulmonary dysplasia, patent ductus arteriosus, pneumothorax and necrotizing enterocolitis. Results: In the SLI group infants needed less intubation in the delivery room (6% vs 21%; p b 0.01), less invasive mechanical ventilation (14% vs 55%; p ≤ 0.001) and shorter duration of ventilation (9.1 days vs 13.8 days; p ≤ 0.001). There wasn’t any difference for nasal continuous positive airway pressure (82% vs 77%; p = 0.43); but there was less surfactant administration (54% vs 85%; p ≤ 0.001) and more infants received INSURE (40% vs 29%; p=0.17). We didn’t found any differences in the outcomes, except for more mild intraventricular hemorrhage in the SLI group (23% vs 14%; p = 0.15; OR= 1.83). Conclusion: SLI is easier to perform even with a single operator, it reduces the necessity of more complicated maneuvers and surfactant without statistically evident adverse effects.

Long-term respiratory consequences of premature birth at less than 32 weeks of gestation

Anne Greenough
Early Human Development 89 (2013) S25–S27
http://dx.doi.org/10.1016/j.earlhumdev.2013.07.004

Chronic respiratory morbidity is a common adverse outcome of very premature birth, particularly in infants who had developed bronchopulmonary dysplasia (BPD). Prematurely born infants who had BPD may require supplementary oxygen at home for many months and affected infants have increased healthcare utilization until school age. Chest radiograph abnormalities are common; computed tomography of the chest gives predictive information in children with ongoing respiratory problems. Readmission to hospital is common, particularly for those who have BPD and suffer respiratory syncytial virus lower respiratory infections (RSV LRTIs). Recurrent respiratory symptoms requiring treatment are common and are associated with evidence of airways obstruction and gas trapping. Pulmonary function improves with increasing age, but children with BPD may have ongoing airflow limitation. Lung function abnormalities may be more severe in those who had RSV LRTIs, although this may partly be explained by worse premorbid lung function. Worryingly, lung function may deteriorate during the first year. Longitudinal studies are required to determine if there is catch up growth.

Long-term pulmonary outcomes of patients with bronchopulmonary dysplasia

Anita Bhandari and Sharon McGrath-Morrow
Seminars in Perinatology 37 (2013)132–137
http://dx.doi.org/10.1053/j.semperi.2013.01.010

Bronchopulmonary dysplasia (BPD) is the commonest cause of chronic lung disease in infancy. The incidence of BPD has remained unchanged despite many advances in neonatal care. BPD starts in the neonatal period but its effects can persist long term. Premature infants with BPD have a greater incidence of hospitalization, and continue to have a greater respiratory morbidity and need for respiratory medications, compared to those without BPD. Lung function abnormalities, especially small airway abnormalities, often persist. Even in the absence of clinical symptoms, BPD survivors have persistent radiological abnormalities and presence of emphysema has been reported on chest computed tomography scans. Concern regarding their exercise tolerance remains. Long-term effects of BPD are still unknown, but given reports of a more rapid decline in lung function and their susceptibility to develop chronic obstructive pulmonary disease phenotype with aging, it is imperative that lung function of survivors of BPD be closely monitored.

Neonatal ventilation strategies and long-term respiratory outcomes

Sandeep Shetty, Anne Greenough
Early Human Development 90 (2014) 735–739
http://dx.doi.org/10.1016/j.earlhumdev.2014.08.020

Long-term respiratory morbidity is common, particularly in those born very prematurely and who have developed bronchopulmonary dysplasia (BPD), but it does occur in those without BPD and in infants born at term. A variety of neonatal strategies have been developed, all with short-term advantages, but meta-analyses of randomized controlled trials (RCTs) have demonstrated that only volume-targeted ventilation and prophylactic high-frequency oscillatory ventilation (HFOV) may reduce BPD. Few RCTs have incorporated long-term follow-up, but one has demonstrated that prophylactic HFOV improves respiratory and functional outcomes at school age, despite not reducing BPD. Results from other neonatal interventions have demonstrated that any impact on BPD may not translate into changes in long-term outcomes. All future neonatal ventilation RCTs should have long-term outcomes rather than BPD as their primary outcome if they are to impact on clinical practice.

Prediction of neonatal respiratory distress syndrome in term pregnancies by assessment of fetal lung volume and pulmonary artery resistance index

Mohamed Laban, GM Mansour, MSE Elsafty, AS Hassanin, SS EzzElarab
International Journal of Gynecology and Obstetrics 128 (2015) 246–250
http://dx.doi.org/10.1016/j.ijgo.2014.09.018

Objective: To develop reference cutoff values for mean fetal lung volume (FLV) and pulmonary artery resistance index (PA-RI) for prediction of neonatal respiratory distress syndrome (RDS) in low-risk term pregnancies. Methods: As part of a cross-sectional study, women aged 20–35 years were enrolled and admitted to a tertiary hospital in Cairo, Egypt, for elective repeat cesarean at 37–40 weeks of pregnancy between January 1, 2012, and July 31, 2013. FLV was calculated by virtual organ computer-aided analysis, and PA-RI was measured by Doppler ultrasonography before delivery. Results: A total of 80 women were enrolled. Neonatal RDS developed in 11 (13.8%) of the 80 newborns. Compared with neonates with RDS, healthy neonates had significantly higher FLVs (P b 0.001) and lower PA-RIs (P b 0.001). Neonatal RDS is less likely with FLV of at least 32 cm³ or PA-RI less than or equal to 0.74. Combining these two measures improved the accuracy of prediction. Conclusion: The use of either FLV or PA-RI predicted neonatal RDS. The predictive value increased when these two measures were combined

Pulmonary surfactant – a front line of lung host defense, 2003 JCI0318650.f2

Pulmonary hypertension in bronchopulmonary dysplasia

Sara K.Berkelhamer, Karen K.Mestan, and Robin H. Steinhorn
Seminars In Perinatology 37 (2013)124–131
http://dx.doi.org/10.1053/j.semperi.2013.01.009

Pulmonary hypertension (PH) is a common complication of neonatal respiratory diseases, including bronchopulmonary dysplasia (BPD), and recent studies have increased aware- ness that PH worsens the clinical course, morbidity and mortality of BPD. Recent evidence indicates that up to 18% of all extremely low-birth-weight infants will develop some degree of PH during their hospitalization, and the incidence rises to 25–40% of the infants with established BPD. Risk factors are not yet well understood, but new evidence shows that fetal growth restriction is a significant predictor of PH. Echocardiography remains the primary method for evaluation of BPD-associated PH, and the development of standardized screening timelines and techniques for identification of infants with BPD-associated PH remains an important ongoing topic of investigation. The use of pulmonary vasodilator medications, such as nitric oxide, sildenafil, and others, in the BPD population is steadily growing, but additional studies are needed regarding their long-term safety and efficacy.
An update on pharmacologic approaches to bronchopulmonary dysplasia

Sailaja Ghanta, Kristen Tropea Leeman, and Helen Christou
Seminars In Perinatology 37 (2013)115–123
http://dx.doi.org/10.1053/j.semperi.2013.01.008

Bronchopulmonary dysplasia (BPD) is the most prevalent long-term morbidity in surviving extremely preterm infants and is linked to increased risk of reactive airways disease, pulmonary hypertension, post-neonatal mortality, and adverse neurodevelopmental outcomes. BPD affects approximately 20% of premature newborns, and up to 60% of premature infants born before completing 26 weeks of gestation. It is characterized by the need for assisted ventilation and/or supplemental oxygen at 36 weeks postmenstrual age. Approaches to prevention and treatment of BPD have evolved with improved understanding of its pathogenesis. This review will focus on recent advancements and detail current research in pharmacotherapy for BPD. The evidence for both current and potential future experimental therapies will be reviewed in detail. As our understanding of the complex and multifactorial pathophysiology of BPD changes, research into these current and future approaches must continue to evolve.

Methylxanthines

Diuretics and bronchodilators

Corticosteroids

Macrolide antibiotics

Recombinant human Clara cell 10-kilodalton protein(rhCC10)

Vitamin A

Surfactant

Leukotriene receptor antagonist

Pulmonary vasodilators

Skeletal and Muscle

Skeletal Stem Cells in Space and Time

Moustapha Kassem and Paolo Bianco
Cell Jan 15, 2015; 160: 17-19
http://dx.doi.org/10.1016/j.cell.2014.12.034

The nature, biological characteristics, and contribution to organ physiology of skeletal stem cells are not completely determined. Chan et al. and Worthley et al. demonstrate that a stem cell for skeletal tissues, and a system of more restricted, downstream progenitors, can be identified in mice and demonstrate its role in skeletal tissue maintenance and regeneration.

The groundbreaking concept that bone, cartilage, marrow adipocytes, and hematopoiesis-supporting stroma could originate from a common progenitor and putative stem cell was surprising at the time when it was formulated (Owen and Friedenstein, 1988). The putative stem cell, nonhematopoietic in nature, would be found in the postnatal bone marrow stroma, generate tissues previously thought of as foreign to each other, and support the turnover of tissues and organs that self-renew at a much slower rate compared to other tissues associated with stem cells (blood, epithelia). This concept also connected bone and bone marrow as parts of a single-organ system, implying their functional interplay. For many years, the evidence underpinning the concept has been incomplete.

While multipotency of stromal progenitors has been demonstrated by in vivo transplantation experiments, self-renewal, the defining property of a stem cell, has not been easily demonstrated until recently in humans (Sacchetti et al., 2007) and mice (Mendez-Ferrer et al., 2010). Meanwhile, a confusing and plethoric terminology has been introduced into the literature, which diverted and confounded the search for a skeletal stem cell and its physiological significance (Bianco et al., 2013).

Two studies in this issue of Cell (Chan et al., 2015; Worthley et al., 2015), using a combination of rigorous single-cell analyses and lineage tracing technologies, mark significant steps toward rectifying the course of skeletal stem cell discovery by making several important points, within and beyond skeletal physiology.

First, a stem cell for skeletal tissues, and a system of more restricted, downstream progenitors can in fact be identified and linked to defined phenotype(s) in the mouse. The system is framed conceptually, and approached experimentally, similar to the hematopoietic system.

Second, based on its assayable functions and potential, the stem cell at the top of the hierarchy is defined as a skeletal stem cell (SSC). As noted earlier (Sacchetti et al., 2007) (Bianco et al., 2013), this term clarifies, well beyond semantics, that the range of tissues that the self-renewing stromal progenitor (originally referred to as an ‘‘osteogenic’’ or ‘‘stromal’’ stem cell) (Owen and Friedenstein, 1988) can actually generate in vivo, overlaps with the range of tissues that make up the skeleton.

Third, these cells are spatially restricted, local residents of the bone/bone marrow organ. The systemic circulation is not a sizable contributor to their recruitment to locally deployed functions.

Fourth, a native skeletogenic potential is inherent to the system of progenitor/ stem cells found in the skeleton, and internally regulated by bone morphogenetic protein (BMP) signaling. This is reflected in the expression of regulators and antagonists of BMP signaling within the system, highlighting potential feedback mechanisms modulating expansion or quiescence of specific cell compartments.

Fifth, in cells isolated from other tissues, an assayable skeletogenic potential is not inherent: it can only be induced de novo by BMP reprogramming. These two studies (Chan et al., 2015, Worthley et al., 2015) corroborate the classical concept of ‘‘determined’’ and ‘‘inducible’’ skeletal progenitors (Owen and Friedenstein, 1988): the former residing in the skeleton, the latter found in nonskeletal tissues; the former capable of generating skeletal tissues, in vivo and spontaneously, the latter requiring reprogramming signals in order to acquire a skeletogenic capacity; the former operating in physiological bone formation, the latter in unwanted, ectopic bone formation in diseases such as fibrodysplasia ossificans progressiva.

To optimize our ability to obtain specific skeletal tissues for medical application, the study by Chan et al. offers a glimpse of another facet of the biology of SSC lineages and progenitors. Chan et al. show that a homogeneous cell population inherently committed to chondrogenesis can alter its output to generate bone if cotransplanted with multipotent progenitors. Conversely, osteogenic cells can be shifted to a chondrogenic fate by blockade of vascular endothelial growth factor receptor, consistent with the avascular and hypoxic milieu of cartilage. This has two important implications:

commitment is flexible in the system;
the choir is as important as the soloist and can modulate the solo tune.

Reversibility and population behavior thus emerge as two features that may be characteristic, albeit not unique, of the stromal system, resonating with conceptually comparable evidence in the human system.

The two studies by Chan et al. and Worthely et al. emphasize the relevance not only of their new data, but also of a proper concept of a skeletal stem cell per se, for proper clinical use. Confusion arising from improper conceptualization of skeletal stem cells has markedly limited clinical development of skeletal stem cell biology.

Gremlin 1 Identifies a Skeletal Stem Cell with Bone, Cartilage, and Reticular Stromal Potential

Daniel L. Worthley, Michael Churchill, Jocelyn T. Compton, Yagnesh Tailor, et al.
Cell, Jan 15, 2015; 160: 269–284
http://dx.doi.org/10.1016/j.cell.2014.11.042

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

Identification and Specification of the Mouse Skeletal Stem Cell

Charles K.F. Chan, Eun Young Seo, James Y. Chen, David Lo, A McArdle, et al.
Cell, Jan 15, 2015; 160: 285–298
http://dx.doi.org/10.1016/j.cell.2014.12.002

How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

Bone mesenchymal development

The bone-remodeling cycle

Nuclear receptor modulation – Role of coregulators in selective estrogen receptor modulator (SERM) actions

Qin Feng, Bert W. O’Malley
Steroids 90 (2014) 39–43
http://dx.doi.org/10.1016/j.steroids.2014.06.008

Selective estrogen receptor modulators (SERMs) are a class of small-molecule chemical compounds that bind to estrogen receptor (ER) ligand binding domain (LBD) with high affinity and selectively modulate ER transcriptional activity in a cell- and tissue-dependent manner. The prototype of SERMs is tamoxifen, which has agonist activity in bone, but has antagonist activity in breast. Tamoxifen can reduce the risk of breast cancer and, at same time, prevent osteoporosis in postmenopausal women. Tamoxifen is widely prescribed for treatment and prevention of breast cancer. Mechanistically the activity of SERMs is determined by the selective recruitment of coactivators and corepressors in different cell types and tissues. Therefore, understanding the coregulator function is the key to understanding the tissue selective activity of SERMs.

Hematopoietic

Hematopoietic Stem Cell Arrival Triggers Dynamic Remodeling of the Perivascular Niche

Owen J. Tamplin, Ellen M. Durand, Logan A. Carr, Sarah J. Childs, et al.
Cell, Jan 15, 2015; 160: 241–252
http://dx.doi.org/10.1016/j.cell.2014.12.032

Hematopoietic stem and progenitor cells (HSPCs) can reconstitute and sustain the entire blood system. We generated a highly specific transgenic reporter of HSPCs in zebrafish. This allowed us to perform high resolution live imaging on endogenous HSPCs not currently possible in mammalian bone marrow. Using this system, we have uncovered distinct interactions between single HSPCs and their niche. When an HSPC arrives in the perivascular niche, a group of endothelial cells remodel to form a surrounding pocket. This structure appears conserved in mouse fetal liver. Correlative light and electron microscopy revealed that endothelial cells surround a single HSPC attached to a single mesenchymal stromal cell. Live imaging showed that mesenchymal stromal cells anchor HSPCs and orient their divisions. A chemical genetic screen found that the compound lycorine promotes HSPC-niche interactions during development and ultimately expands the stem cell pool into adulthood. Our studies provide evidence for dynamic niche interactions upon stem cell colonization.

Neonatal anemia

Sanjay Aher, Kedar Malwatkar, Sandeep Kadam
Seminars in Fetal & Neonatal Medicine (2008) 13, 239e247
http://dx.doi.org:/10.1016/j.siny.2008.02.009

Neonatal anemia and the need for red blood cell (RBC) transfusions are very common in neonatal intensive care units. Neonatal anemia can be due to blood loss, decreased RBC production, or increased destruction of erythrocytes. Physiologic anemia of the newborn and anemia of prematurity are the two most common causes of anemia in neonates. Phlebotomy losses result in much of the anemia seen in extremely low birthweight infants (ELBW). Accepting a lower threshold level for transfusion in ELBW infants can prevent these infants being exposed to multiple donors.

Management of anemia in the newborn

Naomi L.C. Luban
Early Human Development (2008) 84, 493–498
http://dx.doi.org:/10.1016/j.earlhumdev.2008.06.007

Red blood cell (RBC) transfusions are administered to neonates and premature infants using poorly defined indications that may result in unintentional adverse consequences. Blood products are often manipulated to limit potential adverse events, and meet the unique needs of neonates with specific diagnoses. Selection of RBCs for small volume (5–20 mL/kg) transfusions and for massive transfusion, defined as extracorporeal bypass and exchange transfusions, are of particular concern to neonatologists. Mechanisms and therapeutic treatments to avoid transfusion are another area of significant investigation. RBCs collected in anticoagulant additive solutions and administered in small aliquots to neonates over the shelf life of the product can decrease donor exposure and has supplanted the use of fresh RBCs where each transfusion resulted in a donor exposure. The safety of this practice has been documented and procedures established to aid transfusion services in ensuring that these products are available. Less well established are the indications for transfusion in this population; hemoglobin or hematocrit alone are insufficient indications unless clinical criteria (e.g. oxygen desaturation, apnea and bradycardia, poor weight gain) also augment the justification to transfuse. Comorbidities increase oxygen consumption demands in these infants and include bronchopulmonary dysplasia, rapid growth and cardiac dysfunction. Noninvasive methods or assays have been developed to measure tissue oxygenation; however, a true measure of peripheral oxygen offloading is needed to improve transfusion practice and determine the value of recombinant products that stimulate erythropoiesis. The development of such noninvasive methods is especially important since randomized, controlled clinical trials to support specific practices are often lacking, due at least in part, to the difficulty of performing such studies in tiny infants.
The Effect of Blood Transfusion on the Hemoglobin Oxygen Dissociation Curve of Very Early Preterm Infants During the First Week of Life

Virginie De HaUeux, Anita Truttmann, Carmen Gagnon, and Harry Bard
Seminars in Perinatology, 2002; 26(6): 411-415
http://dx.doi.org:/10.1053/sper.2002.37313

This study was conducted during the first week of life to determine the changes in Ps0 (PO2 required to achieve a saturation of 50% at pH 7.4 and 37~ and the proportions of fetal hemoglobin (I-IbF) and adult hemoglobin (HbA) prior to and after transfusion in very early preterm infants. Eleven infants with a gestational age <–27 weeks have been included in study. The hemoglobin dissociation curve and the Ps0 was determined by Hemox-analyser. Liquid chromatography was also performed to determine the proportions of HbF and HbA. The mean gestational age of the 11 infants was 25.1 weeks (-+1 weeks) and their mean birth weight was 736 g (-+125 g). They received 26.9 mL/kg of packed red cells. The mean Ps0 prior and after transfusion was 18.5 +- 0.8 and 21.0 + 1 mm Hg (P = .0003) while the mean percentage of HbF was 92.9 -+ 1.1 and 42.6 -+ 5.7%, respectively. The data of this study show a decrease of hemoglobin oxygen affinity as a result of blood transfusion in very early preterm infants prone to O 2 toxicity. The shift in HbO 2 curve after transfusion should be taken into consideration when oxygen therapy is being regulated for these infants.

Effect of neonatal hemoglobin concentration on long-term outcome of infants affected by fetomaternal hemorrhage

Mizuho Kadooka, H Katob, A Kato, S Ibara, H Minakami, Yuko Maruyama
Early Human Development 90 (2014) 431–434
http://dx.doi.org/10.1016/j.earlhumdev.2014.05.010

Background: Fetomaternal hemorrhage (FMH) can cause severe morbidity. However, perinatal risk factors for long-term poor outcome due to FMH have not been extensively studied. Aims: To determine which FMH infants are likely to have neurological sequelae.
Study design: A single-center retrospective observational study. Perinatal factors, including demographic characteristics, Kleihauer–Betke test, blood gas analysis, and neonatal blood hemoglobin concentration ([Hb]), were analyzed in association with long-term outcomes.
Subjects: All 18 neonates referred to a Neonatal Intensive Care Unit of Kagoshima City Hospital and diagnosed with FMH during a 15-year study period. All had a neonatal [Hb] b7.5 g/dL and 15 of 17 neonates tested had Kleihauer–Betke test result N4.0%.
Outcome measures: Poor long-term outcome was defined as any of the following determined at 12 month old or more: cerebral palsy, mental retardation, attention deficit/hyperactivity disorder, and epilepsy.
Results: Nine of the 18 neonates exhibited poor outcomes. Among demographic characteristics and blood variables compared between two groups with poor and favorable outcomes, significant differences were observed in [Hb] (3.6 ± 1.4 vs. 5.4 ± 1.1 g/dL, P = 0.01), pH (7.09 ± 0.11 vs. 7.25 ± 0.13, P = 0.02) and base deficits (17.5 ± 5.4 vs. 10.4 ± 6.0 mmol/L, P = 0.02) in neonatal blood, and a number of infants with [Hb] ≤ 4.5 g/dL (78%[7/9] vs. 22%[2/9], P= 0.03), respectively. The base deficit in neonatal arterial blood increased significantly with decreasing neonatal [Hb].
Conclusions: Severe anemia causing severe base deficit is associated with neurological sequelae in FMH infants

Clinical and hematological presentation among Indian patients with common hemoglobin variants

Khushnooma Italia, Dipti Upadhye, Pooja Dabke, Harshada Kangane, et al.
Clinica Chimica Acta 431 (2014) 46–51
http://dx.doi.org/10.1016/j.cca.2014.01.028

Background: Co-inheritance of structural hemoglobin variants like HbS, HbD Punjab and HbE can lead to a variable clinical presentation and only few cases have been described so far in the Indian population.
Methods: We present the varied clinical and hematological presentation of 22 cases (HbSD Punjab disease-15, HbSE disease-4, HbD Punjab E disease-3) referred to us for diagnosis.
Results: Two of the 15 HbSDPunjab disease patients had moderate crisis, one presented with mild hemolytic anemia; however, the other 12 patients had a severe clinical presentation with frequent blood transfusion requirements, vaso occlusive crisis, avascular necrosis of the femur and febrile illness. The 4 HbSE disease patients had a mild to moderate presentation. Two of the 3 HbD Punjab E patients were asymptomatic with one patient’s sibling having a mild presentation. The hemoglobin levels of the HbSD Punjab disease patients ranged from 2.3 to 8.5 g/dl and MCV from 76.3 to 111.6 fl. The hemoglobin levels of the HbD Punjab E and HbSE patients ranged from 10.8 to 11.9 and 9.8 to 10.0 g/dl whereas MCV ranged from 67.1 to 78.2 and 74.5 to 76.0 fl respectively.
Conclusions: HbSD Punjab disease patients should be identified during newborn screening programs and managed in a way similar to sickle cell disease. Couple at risk of having HbSD Punjab disease children may be given the option of prenatal diagnosis in subsequent pregnancies.

Sickle cell anemia is the most common hemoglobinopathy seen across the world. It is caused by a point mutation in the 6th codon of the beta (β) globin gene leading to the substitution of the amino acid glutamic acid to valine. The sickle gene is frequently seen in Africa, some Mediterranean countries, India, Middle East—Saudi Arabia and North America. In India the prevalence of hemoglobin S (HbS) carriers varies from 2 to 40% among different population groups and HbS is mainly seen among the scheduled tribe, scheduled caste and other backward class populations in the western, central and parts of eastern and southern India. Sickle cell anemia has a variable clinical presentation in India with the most severe clinical presentation seen in central India whereas patients in the western region show a mild to moderate clinical presentation.

Hemoglobin D Punjab (HbD Punjab) (also known as HbD Los-Angeles, HbD Portugal, HbD North Carolina, D Oak Ridge and D Chicago) is another hemoglobin variant due to a point mutation in codon 121 of the β globin gene resulting in the substitution of the amino acid glutamic acid to glycine. It is a widely distributed hemoglobin with a relatively low prevalence of 0.86% in the Indo-Pak subcontinent, 1–3% in north-western India, 1–3% in the Black population in the Caribbean and North America and has also been reported among the English. It accounts for 55.6% of all the Hb variants seen in the Xenjiang province of China.

Hemoglobin E (HbE) is the most common abnormal hemoglobin in Southeast Asia. In India, the frequency ranges from 4% to 51% in the north eastern region and 3% to 4% in West Bengal in the east. The HbE mutation (β26 GAG→AAG) creates an alternative splice site and the βE chain is insufficiently synthesized, hence the phenotype of this disorder is that of a mild form of β thalassemia.

Though these 3 structural variants are prevalent in different regions of India, their interaction is increasingly seen in all states of the country due to migration of people to different regions for a better livelihood. There are very few reports on interaction of these commonly seen Hb variants and the phenotypic–genotypic presentation of these cases is important for genetic counseling and management.

HbF of patients with HbSD Punjab disease with variable clinical severity. The HbF values of 4 patients are not included as they were post blood transfusion

The genotypes of the patients were confirmed by restriction enzyme digestion and ARMS (Fig). Patients 1 to 15 were characterized as compound heterozygous for HbS and HbD Punjab whereas patients 16 to 19 were characterized as compound heterozygous for HbS and HbE. Patient nos. 20 to 22 were characterized as compound heterozygous for HbE and HbD Punjab.

Molecular characterization of HbS and HbDPunjab by restriction enzyme digestion and of HbE by ARMS.

The 3 common β globin gene variants of hemoglobin, HbS, HbE and HbD Punjab are commonly seen in India, with HbS having a high prevalence in the central belt and some parts of western, eastern and southern India, HbE in the eastern and north eastern region whereas HbD is mostly seen in the north western part of India. These hemoglobin variants have been reported in different population groups. However, with migration and intermixing of the different populations from different geographic regions, occasional cases of HbSD Punjab and HbSE are being reported. There are several HbD variants like HbD Punjab, HbD Iran, HbD Ibadan. However, of these only HbD Punjab interacts with HbS to form a clinically significant condition as the glutamine residue facilitates polymerization of HbS. HbD Iran and HbD Ibadan are non-interacting and produce benign conditions like the sickle cell trait. The first case of HbSD Punjab disease was a brother and sister considered to have atypical sickle cell disease in 1934. This family was further reinvestigated and reported as the first case of HbD Los Angeles which has the same mutation as the HbD Punjab. Serjeant et al. reported HbD Punjab in an English parent in 6 out of 11 HbSD-Punjab disease cases. This has been suggested to be due to the stationing of nearly 50,000 British troops on the Indian continent for a period of 200 y and the introduction into Britain of their Anglo-Indian children.

HbSD Punjab disease shows a similar pattern to HbS homozygous on alkaline hemoglobin electrophoresis but can be differentiated on acid agar gel electrophoresis and on HPLC. In HbSD Punjab disease cases, the peripheral blood films show anisocytosis, poikilocytosis, target cells and irreversibly sickled cells. Values of HbF and HbA2 are similar to those in sickle homozygous cases. HbSD Punjab disease is characterized by a moderately severe hemolytic anemia.

Twenty-one cases of HbSDPunjab were reported by Serjeant of which 16 were reported by different workers among patients originating from Caucasian, Spanish, Australian, Irish, English, Portuguese, Black, American, Venezuelan, Caribbean, Mexican, Turkish and Jamaican backgrounds. Yavarian et al. 2009 reported a multi centric origin of HbD Punjab which in combination with HbS results in sickle cell disease. Patel et al. 2010 have also reported 12 cases of HbSD Punjab from the Orissa state of eastern India. Majority of these cases were symptomatic, presenting with chronic hemolytic anemia and frequent painful crises.

HbF levels >20% were seen in 4 out of our 11 clinically severe patients of HbSD-Punjab disease with the mean HbF levels of 16.8% in 8 clinically severe patients, while 3 clinically severe patients were post transfused. However, the 3 patients with a mild to moderate clinical presentation showed a mean HbF level of 8.6%. This is in contrast to the relatively milder clinical presentation associated with high HbF seen in patients with sickle cell anemia. This was also reported by Adekile et al. 2010 in 5 cases of HbS-DLos Angeles where high HbF did not ameliorate the severe clinical presentation seen in these patients.

These 15 cases of HbSD^Punjab disease give us an overall idea of the severe clinical presentation of the disease in different regions of India. However the HbD^PunjabE cases were milder or asymptomatic and the HbSE cases were moderately symptomatic. Since most of the cases of HbSD^Punjab disease were clinically severe, it is important to pick up these cases during newborn screening and enroll them into a comprehensive care program with the other sickle cell disease patients with introduction of therapeutic interventions such as penicillin prophylaxis if required and pneumococcal immunization. In fact, 2 of our cases (No. 6 and 7) were identified during newborn screening for sickle cell disorders. The parents can be given information on home care and educated to detect symptoms that may lead to serious medical emergencies. The parents of these patients as well as the couples who are at risk of having a child with HbSD^Punjab disease could also be counseled about the option of prenatal diagnosis in subsequent pregnancies. It is thus important to document the clinical and hematological presentation of compound heterozygotes with these common β globin chain variants.

Common Hematologic Problems in the Newborn Nursery

Jon F. Watchko
Pediatr Clin N Am – (2015) xxx-xxx
http://dx.doi.org/10.1016/j.pcl.2014.11.011

Common RBC disorders include hemolytic disease of the newborn, anemia, and polycythemia. Another clinically relevant hematologic issue in neonates to be covered herein is thrombocytopenia. Disorders of white blood cells will not be reviewed.

KEY POINTS

(1) Early clinical jaundice or rapidly developing hyperbilirubinemia are often signs of hemolysis, the differential diagnosis of which commonly includes immune-mediated disorders, red-cell enzyme deficiencies, and red-cell membrane defects.

(2) Knowledge of the maternal blood type and antibody screen is critical in identifying non-ABO alloantibodies in the maternal serum that may pose a risk for severe hemolytic disease in the newborn.

(3) Moderate to severe thrombocytopenia in an otherwise well-appearing newborn strongly suggests immune-mediated (alloimmune or autoimmune) thrombocytopenia.

Hemolytic conditions in the neonate

1. Immune-mediated (positive direct Coombs test) a. Rhesus blood group: Anti-D, -c, -C, -e, -E, CW, and several others

b. Non-Rhesus blood groups: Kell, Duffy, Kidd, Xg, Lewis, MNS, and others

c. ABO blood group: Anti-A, -B

2. Red blood cell (RBC) enzyme defects

a. Glucose-6-phosphate dehydrogenase (G6PD) deficiency

b. Pyruvate kinase deficiency

c. Others

3. RBC membrane defects

a. Hereditary spherocytosis

b. Elliptocytosis

c. Stomatocytosis

d. Pyknocytosis

e. Others

4. Hemoglobinopathies

a. alpha-thalassemia

b. gamma-thalassemia

Standard maternal antibody screeningAlloantibody Blood Group

D, C, c, E, e, f, CW, V Rhesus

K, k, Kpa, Jsa Kell

Fya, Fyb Duffy

Jka, Jkb Kidd

Xga Xg

Lea, Leb Lewis

S, s, M, N MNS

P1 P

Lub Lutheran

Non-ABO alloantibodies reported to cause moderate to severe hemolytic disease of the newbornWithin Rh system: Anti-D, -c, -C, -Cw, -Cx, -e, -E, -Ew, -ce, -Ces, -Rh29, -Rh32, -Rh42, -f, -G, -Goa, -Bea, -Evans, -Rh17, -Hro, -Hr, -Tar, -Sec, -JAL, -STEM

Outside Rh system: Anti-LW, -K, -k, -Kpa, -Kpb, -Jka, -Jsa, -Jsb, -Ku, -K11, -K22, -Fya, -M, -N, -S, -s, -U, -PP1 pk, -Dib, -Far, -MUT, -En3, -Hut, -Hil, -Vel, -MAM, -JONES, -HJK, -REIT

Red Blood Cell Enzymopathies

G6PD9 and pyruvate kinase (PK) deficiency are the 2 most common red-cell enzyme disorders associated with marked neonatal hyperbilirubinemia. Of these, G6PD deficiency is the more frequently encountered and it remains an important cause of kernicterus worldwide, including the United States, Canada, and the United Kingdom, the prevalence in Western countries a reflection in part of immigration patterns and intermarriage. The risk of kernicterus in G6PD deficiency also relates to the potential for unexpected rapidly developing extreme hyperbilirubinemia in this disorder associated with acute severe hemolysis.

Red Blood Cell Membrane Defects

Establishing a diagnosis of RBC membrane defects is classically based on the development of Coombs-negative hyperbilirubinemia, a positive family history, and abnormal RBC smear, albeit it is often difficult because newborns normally exhibit a marked variation in red-cell membrane size and shape. Spherocytes, however, are not often seen on RBC smears of hematologically normal newborns and this morphologic abnormality, when prominent, may yield a diagnosis of hereditary spherocytosis (HS) in the immediate neonatal period. Given that approximately 75% of families affected with hereditary spherocytosis manifest an autosomal dominant phenotype, a positive family history can often be elicited and provide further support for this diagnosis. More recently, Christensen and Henry highlighted the use of an elevated mean corpuscular hemoglobin concentration (MCHC) (>36.0 g/dL) and/or elevated ratio of MCHC to mean corpuscular volume, the latter they term the “neonatal HS index” (>0.36, likely >0.40) as screening tools for HS. An index of greater than 0.36 had 97% sensitivity, greater than 99% specificity, and greater than 99% negative predictive value for identifying HS in neonates. Christensen and colleagues also provided a concise update of morphologic RBC features that may be helpful in diagnosing this and other underlying hemolytic conditions in newborns.

The diagnosis of HS can be confirmed using the incubated osmotic fragility test when coupled with fetal red-cell controls or eosin-5-maleimide flow cytometry. One must rule out symptomatic ABO hemolytic disease by performing a direct Coombs test, as infants so affected also may manifest prominent micro-spherocytosis. Moreover, HS and symptomatic ABO hemolytic disease can occur in the same infant and result in severe hyperbilirubinemia and anemia. Of other red-cell membrane defects, only hereditary elliptocytosis, stomato-cytosis, and infantile pyknocytosis have been reported to exhibit significant hemolysis in the newborn period. Hereditary elliptocytosis and stomatocytosis are both rare. Infantile pyknocytosis, a transient red-cell membrane abnormality manifesting itself during the first few months of life, is more common.

Risk factors for bilirubin neurotoxicityIsoimmune hemolytic disease

G6PD deficiency

Asphyxia

Sepsis

Acidosis

Albumin less than 3.0 g/dL
Data from Maisels MJ, Bhutani VK, Bogen D, et al. Hyperbilirubinemia in the newborn infant > or 535 weeks’ gestation: an update with clarifications. Pediatrics 2009; 124:1193–8.

Polycythemia

Polycythemia (venous hematocrit 65%) in seen in infants across a range of conditions associated with active erythropoiesis or passive transfusion.76,77 They include, among others, placental insufficiency, the infant of a diabetic mother, recipient in twin-twin transfusion syndrome, and several aneuploidies, including trisomy. The clinical concern related to polycythemia is the risk for microcirculatory complications of hyperviscosity. However, determining which polycythemic infants are hyperviscous and when to intervene is a challenge.

Liver

Metabolic disorders presenting as liver disease

Germaine Pierre, Efstathia Chronopoulou
Paediatrics and Child Health 2013; 23(12): 509-514
The liver is a highly metabolically active organ and many inherited metabolic disorders have hepatic manifestations. The clinical presentation in these patients cannot usually be distinguished from liver disease due to acquired causes like infection, drugs or hematological disorders. Manifestations include acute and chronic liver failure, cholestasis and hepatomegaly. Metabolic causes of acute liver failure in childhood can be as high as 35%. Certain disorders like citrin deficiency and Niemann-Pick C disease may present in infancy with self-limiting cholestasis before presenting in later childhood or adulthood with irreversible disease. This article reviews important details from the history and clinical examination when evaluating the pediatric patient with suspected metabolic disease, the specialist and genetic tests when investigating, and also discusses specific disorders, their clinical course and treatment. The role of liver transplantation is also briefly discussed. Increased awareness of this group of disorders is important as in many cases, early diagnosis leads to early intervention with improved outcome. Diagnosis also allows genetic counselling and future family planning.

Adult liver disorders caused by inborn errors of metabolism: Review and update

Sirisak Chanprasert, Fernando Scaglia
Molecular Genetics and Metabolism 114 (2015) 1–10
http://dx.doi.org/10.1016/j.ymgme.2014.10.011

Inborn errors of metabolism (IEMs) are a group of genetic diseases that have protean clinical manifestations and can involve several organ systems. The age of onset is highly variable but IEMs afflict mostly the pediatric population. However, in the past decades, the advancement in management and new therapeutic approaches have led to the improvement in IEM patient care. As a result, many patients with IEMs are surviving into adulthood and developing their own set of complications. In addition, some IEMs will present in adulthood. It is important for internists to have the knowledge and be familiar with these conditions because it is predicted that more and more adult patients with IEMs will need continuity of care in the near future. The review will focus on Wilson disease, alpha-1 antitrypsin deficiency, citrin deficiency, and HFE-associated hemochromatosis which are typically found in the adult population. Clinical manifestations and pathophysiology, particularly those that relate to hepatic disease as well as diagnosis and management will be discussed in detail.

Inborn errors of metabolism (IEMs) are a group of genetic diseases characterized by abnormal processing of biochemical reactions, resulting in accumulation of toxic substances that could interfere with normal organ functions, and failure to synthesize essential compounds. IEMs are individually rare, but collectively numerous. The clinical presentations cover a broad spectrum and can involve almost any organ system. The age of onset is highly variable but IEMs afflict mostly the pediatric population.

Wilson disease is an autosomal recessive genetic disorder of copper metabolism. It is characterized by an abnormal accumulation of inorganic copper in various tissues, most notably in the liver and the brain, especially in the basal ganglia. The disease was first described in 1912 by Kinnier Wilson, and affects between 1 in 30,000 and 1 in 100,000 individuals. Clinical features are variable and depend on the extent and the severity of copper deposition. Typically, patients tend to develop hepatic disease at a younger age than the neuropsychiatric manifestations. Individuals withWilson disease eventually succumb to complications of end stage liver disease or become debilitated from neurological problems, if they are left untreated.

The clinical presentations of Wilson disease are varied affecting many organ systems. However, the overwhelming majority of cases display hepatic and neurologic symptoms. In general, patients with hepatic disease present between the first and second decades of life although patients as young as 3 years old or over 50 years old have also been reported. The most common modes of presentations are acute self-limited hepatitis and chronic active hepatitis that are indistinguishable from other hepatic disorders although liver aminotransferases are generally much lower than in autoimmune or viral hepatitis. Acute fulminant hepatic failure is less common but is observed in approximately 3% of all cases of acute liver failure. Symptoms of acute liver failure include jaundice, coagulopathy, and hepatic encephalopathy. Cirrhosis can develop over time and may be clinically silent. Hepatocellular carcinoma (HCC) is rarely associated with Wilson disease, but may occur in the setting of cirrhosis and chronic inflammation.

Copper is an essential element, and is required for the proper functioning of various proteins and enzymes. The total body content of copper in a healthy adult individual is approximately 70–100 mg, while the daily requirements are estimated to be between 1 and 5 mg. Absorption occurs in the small intestine. Copper is taken up to the hepatocytes via the copper transporter hTR1. Once inside the cell, copper is bound to various proteins including metallothionein and glutathione, however, it is the metal chaperone, ATOX1 that helps direct copper to the ATP7B protein for intracellular transport and excretion. At the steady state, copper will be bound to ATP7B and is then incorporated to ceruloplasmin and secreted into the systemic circulation. When the cellular copper concentration arises, ATP7B protein will be redistributed from the trans-Golgi network to the prelysosomal vesicles facilitating copper excretion into the bile. The molecular defects in ATP7B lead to a reduction of copper excretion. Excess copper is accumulated in the liver causing tissue injury. The rate of accumulation of copper varies among individuals, and it may depend on other factors such as alcohol consumption, or viral hepatitis infections. If the liver damage is not severe, patients will accumulate copper in various tissues including the brain, the kidney, the eyes, and the musculoskeletal system leading to clinical disease. A failure of copper to incorporate into ceruloplasmin leads to secretion of the unsteady protein that has a shorter half-life, resulting in the reduced concentrations of ceruloplasmin seen in most patients with Wilson disease.

Wilson disease used to be a progressive fatal condition during the first half of the 20th century because there was no effective treatment available at that time. Penicillamine was the first pharmacologic agent introduced in 1956 for treating this condition. Penicillamine is a sulfhydryl-bearing amino acid cysteine doubly substituted with methyl groups. This drug acts as a chelating agent that promotes the urinary excretion of copper. It is rapidly absorbed in the gastrointestinal track, and over 80% of circulating penicillamine is excreted via the kidneys. Although it is very effective, approximately 10%–50% of Wilson disease patients with neuropsychiatric presentations may experience worsening of their symptoms, and often times the worsening symptoms may not be reversible.

Alpha1-antitrypsin deficiency

Alpha1-antitrypsin deficiency (AATD) is one of the most common genetic liver diseases in children and adults, affecting 1 in 2000 to 1 in 3000 live births worldwide. It is transmitted in an autosomal co-dominant fashion with variable expressivity. Alpha1 antitrypsin (A1AT) is a member of the serine protease inhibitor (SERPIN) family. Its function is to counteract the proteolytic effect of neutrophil elastase and other neutrophil proteases. Mutations in the SERPINA1, the gene encoding A1AT, result in changes in the protein structure with the PiZZ phenotype being the most common cause of liver and lung disease-associated AATDs. Although, it classically causes early onset chronic obstructive pulmonary disease (COPD) in adults, liver disease characterized by chronic inflammation, hepatic fibrosis, and cirrhosis is not uncommon in the adult population. Decreased plasma concentration of A1AT predisposes lung tissue to be more susceptible to injury from protease enzymes. However, the underlying mechanism of liver injury is different, and is believed to be caused by accumulation of polymerized mutant A1AT in the hepatocyte endoplasmic reticulum (ER). Currently, there is no specific treatment for liver disease-associated AATD, but A1AT augmentation therapy is available for patients affected with pulmonary involvement.

A1AT is a single-chain, 52-kDa polypeptide of approximately 394 amino acids [56]. It is synthesized in the liver, circulates in the plasma, and functions as an inhibitor of neutrophil elastase and other proteases such as cathepsin G, and proteinase 3. A1AT has a globular shape composed of two central β sheets surrounded by a small β sheet and nine α helices. The pathophysiology underlying liver disease is thought to be a toxic gain-of-function mutation associated with the PiZZ phenotypes. This hypothesis has been supported by the fact that null alleles which produce no detectable plasma A1AT, are not associated with liver disease. In addition, the transgenic mouse model of AATD PiZZ developed periodic acid-Schiff-positive diastase-resistant intrahepatic globule early in life similar to AATD patients. The PiZZ phenotype results in the blockade of the final processing of A1AT in the liver, as only 15% of the A1AT reaches the circulation whereas 85% of non-secreted protein is accumulated in the hepatocytes.

Citrin deficiency

Citrin deficiency is a relatively newly-defined autosomal recessive disease. It encompasses two different sub-groups of patients, neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and adult onset citrullinemia type 2 (CTLN 2).

AGC2 exports aspartate out of the mitochondrial matrix in exchange for glutamate and a proton. Thus, this protein has an important role in ureagenesis and gluconeogenesis. In CTLN2, a defect in this protein is believed to limit the supply of aspartate for the formation of argininosuccinate in the cytosol resulting in impairment of ureagenesis. Interestingly, the mouse model of citrin deficiency (Ctrn−/−) fails to develop symptoms of CTLN2 suggesting that the mitochondrial aspartate is not the only source of ureagenesis. However, it should be noted that the rodent liver expresses higher glycerol-phosphate shuttle activity than the human counterpart. With the intact glycerol-phosphate dehydrogenase, it can compensate for the deficiency of AGC2, as demonstrated by the AGC2 and glycerol-phosphate dehydrogenase double knock-out mice that exhibit similar features to those observed in human CTLN2.

HFE-associated hemochromatosis

HFE-associated hemochromatosis is an inborn error of iron metabolism characterized by excessive iron storage resulting in tissue and organ damage. It is the most common autosomal recessive disorder in the Caucasian population, affecting 0.3%–0.5% of individuals of Northern European descent. The term “hemochromatosis” was coined in 1889 by the German pathologist Friedrich Daniel Von Recklinghausen, who described it as bronze stain of organs caused by a blood borne pigment.

The classic clinical triad of cirrhosis, diabetes, and bronze skin pigmentation is rarely observed nowadays given the early recognition, diagnosis, and treatment of this condition. The most common presenting symptoms are nonspecific including weakness, lethargy, and arthralgia.

The liver is a major site of iron storage in healthy individuals and as such it is the organ that is universally affected in HFE-associated hemochromatosis. Elevation of liver aminotransferases indicative of hepatocyte injury is the most common mode of presentation and it can be indistinguishable from other causes of hepatitis. Approximately 15%–40% of patients with HFE-associated hemochromatosis have other liver conditions, including chronic viral hepatitis B or C infection, nonalcoholic fatty liver disease, and alcoholic liver disease.

The liver in haemochromatosis

Rune J. Ulvik
Journal of Trace Elements in Medicine and Biology xxx (2014) xxx–xxx
http://dx.doi.org/10.1016/j.jtemb.2014.08.005

The review deals with genetic, regulatory and clinical aspects of iron homeostasis and hereditary hemochromatosis. Hemochromatosis was first described in the second half of the 19th century as a clinical entity characterized by excessive iron overload in the liver. Later, increased absorption of iron from the diet was identified as the pathophysiological hallmark. In the 1970s genetic evidence emerged supporting the apparent inheritable feature of the disease. And finally in 1996 a new “hemochromato-sis gene” called HFE was described which was mutated in about 85% of the patients. From the year2000 onward remarkable progress was made in revealing the complex molecular regulation of iron trafficking in the human body and its disturbance in hemochromatosis. The discovery of hepcidin and ferroportin and their interaction in regulating the release of iron from enterocytes and macrophages to plasma were important milestones. The discovery of new, rare variants of non-HFE-hemochromatosis was explained by mutations in the multicomponent signal transduction pathway controlling hepcidin transcription. Inhibited transcription induced by the altered function of mutated gene products, results in low plasma levels of hepcidin which facilitate entry of iron from enterocytes into plasma. In time this leads to progressive accumulation of iron and subsequently development of disease in the liver and other parenchymatous organs. Being the major site of excess iron storage and hepcidin synthesis the liver is a cornerstone in maintaining normal systemic iron homeostasis. Its central pathophysiological role in HFE-hemochromatosis with downgraded hepcidin synthesis, was recently shown by the finding that liver transplantation normalized the hepcidin levels in plasma and there was no sign of iron accumulation in the new liver.

Gastrointestinal

Decoding the enigma of necrotizing enterocolitis in premature infants

Roberto Murgas Torrazza, Nan Li, Josef Neu
Pathophysiology 21 (2014) 21–27
http://dx.doi.org/10.1016/j.pathophys.2013.11.011

Necrotizing enterocolitis (NEC) is an enigmatic disease that affects primarily premature infants. It often occurs suddenly and when it occurs, treatment attempts at treatment often fail and results in death. If the infant survives, there is a significant risk of long term sequelae including neurodevelopmental delays. The pathophysiology of NEC is poorly understood and thus prevention has been difficult. In this review, we will provide an overview of why progress may be slow in our understanding of this disease, provide a brief review diagnosis, treatment and some of the current concepts about the pathophysiology of this disease.

Necrotizing enterocolitis (NEC) has been reported since special care units began to house preterm infants .With the advent of modern neonatal intensive care approximately 40 years ago, the occurrence and recognition of the disease markedly increased. It is currently the most common and deadly gastro-intestinal illness seen in preterm infants. Despite major efforts to better understand, treat and prevent this devastating disease, little if any progress has been made during these 4 decades. Underlying this lack of progress is the fact that what is termed “NEC” is likely more than one disease, or mimicked by other diseases, each with a different etiopathogenesis.

Human gut microbiome

Term or near term infants with “NEC” when compared to matched controls usually have occurrence of their disease in the first week after birth, have a significantly higher frequency of prolonged rupture of membranes, chorio-amnionitis, Apgar score <7 at 1 and 5 min, respiratory problems, congenital heart disease, hypoglycemia, and exchange transfusions. When a “NEC” like illness presents in term or near term infants, it should be noted that these are likely to be distinct in pathogenesis than the most common form of NEC and should be differentiated as such.

The infants who suffer primary ischemic necrosis are term or near term infants (although this can occur in preterms) who have concomitant congenital heart disease, often related to poor left ventricular output or obstruction. Other factors that have been associated with primary ischemia are maternal cocaine use, hyperviscosity caused by polycythemia or a severe antecedent hypoxic–ischemic event. Whether the dis-ease entity that results from this should be termed NEC can be debated on historical grounds, but the etiology is clearly different from the NEC seen in most preterm infants.

The pathogenesis of NEC is uncertain, and the etiology seems to be multifactorial. The “classic” form of NEC is highly associated with prematurity; intestinal barrier immaturity, immature immune response, and an immature regulation of intestinal blood flow (Fig.). Although genetics appears to play a role, the environment, especially a dysbiotic intestinal microbiota acting in concert with host immaturities predisposes the preterm infant to disruption of the intestinal epithelia, increased permeability of tight junctions, and release of inflammatory mediators that leads to intestinal mucosa injury and therefore development of necrotizing enterocolitis.

NEC is a multifactorial disease

What causes NEC? NEC is a multifactorial disease with an interaction of several etiophathologies

It is clear from this review that there are several entities that have been described as NEC. What is also clear is that despite having some overlap in the final parts of the pathophysiologic cascade that lead to necrosis, the disease that is most commonly seen in the preterm infant is likely to have an origin that differs markedly from that seen in term infants with congenital heart disease or severe hypoxic–ischemic injury. Thus, epidemiologic studies will need to differentiate these entities, if the aim is to dissect common features that are most highly associated with development of the disease. At this juncture, we areleft with more of a population based preventative approach, where the use of human milk, evidence based feeding guide-lines, considerations for microbial therapy once these are proved safe and effective and approved as such by regulatory authorities, and perhaps even measures that prevent prematurity will have a major impact on this devastating disease.

Influenced by the microbiota, intestinal epithelial cells (IECs) elaborate cytokines

Influenced by the microbiota, intestinal epithelial cells (IECs) elaborate cytokines, including thymic stromal lymphoprotein (TSLP), transforming growthfactor (TGF), and interleukin-10 (IL-10), that can influence pro-inflammatory cytokine production by dendritic cells (DC) and macrophages present in the laminapropria (GALT) and Peyer’s patches. Signals from commensal organisms may influence tissue-specific functions, resulting in T-cell expansion and regulation of the numbers of Th-1,
Th-2, and Th-3 cells. Also modulated by the microbiota, other IEC derived factors, including APRIL (a proliferation-inducing ligand),B-cell activating factor (BAFF), secretory leukocyte peptidase inhibitor (SLPI), prostaglandin E2(PGE2), and other metabolites, directly regulate functions ofboth antigen presenting cells and lymphocytes in the intestinal ecosystem. NK: natural killer cell; LN: lymph node; DC: dendritic cells.Modified from R. Sharma, C. Young, M. Mshvildadze, J. Neu, Intestinal microbiota does it play a role in diseases of the neonate? NeoReviews 10 (4) (2009)e166, with permission

Cross-talk between monocyte.macrophage cells and T.NK lymphocytes

Current Issues in the Management of Necrotizing Enterocolitis

Marion C. W. Henry and R. Lawrence Moss
Seminars in Perinatology, 2004; 28(3): 221-233
http://dx.doi.org:/10.1053/j.semperi.2004.03.010

Necrotizing enterocolitis is almost exclusively a disease of prematurity, with 90% of all cases occurring in premature infants and 90% of those infants weighing less than 2000 g. Prematurity is the only risk factor for necrotizing enterocolitis consistently identified in case control studies and the disease is rare in countries where prematurity is uncommon such as Japan and Sweden. When necrotizing enterocolitis does occur in full-term infants, it appears to by a somewhat different disease, typically associated with some predisposing condition.

NEC occurs in one to three in 1,000 live births and most commonly affects babies born between 30-32 weeks. It is most often diagnosed during the second week of life and occurs more often in previously fed infants. The mortality from NEC has been cited as 10% to 50% of all NEC cases. Surgical mortality has decreased over the last several decades from 70% to between 20 and 50%. The incremental cost per case of acute hospital care is estimated at $74 to 186 thousand compared to age matched controls, not including additional costs of long term care for the infants’ with lifelong morbidity. Survivors may develop short bowel syndrome, recurrent bouts of catheter-related sepsis, malabsorption, malnutrition, and TPN induced liver failure.

Although extensive research concerning the pathophysiology of necrotizing enterocolitis has occurred, a complete understanding has not been fully elucidated. The classic histologic finding is coagulation necrosis; present in over 90% of specimens. This finding suggests the importance of ischemia in the pathogenesis of NEC. Inflammation and bacterial overgrowth also are present. These findings support the assumptions by Kosloske that NEC occurs by the interaction of 3 events:

intestinal ischemia,
colonization by pathogenic bacteria and
excess protein substrate in the intestinal lumen.

Additionally, the immunologic immaturity of the neonatal gut has been implicated in the development of NEC. Reparative tissue changes including epithelial regeneration, formation of granulation tissue and fibrosis, and mixed areas of acute and chronic inflammatory changes suggest that the pathogenesis of NEC may involve a chronic process of injury and repair.

Premature newborns born prior to the 32^nd week of gestational age may have compromised intestinal peristalsis and decreased motility. These motility problems may lead to poor clearance of bacteria, and subsequent bacterial overgrowth. Premature infants also have an immature intestinal tract in terms of immunologic immunity.

There are fewer functional B lymphocytes present and the ability to produce sufficient secretory IgA is reduced. Pepsin, gastric acid and mucus are also not produced as well in prematurity. All of these factors may contribute to the limited proliferation of intestinal flora and the decreased binding of these flora to mucosal cells (Fig).

Role of nitric oxide in the pathogenesis of NEC

Role of nitric oxide in the pathogenesis of NEC.

Characteristics of the immature gut leading to increased risk of necrotizing enterocolitis

Characteristics of the immature gut leading to increased risk of necrotizing enterocolitis.

As understanding of the pathophysiology of necrotizing enterocolitis continues to evolve, a unifying concept is emerging. Initially, there is likely a subclinical insult leading to NEC. This may arise from a brief episode of hypoxia or infection. With colonization of the intestines, bacteria bind to the injured mucosa eliciting an inflammatory response which leads to further inflammation.

Intestinal Microbiota Development in Preterm Neonates and Effect of Perinatal Antibiotics

Silvia Arboleya, Borja Sanchez,, Christian Milani, Sabrina Duranti, et al.
Pediatr 2014;-:—). http://dx.doi.org/10.1016/j.jpeds.2014.09.041

Objectives Assess the establishment of the intestinal microbiota in very low birth-weight preterm infants and to evaluate the impact of perinatal factors, such as delivery mode and perinatal antibiotics.
Study design We used 16S ribosomal RNA gene sequence-based microbiota analysis and quantitative polymerase chain reaction to evaluate the establishment of the intestinal microbiota. We also evaluated factors affecting the microbiota, during the first 3 months of life in preterm infants (n = 27) compared with full-term babies (n = 13).
Results Immaturity affects the microbiota as indicated by a reduced percentage of the family Bacteroidaceae during the first months of life and by a higher initial percentage of Lactobacillaceae in preterm infants compared with full term infants. Perinatal antibiotics, including intrapartum antimicrobial prophylaxis, affects the gut microbiota, as indicated by increased Enterobacteriaceae family organisms in the infants.

Human gut microbiome

Conclusions Prematurity and perinatal antibiotic administration strongly affect the initial establishment of microbiota with potential consequences for later health.

Ischemia and necrotizing enterocolitis: where, when, and how

Philip T. Nowicki
Seminars in Pediatric Surgery (2005) 14, 152-158
http://dx.doi.org:/10.1053/j.sempedsurg.2005.05.003

While it is accepted that ischemia contributes to the pathogenesis of necrotizing enterocolitis (NEC), three important questions regarding this role subsist. First, where within the intestinal circulation does the vascular pathophysiology occur? It is most likely that this event begins within the intramural microcirculation, particularly the small arteries that pierce the gut wall and the submucosal arteriolar plexus insofar as these represent the principal sites of resistance regulation in the gut. Mucosal damage might also disrupt the integrity or function of downstream villous arterioles leading to damage thereto; thereafter, noxious stimuli might ascend into the submucosal vessels via downstream venules and lymphatics. Second, when during the course of pathogenesis does ischemia occur? Ischemia is unlikely to the sole initiating factor of NEC; instead, it is more likely that ischemia is triggered by other events, such as inflammation at the mucosal surface. In this context, it is likely that ischemia plays a secondary, albeit critical role in disease extension. Third, how does the ischemia occur? Regulation of vascular resistance within newborn intestine is principally determined by a balance between the endothelial production of the vasoconstrictor peptide endothelin-1 (ET-1) and endothelial production of the vasodilator free radical nitric oxide (NO). Under normal conditions, the balance heavily favors NO-induced vasodilation, leading to a low resting resistance and high rate of flow. However, factors that disrupt endothelial cell function, eg, ischemia-reperfusion, sustained low-flow perfusion, or proinflammatory mediators, alter the ET-1:NO balance in favor of constriction. The unique ET-1–NO interaction thereafter might facilitate rapid extension of this constriction, generating a viscous cascade wherein ischemia rapidly extends into larger portions of the intestine.

Schematic representation of the intestinal microcirculation

Schematic representation of the intestinal microcirculation. Small mesenteric arteries pierce the muscularis layers and terminate in the submucosa where they give rise to 1A (1st order) arterioles. 2A (2nd order) arterioles arise from the 1A. Although not shown here, these 2A arterioles connect merge with several 1A arterioles, thus generating an arteriolar plexus, or manifold that serves to pressurize the terminal downstream microvasculature. 3A (3rd order) arterioles arise from the 2A and proceed to the mucosa, giving off a 4A branch just before descent into the mucosa. This 4A vessel travels to the muscularis layers. Each 3A vessel becomes the single arteriole perfusing each villus.

Collectively, these studies indicate that disruption of endothelial cell function has the potential to disrupt the normal balance between NO and ET-1 within the newborn intestinal circulation, and that such an event can generate significant ischemia. In this context, it is important to note that NO and ET-1 each regulate the expression and activity of the other. An increased [NO] within the microvascular environment reduces ET-1 expression and compromises ligand binding to the ETA receptor (thus decreasing its contractile efficacy), while ET-1 compromises eNOS expression. Thus, factors that upset the balance between NO and ET-1 will have an immediate and direct effect on vascular tone, but also exert an additional indirect effect by extenuating the disruption of balance between these two factors.

It is not difficult to construct a hypothesis that links the perturbations of I/R and sustained low-flow perfusion with an initial inflammatory insult. Initiation of an inflammatory process at the mucosal–luminal interface could have a direct impact on villus and mucosal 3A arterioles, damaging arteriolar integrity and disrupting villus hemodynamics. Ascent of proinflammatory mediators to the submucosal 1A–2A arteriolar plexus could occur via draining venules and lymphatics, generating damage to vascular effector systems therein; these mediators might include cytokines and platelet activating factor, as these elements have been recovered from human infants with NEC. This event, coupled with a generalized loss of 3A flow throughout a large portion of the mucosal surface, could compromise flow rate within the submucosal arteriolar plexus.

Necrotizing enterocolitis: An update

Loren Berman, R. Lawrence Moss
Seminars in Fetal & Neonatal Medicine 16 (2011) 145e150
http://dx.doi.org:/10.1016/j.siny.2011.02.002

Necrotizing enterocolitis (NEC) is a leading cause of death among patients in the neonatal intensive care unit, carrying a mortality rate of 15e30%. Its pathogenesis is multifactorial and involves an over reactive response of the immune system to an insult. This leads to increased intestinal permeability, bacterial translocation, and sepsis. There are many inflammatory mediators involved in this process, but thus far none has been shown to be a suitable target for preventive or therapeutic measures. NEC usually occurs in the second week of life after the initiation of enteral feeds, and the diagnosis is made based on physical examination findings, laboratory studies, and abdominal radiographs. Neonates with NEC are followed with serial abdominal examinations and radiographs, and may require surgery or primary peritoneal drainage for perforation or necrosis. Many survivors are plagued with long term complications including short bowel syndrome, abnormal growth, and neurodevelopmental delay. Several evidence-based strategies exist that may decrease the incidence of NEC including promotion of human breast milk feeding, careful feeding advancement, and prophylactic probiotic administration in at-risk patients. Prevention is likely to have the greatest impact on decreasing mortality and morbidity related to NEC, as little progress has been made with regard to improving outcomes for neonates once the disease process is underway.

Immune Deficiencies

Primary immunodeficiencies: A rapidly evolving story

Nima Parvaneh, Jean-Laurent Casanova, LD Notarangelo, ME Conley
J Allergy Clin Immunol 2013;131:314-23.
http://dx.doi.org/10.1016/j.jaci.2012.11.051

The characterization of primary immunodeficiencies (PIDs) in human subjects is crucial for a better understanding of the biology of the immune response. New achievements in this field have been possible in light of collaborative studies; attention paid to new phenotypes, infectious and otherwise; improved immunologic techniques; and use of exome sequencing technology. The International Union of Immunological Societies Expert Committee on PIDs recently reported on the updated classification of PIDs. However, new PIDs are being discovered at an ever-increasing rate. A series of 19 novel primary defects of immunity that have been discovered after release of the International Union of Immunological Societies report are discussed here. These new findings highlight the molecular pathways that are associated with clinical phenotypes and suggest potential therapies for affected patients.

Combined Immunodeficiencies

T-cell receptor a gene mutation: T-cell receptor ab1 T-cell depletion

T cells comprise 2 distinct lineages that express either ab or gd T-cell receptor (TCR) complexes that perform different tasks in immune responses. During T-cell maturation, the precise order and efficacy of TCR gene rearrangements determine the fate of the cells. Productive β-chain gene rearrangement produces a pre-TCR on the cell surface in association with pre-Tα invariant peptide (β-selection). Pre-TCR signals promote α-chain recombination and transition to a double-positive stage (CD41CD81). This is the prerequisite for central tolerance achieved through positive and negative selection of thymocytes.

Ras homolog gene family member H deficiency: Loss of naive T cells and persistent human papilloma virus infections
MST1 deficiency: Loss of naive T cells

New insight into the role of MST1 as a critical regulator of T-cell homing and function was provided by the characterization of 8 patients from 4 unrelated families who had homozygous nonsense mutations in STK4, the gene encoding MST1. MST1 was originally identified as an ubiquitously expressed kinase with structural homology to yeast Ste. MST1 is the mammalian homolog of the Drosophila Hippo protein, controlling cell growth, apoptosis, and tumorigenesis. It has both proapoptotic and antiapoptotic functions.

Lymphocyte-specific protein tyrosine kinase deficiency: T-cell deficiency with CD41 lymphopenia

Defects in pre-TCR– and TCR-mediated signaling lead to aberrant T-cell development and function (Fig). One of the earliest biochemical events occurring after engagement of the (pre)-TCR is the activation of lymphocyte-specific protein tyrosine kinase (LCK), a member of the SRC family of protein tyrosine kinases. This kinase then phosphorylates immunoreceptor tyrosine-based activation motifs of intracellular domains of CD3 subunits. Phosphorylated immunoreceptor tyrosine-based activation motifs recruit z-chain associated protein kinase of 70 kDa, which, after being phosphorylated by LCK, is responsible for activation of critical downstream events. Major consequences include activation of the membrane-associated enzyme phospholipase Cg1, activation of the mitogen-activated protein kinase, nuclear translocation of nuclear factor kB (NFkB), and Ca21/Mg21 mobilization. Through these pathways, LCK controls T-cell development and activation. In mice lacking LCK, T-cell development in the thymus is profoundly blocked at an early double-negative stage.

TCR signaling

TCR signaling. Multiple signal transduction pathways are stimulated through the TCR. These pathways collectively activate transcription factors that organize T-cell survival, proliferation, differentiation, homeostasis, and migration. Mutant molecules in patients with TCR-related defects are indicated in red.

Uncoordinated 119 deficiency: Idiopathic CD41 lymphopenia

Idiopathic CD41 lymphopenia (ICL) is a very heterogeneous clinical entity that is defined, by default, by persistent CD41 T-cell lymphopenia (<300 cells/mL or <20% of total T cells) in the absence of HIV infection or any other known cause of immunodeficiency.

Well-Defined Syndromes with Immunodeficiency

Wiskott-Aldrich syndrome protein–interacting protein deficiency: Wiskott-Aldrich syndrome-like phenotype

In hematopoietic cells Wiskott-Aldrich syndrome protein (WASP) is stabilized through forming a complex with WASP interacting protein (WIP).

Phospholipase Cg2 gain-of-function mutations: Cold urticaria, immunodeficiency, and autoimmunity/autoinflammatory

This is a unique phenotype, sharing features of antibody deficiency, autoinflammatory diseases, and immune dysregulatory disorders, making its classification difficult. Two recent studies validated the pleiotropy of genetic alterations in the same gene.

Predominantly Antibody Defects

Defect in the p85a subunit of phosphoinositide 3-kinase: Agammaglobulinemia and absent B cells
CD21 deficiency: Hypogammaglobulinemia
LPS-responsive beige-like anchor deficiency:
Hypogammaglobulinemia with autoimmunity and

early colitis

Defects Of Immune Dysregulation

Pallidin deficiency: Hermansky-Pudlak syndrome type 9
CD27 deficiency: Immune dysregulation and
persistent EBV infection

Congenital Defects Of Phagocyte Number, Function, Or Both

Interferon-stimulated gene 15 deficiency: Mendelian susceptibility to mycobacterial diseases

Defects In Innate Immunity

NKX2-5 deficiency: Isolated congenital asplenia
Toll/IL-1 receptor domain–containing adaptor inducing IFN-b and TANK-binding kinase 1 deficiencies: Herpes simplex encephalitis
Minichromosome maintenance complex component 4 deficiency: NK cell deficiency associated with growth retardation and adrenal insufficiency

Autoinflammatory Disorders

A disintegrin and metalloproteinase 17 deficiency: Inflammatory skin and bowel disease

Cross-talk between monocyte.macrophage cells and T.NK lymphocytes

Cross-talk between monocyte/macrophage cells and T/NK lymphocytes. Genes in the IL-12/IFN-g pathway are particularly important for protection against mycobacterial disease. IRF8 is an IFN-g–inducible transcription factor required for the induction of various target genes, including IL-12. The NF-kB essential modulator (NEMO) mutations in the LZ domain impair CD40-NEMO–dependent pathways. Some gp91phox mutations specifically abolish the respiratory burst in monocyte-derived macrophages. ISG15 is secreted by neutrophils and potentiates IFN-g production by NK/T cells. Genetic defects that preclude monocyte development (eg, GATA2) can also predispose to mycobacterial infections (not shown). Mutant molecules in patients with unusual susceptibility to infection are indicated in red.

The field of PIDs is advancing at full speed in 2 directions. New genetic causes of known PIDs are being discovered (eg, CD21 and TRIF). Moreover, new phenotypes qualify as PIDs with the identification of a first genetic cause (eg, generalized pustular psoriasis). Recent findings contribute fundamental knowledge about immune system biology and its perturbation in disease. They are also of considerable clinical benefit for the patients and their families. A priority is to further translate these new discoveries into improved diagnostic methods and more effective therapeutic strategies, promoting the well-being of patients with PIDs.

Primary immunodeficiencies

Luigi D. Notarangelo
J Allergy Clin Immunol 2010; 125(2): S182-194
http://dx.doi.org:/10.1016/j.jaci.2009.07.053

In the last years, advances in molecular genetics and immunology have resulted in the identification of a growing number of genes causing primary immunodeficiencies (PIDs) in human subjects and a better understanding of the pathophysiology of these disorders. Characterization of the molecular mechanisms of PIDs has also facilitated the development of novel diagnostic assays based on analysis of the expression of the protein encoded by the PID-specific gene. Pilot newborn screening programs for the identification of infants with severe combined immunodeficiency have been initiated. Finally, significant advances have been made in the treatment of PIDs based on the use of subcutaneous immunoglobulins, hematopoietic cell transplantation from unrelated donors and cord blood, and gene therapy. In this review we will discuss the pathogenesis, diagnosis, and treatment of PIDs, with special attention to recent advances in the field.

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Thyroid Function and Disorders

Posted in Amino acids, Calcium Signaling, Clinical Diagnostics, Curation, Developmental biology, Disease Biology, Endocrine Diseases, Genomic Endocrinology, Metabolism, Neurological Diseases, Proteins, Translational Science, tagged blood brain barrier, brain, hyperthyroidism, hypothyroid, MCT8, retinoids, T3, T4, thyroglobulin, Thyroid, thyroid nodule on February 5, 2015| Leave a Comment »

Thyroid Function and Disorders

Writer and Curator: Larry H. Bernstein, MD, FCAP

Normal thyroid function is maintained by endocrine interactions between the hypothalamus, anterior pituitary and thyroid gland [Matfin, 2009]. Iodide is transported across the basement membrane of the thyroid cells by an intrinsic membrane protein called the Na/I symporter (NIS). At the apical border, a second iodide transport protein called pendrin moves iodide into the colloid, where it is involved in hormono-genesis. Once inside the follicle, most of the iodide is oxidized by the enzyme thyroid peroxidase (TPO) in a reaction that facilitates combination with a tyrosine molecule to ultimately form thyroxine (T4) and triiodothyronine (T3). Thyroxine is the major thyroid hormone secreted into the circulation (90%, with T3 composing the other 10%). There is evidence that T3 is the active form of the hormone and that T4 is converted into T3 before it can act physiologically.

All of the major organs in the body are affected by altered levels of thyroid hormone. These actions are mainly mediated by T3. In the cell, T3 binds to a nuclear receptor, resulting in transcription of specific thyroid hormone response genes.

Maternal thyroid hormones are essential for neural development in zebrafish.

Marco A Campinho, João Saraiva, Claudia Florindo, Deborah M Power Molecular endocrinology (Baltimore, Md.) 05/2014;
http://dx.doi.org:/10.1210/me.2014-1032

ABSTRACT Teleost eggs contain an abundant store of maternal thyroid hormones (THs) and early in zebrafish embryonic development all the genes necessary for TH signalling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development an MCT8 knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7 and pax8 genes but downstream of shha and fgf8a signalling. The lack of inhibitory spinal cord interneurons and increased motorneurons in the MCT8 morphants is consistent with their stiff axial body and impaired mobility. MCT8 mutations are associated with X-linked mental retardation in humans and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition.
Relationship between thyroid status and renal function in a general population of unselected outpatients

Giuseppe Lippi, Martina Montagnana, Giovanni Targher, Gian Luca Salvagno, Gian Cesare Guidi
Clin Biochem May 2008; 41(7–8): 625-627

When compared with euthyroid subjects, those with TSH < 0.2 mIU/L and > 2.5 mIU/L had increased and decreased estimated glomerular filtration rate (e-GFR), respectively. TSH levels were an independent predictor of e-GFR.

Serum Thyroid-Stimulating Hormone Measurement for Assessment of Thyroid Function and Disease

Douglas S. Ross
Endocr and Metab Clinics of N Am, Jun 2001; 30(2, 1): 245-264

Thyrotropin, or thyroid-stimulating hormone (TSH), is one of a family of glycoprotein hormones including luteinizing hormone (LH), follicle-stimulating hormone (FSH), and human chorionic gonadotropin (hCG) that share a common α-subunit and a unique β-subunit. Pituitary TSH regulates the secretion of the thyroid hormones T4 (thyroxine) and T3 (triiodothyronine). TSH secretion, in turn, is controlled through negative feedback by thyroid hormone on the pituitary thyrotrope. This relationship is negative log-linear. Small changes in serum free thyroid hormone concentrations result in large changes in serum TSH concentrations, and even subtle changes in thyroid hormone production are best assessed by measurement of serum TSH . Until the late 1980s, the detection limit of TSH assays was within the normal range, and these first-generation TSH assays were useful only for the detection of hypothyroidism. Free T4 measurements were primarily used for assessing thyroid function despite the technical difficulties in free thyroid hormone measurements owing to abnormal binding proteins, changes in binding protein concentrations, and the effects of drugs and illness on thyroid hormone binding. With the use of sensitive second- and third-generation TSH assays, TSH measurement has emerged as the single most useful test of thyroid function. It is widely and appropriately used as a screening test. Unfortunately, the trend has been to rely on TSH measurements alone for the assessment of complicated thyroid disease and patients undergoing treatment for thyroid dysfunction. This article focuses on the potential and real limitations of TSH measurement.
Correlation of creatinine with TSH levels in overt hypothyroidism — A requirement for monitoring of renal function in hypothyroid patients?

Vandana Saini, Amita Yadav, Megha Kataria Arora, Sarika Arora, Ritu Singh, Jayashree Bhattacharjee
Clin Biochem Feb 2012; 45(3): 212-214

Highlights
► Increase serum creatinine levels in both subclinical and overt hypothyroidism. ► Creatinine levels progressively increase with increasing degree of hypothyroidism. ► Increase in creatinine correlated with TSH levels in overt hypothyroid subjects. ► Regular monitoring of renal function is required in hypothyroid patients.

Renal function is influenced by thyroid status. Therefore, this study was done to determine the relationship between renal function and different degrees of thyroid dysfunction.
Design and methods
Thyroid and kidney function tests were analyzed in 47 patients with overt (TSH ≥ 10.0 μIU/L) and 77 patients with subclinical hypothyroidism (TSH 6.0–9.9 μIU/L) in a cross-sectional study. These were compared with 120 age- and sex-matched euthyroid controls.
Results
Overt hypothyroid subjects showed significantly raised serum urea, creatinine and uric acid levels as compared to controls whereas subclinical hypothyroid patients showed significant increased levels of serum urea and creatinine levels. TSH showed significant positive correlation with creatinine and uric acid values and, fT4 had a negative correlation with uric acid in overt hypothyroidism.
Conclusion
Hypothyroid state is associated with significant derangement in biochemical parameters of renal function. Hence the renal function should be regularly monitored in hypothyroid patients.

Ability of Serum Thyroid-Stimulating Hormone Levels to Reflect Peripheral and Central Thyroid Hormone Action Appropriately

Uncertainty Owing to Heterogeneity of T4 Deiodinases
Uncertainty Owing to Heterogeneity of T3 Receptors
Uncertainty Owing to Resetting of the Threshold for Negative Feedback

Clinical Utility of Thyroid-Stimulating Hormone Measurement
Screening for Thyroid Disease and Assessment of Patients Suspected of Having Thyroid Disease

Limitations of Thyroid-Stimulating Hormone Testing in Patients with Known Thyroid Disease Central Hypothyroidism
Thyrotoxicosis Owing to Inappropriate Thyroid-Stimulating Hormone Secretion
Monitoring Thyroid Hormone Therapy
Patients Treated for Hyperthyroidism

The Pituitary-Thyroid Axis in Nonthyroidal Illness

Measurement of Thyroid-Stimulating Hormone
Drugs that Affect Serum Thyroid-Stimulating Hormone Concentrations

Investigations into the etiology of elevated serum T3 levels in protein-malnourished rats

Robert C. Smallridge, Allan R. Glass, Leonard Wartofsky, Keith R. Latham, Kenneth D. Burman
Metabolism, V June 1982; 31(6): 538-542

Thyroid function studies and the peripheral metabolism of thyroid hormone were examined in rats fed a low protein diet (9% casein) for 4–8 wk. Compared to animals fed a normal protein diet ad libitum, both the low protein rats and a pair-fed control group weighed less at the end of the study. However, serum total T3 levels were significantly higher only in the protein deficient rats. The elevated serum T3 was not explainable by enhanced peripheral T4 to T3 conversion, as there was no evidence of any change in hepatic or renal 5′-deiodinase activity when homogenates were examined for conversion of T4 to T3, reverse T3 to 3,3′-diiodothyronine, or 3′,5′-diiodothyronine to 3′-monoiodothyronine. Neither was there an effect on hepatic T3 receptor maximal binding capacity (204 ± 24 versus 168 ± 15 fmol/mg DNA control) or binding affinity (2.07 ± 0.38 versus 2.49 ± 0.24 × 10−10 M control). In two separate experiments the dialyzable fraction of T3 was significantly lower in the low protein group while free T3 concentrations were unchanged or reduced. In contrast, serum total and free T4 were either normal or reduced and dialyzable T4 was unaffected by protein deficiency. We conclude that while serum total T3 is elevated in rats chronically fed a low protein diet, this elevation is not due to enhanced T4 to T3 conversion. Rather, the increased T3 levels can be accounted for by a striking alteration in protein binding to T3. Moreover, the failure to demonstrate similar changes in serum total and dialyzable T4 suggests that in the rat, protein deficiency has different effects on binding to the two major thyroid hormones. Dietary induced changes in serum thyroid hormone binding must be kept in mind in nutrition studies in the rat.

Role of thyrotropin in metabolism of thyroid hormones in nonthyroidal tissues

Udaya M. Kabadi
Metabolism, Jun 2006; 55(6): 748-750

T4 conversion into T3 in peripheral tissues is the major source of circulating T3. However, the exact mechanism of this process is ill defined. Several in vitro studies have demonstrated that thyrotropin facilitates deiodination of T4 into T3 in liver and kidneys. However, there is a paucity of in vitro studies confirming this activity of thyrotropin. Therefore, this study was conducted to examine the influence of thyrotropin on thyroid hormone metabolism in nonthyroidal tissues. We assessed T4, T3, reverse T3 (rT3), and T3 resin uptake (T3RU) responses up to 12 hours at intervals of 4 hours in 6 thyroidectomized female mongrel dogs rendered euthyroid with LT4 replacement therapy before and after subcutaneous (SC) administration of bovine thyrotropin (5 U) on one day and normal saline (0.5 mL) on another in a randomized sequence between 08:00 and 09:00 am. Euthyroid state after LT4 replacement was confirmed before thyrotropin administration. Serum T4, T3, rT3, and T3RU all remained unaltered after SC administration of normal saline. No significant alteration was noted in serum T3RU values on SC administration of thyrotropin. However, serum T3 rose progressively reaching a peak at 12 hours with simultaneous declines being noted in both serum T4 and rT3 concentrations (P < .05 vs prethyrotropin values for all determinations). The changes after SC administration were significantly different (P < .001) in comparison to those noted on SC administration of normal saline. Thyrotropin may promote both the conversion of T4 to T3 and metabolism of rT3 into T2 in nonthyroidal tissues via enhancement of the same monodeionase.

Effects of growth hormone administration on fuel oxidation and thyroid function in normal man

Jens Møller, Jens O.L. Jørgensen, Niels Møller, Jens S. Christiansen, Jørgen Weeke
Metabolism, Jul 1992; 41(7): 728-731

In a randomized, double-blind, placebo-controlled, cross-over study, we examined the effects of 14 days of growth hormone (GH) administration (12 IU/d subcutaneously) on energy expenditure (EE), respiratory exchange ratio (RER), and thyroid function in 14 normal adults of normal weight (eight men and six women). EE (kcal/24 h) was significantly elevated after GH administration (2,073 ± 392, [GH], 1,900 ± 310, [placebo], P = .01). RER was significantly lowered during GH administration (0.73 ± 0.04 v 0.78 ± 0.06, P = .02), reflecting increased oxidation of lipids. Total triiodothyronine (TT3) (nmol/L) and free T3 (FT3) (pmol/L) increased significantly during GH (TT3: 1.73 ± 0.06 [GH], 1.48 ± 0.08 [placebo], P = .01; FT3: 6.19 ± 0.56 [GH], 5.49 ± 0.56 [placebo], P = .01). Concomitantly, an insignificant decrease in reverse T3 (rT3) (nmol/L) was observed (0.07 ± 0.01 [GH], 0.15 ± 0.01 [placebo], P = .08). GH caused a highly significant increase in T3/thyroxine (T4 (×100) ratio (1.84 ± 0.12 [GH], 1.37 ± 0.06 [placebo]). Serum thyrotropin (TSH) was not significantly changed by GH. No changes in total thyroxine (TT4) (nmol/L) (98 ± 6 [GH], 111 ± 8 [placebo], P = .40) and free thyroxine (FT4) (pmol/L) (17.4 ± 1.3 [GH], 18.6 ± 1.1 [placebo], P = .37) after 14 days of GH administration were observed. In conclusion, 2 weeks of GH administration increases EE and lipidoxidation. This finding may partly be mediated by an increase in peripheral T4 to T3 conversion.

Studies on the deiodination of thyroid hormones in Xenopus laevis tadpoles

Helen Robinson, Valerie Anne Galton
Gen Compar Endocr, Sept 1976; 30(1): 83-90

Liver and tail tissues from Xenopus laevis tadpoles possess deiodinating systems capable of degrading both thyroxine (T4) and 3,5,3′-triiodothyronine (T3). Deiodinating activity in liver remains at a constant level throughout late development and metamorphosis with the exception of a transient increase at stage 59, the onset of metamorphosis. Tail activity remains constant during development but rises sharply during metamorphosis when the tail is undergoing regression. In contrast to these findings on spontaneously metamorphosing tadpoles, tail tips induced to regress in vitro do not exhibit any rise in deiodinating activity, even when the tail tips are undergoing extensive autolysis. These results indicate that, while a rise in deiodinating activity may coincide temporarily with hormone action during metamorphosis, the two phenomena may be separated. The deiodinating activity present in tadpole tissues appears to be enzymic and possesses properties characteristic of peroxidase activity. The reaction catalyzed by this mechanism does not appear to involve monodeiodination and hence cannot be considered a mechanism for the peripheral conversion of T4 to T3.

Mechanisms governing the relative proportions of thyroxine and 3,5,3′-triiodothyronine in thyroid secretion

Peter Laurberg
Metabolism, Apr 1984; 33(4): 379-392

In subjects with normal thyroid function only a minor part of circulating 3,5,3′-triiodothyronine (T3) originates directly from the thyroid; the majority is produced in the peripheral tissues by deiodination of thyroxine (T4). However, T3 of thyroidal origin constitutes a relatively high fraction of the total T3 produced in many patients with thyroid hyperfunction or hypofunction. Such a relatively high T3 content in the secretion of the thyroid could be caused by a low T4T3 ratio in thyroglobulin. Severe iodine deficiency is a well-known inducer of a low T4T3 ratio, but a low T4T3 ratio can also be produced independent of the iodine content. This is seen in in vitro studies of thyroglobulin iodination when small amounts of DIT are added to the incubation mixture and in vivo in TSH-treated animals and in patients with Graves’ disease. Another mechanism for high thyroidal secretion of T3 could be an enhanced fractional deiodination of T4 to T3 in the thyroid. In vitro thyroid perfusion studies have shown that the T3 content of thyroid secretions is higher than would be expected from the T4T3 ratio of thyroid hydrolysate and that the major mechanism is deiodination of T4 to T3. Thyroxine deiodinases are also present in the human thyroid, and the amount of T4 deiodinase is enhanced in the thyroids from patients with medically treated Graves’ disease and in the hyperstimulated thyroids of rats. Other factors of possible importance for the mixture of T3 and T4 secreted by the thyroid are a relatively faster liberation of T3 than of T4 from thyroglobulin during partial hydrolysis (this faster release of T3 is probably the mechanism behind the more “rapid” secretion of T3 than of T4), or some kind of thyroid heterogeneity leading to pinocytosis and hydrolysis of thyroglobulin with a lower T4T3 ratio than that of average thyroglobulin.

Starvation-induced alterations of circulating thyroid hormone concentrations in man

Thomas J. Merimee, E.S. Fineberg
Metabolism Jan 1976; 25(1): 79-83

Serum concentrations of triiodothyronine (T3), thyroxine (T4), and TSH were examined in seven men and seven women of normal weight during a 60-hr fast. Similar studies were conducted in two women who received daily for 1 mo before and during a similar fast, 0.4 mg and 0.5 mg of l-thyroxine.
The serum concentrations of T3 decreased in each of the untreated normal subjects (sign test of significance, p < 0.001). The mean control concentration of T3 in women was 152 ± 9 ng100 ml (X ± SEM); after 24 hr of fasting, 131 ± 31 ng100 ml; and at the termination of the fast, 90 ± 15 ng100 ml. The latter value differed from the control value with a p value of < 0.01. Similar changes of T3 concentration occurred in men (mean basal T = 160 ± 11 ng100 ml; mean at termination of fast = 87 ± 16 ng100 ml). The range of decrease for T3 in all subjects varied from 24% to 55%.
The mean T4 concentration at the beginning of the fast was 6.9 ± 0.9, and at the termination of the fast, 7.5 ± 0.6 (p = NS). TSH concentrations remained unchanged (Control, 3.8 ± 0.45 μU/ml; at 60 hr, 4.0 ± 0.26 μU/ml, p = NS).
Studies in two women who received, before and during a fast, T4, indicate that a decreased peripheral conversion of T4 to T3 is the most likely mechanism responsible for this change.

Effect of estrogens on thyroid function. II. Alterations in plasma thyroid hormone levels and their metabolism

Ramesh C. Sawhney, Indra Rastogi, Gopal K. Rastogi
Metabolism Mar 1978; 27(3): 279-288

The circulating levels of total triiodothyronine (TT3), thyroxine (TT4, and T4-bbinding globulin (TBG) and the kinetics of T3 and T4 were studied in five menstruating rhesus monkeys before, during, and after prolonged treatment with estradiol monobenzoate (E2B, 50 μg/kg body weight/day subcutaneously). A significant increase over pretreatment (p < 0.01) plasma TT3, TT4, and TBG was recorded on day 6 of E2B therapy. A further significant stepwise increase in these parameters was noted up to day 19 of E2B, when the levels plateaued for the rest of the period of E2B treatment. Two weeks after discontinuation of E2B, plasma TT3, TT4, and TBG had returned to the pretreatment range and remained so up to 40 days of observation. Although the percent free T3 and percent free T4 were significantly decreased (p < 0.01) during E2B therapy, the absolute concentrations of free T3 and free T4 were not altered. After prolonged E2B treatment the metabolic clearance rate, distribution space, and production rate (PR) of both T3 and T4 were decreased (p < 0.01). The extrathyroidal T4 pool (ETT4P) was significantly increased (p < 0.01), whereas ETT3P did not show any significant alterations (p > 0.05). The decreased PR of T4 might have been due to a direct inhibitory effect of E2B on the thyroid, whereas the decrease in PR of T3 might have been due to either decreased conversion of T4 to T3, to decreased secretion by the thyroid, or both.
Zebrafish as a model to study peripheral thyroid hormone metabolism in vertebrate development

Marjolein Heijlen, Anne M. Houbrechts, Veerle M. Darras
Gen Compar Endocr 1 Jul 2013; 188: 289-296

To unravel the role of thyroid hormones (THs) in vertebrate development it is important to have suitable animal models to study the mechanisms regulating TH availability and activity. Zebrafish (Danio rerio), with its rapidly and externally developing transparent embryo has been a widely used model in developmental biology for some time. To date many of the components of the zebrafish thyroid axis have been identified, including the TH transporters MCT8, MCT10 and OATP1C1, the deiodinases D1, D2 and D3, and the receptors TRα and TRβ. Their structure and function closely resemble those of higher vertebrates. Interestingly, due to a whole genome duplication in the early evolution of ray-finned fishes, zebrafish possess two genes for D3 (dio3 and dio3a) and for TRα (thraa and thrab). Transcripts of all identified genes are present during embryonic development and several of them show dynamic spatio-temporal distribution patterns. Transient morpholino-knockdown of D2, D3 or MCT8 expression clearly disturbs embryonic development, confirming the importance of each of these regulators during early life stages. The recently available tools for targeted stable gene knockout will further increase the value of zebrafish to study the role of peripheral TH metabolism in pre- and post-hatch/post-natal vertebrate development.

The consequences of inappropriate treatment because of failure to recognize the syndrome of pituitary and peripheral tissue resistance to thyroid hormone

Samuel Refetoff, Angel Salazar, Terry J. Smith, Neal H. Scherberg
Metabolism Aug 1983; 32(8); 822-834

Since the description of the syndrome of global (peripheral tissues and pituitary) resistance to thyroid hormone, new cases are being recognized with increasing frequency. The patient described herein had a markedly elevated serum TSH concentration of 260 μU/mL at the time of diagnosis. Studies suggest that elevations of serum TSH levels in this and other patients with the syndrome are most likely iatrogenic in origin. The patient was 312 years old when a goiter and a high serum T4 concentration were detected. Despite subtotal thyroidectomy, antithyroid drugs were required to maintain her T4 level in the normal range. She was referred at age 1112 years because of recurrent goiter. Her parents and five older siblings had normal thyroid function. Off therapy, her serum T4 level was 14.9 μg/dL, FT4I was 17.0, T3 was 362 ng/dL, TSH was 260 μU/mL, and antibodies were negative. There were no signs of thyrotoxicosis, her bone age was 7 years, her growth was stunted (third percentile), her intellectual quotient (IQ) was 67, and there was a 30–50 dB sensorineural hearing loss. The presence of a pituitary adenoma was ruled out. Her TSH had normal bioreactivity and rose to 540 μU/mL in response to TRH. Triiodothyronine was given in incremental doses of 50, 100, 200, and 400 μg/d over 28 days. The log concentrations of serum TSH showed an inverse linear correlation with serum T3. While receiving the highest dose of T3, on which the level of serum T3 ranged from 1400 to 2500 ng/dL, the TSH response to TRH normalized (basal 4.2 and peak 20 μU/mL), as did the high levels of serum cholesterol, carotene, and T4. Her BMR rose from +5 to +22%, her IQ rose to 77, and she gained weight without an increase in caloric intake. Only minimal changes were observed in levels of urinary cAMP, hydroxyproline, magnesium, and nitrogen. All values, with the exception of the weight gain, returned to baseline 2 months after T3 treatment was discontinued. The TSH level was suppressed by l-dopa and by prednisone. Long-term therapy with equivalent doses of T4 (from 300 to 1000 μg/d) produced a growth of 3 cm during the initial 6 weeks, 10.5 cm over the ensuring year (above the 10th percentile), and regression of goiter without thyrotoxicosis. The patient exhibited resistance to thyroid hormone in pituitary and peripheral tissues. The optimal dose of T4 replacement could be predicted by studying tissue responses to incremental doses of T3. The marked elevation in serum TSH concentration, stunted growth, and laboratory evidence of hypothyroidism were due to the limited thyroidal reserve caused by thyroidectomy. All patients with an impaired ability to compensate for the defect as a result of inappropriate treatment should be given thyroid hormone in amounts short of producing catabolic effects. Such a dose is expected to normalize the basal serum TSH concentration and its response to TRH.

Solving the mystery of iodine uptake

Valda Vinson
Science 20 Jun 2014; 344(6190), p. 1355
http://dx.doi.org:/10.1126/science.344.6190.1355-a

The thyroid gland produces iodine-containing hormones that regulate metabolism. The cell membrane protein NIS (sodium/iodine symporter) transports iodine into thyroid cells, but because iodine concentrations outside of the cell are so low, how it does so is a mystery. The key? Moving two sodium ions along with the iodine ion, Nicola et al found. NIS also does not bind sodium very tightly, but the high concentrations of sodium outside the cell allow one sodium ion to bind. This binding increases the affinity of NIS for a second sodium ion and also for iodine. With the three ions bound, NIS changes its conformation so that it opens to the inside of the cell, where the sodium concentration is low enough for NIS to release its sodium ions. When the sodium goes away, so does NIS’s affinity for iodine, leading NIS to release it.

Unliganded Thyroid Hormone Receptor α Regulates Developmental Timing via Gene Repression in Xenopus tropicalis

Jinyoung Choi, Ken-ichi T. Suzuki, Tetsushi Sakuma, Leena Shewade, Takashi Yamamoto, and Daniel R. Buchholz
Endocr Feb 2015; 156(2): 735–744 http://dx.doi.org:/10.1210/en.2014-1554

Thyroid hormone (TH) receptor (TR) expression begins early in development in all vertebrates when circulating TH levels are absent or minimal, yet few developmental roles for unliganded TRs have been established. Unliganded TRs are expected to repress TH-response genes, increase tissue responsivity to TH, and regulate the timing of developmental events. Here we examined the role of unliganded TRα in gene repression and development in Xenopus tropicalis. We used transcription activator-like effector nuclease gene disruption technology to generate founder animals with mutations in the TRα gene and bred them to produce F1 offspring with a normal phenotype and a mutant phenotype, characterized by precocious hind limb development. Offspring with a normal phenotype had zero or one disrupted TRα alleles , and tadpoles with the mutant hind limb phenotype had two truncated TRα alleles with frame shift mutations between the two zinc fingers followed by 40–50 mutant amino acids and then an out-of-frame stop codon. We examined TH-response gene expression and early larval development with and without exogenous TH in F1 offspring. As hypothesized, mutant phenotype tadpoles had increased expression of TH-response genes in the absence of TH and impaired induction of these same genes after exogenous TH treatment, compared with normal phenotype animals. Also, mutant hind limb phenotype animals had reduced hind limb and gill responsivity to exogenous TH. Similar results in methimazole-treated tadpoles showed that increased TH-response gene expression and precocious development were not due to early production of TH. These results indicate that unliganded TRα delays developmental progression by repressing TH-response genes.
The discovery of thyroid replacement therapy. Part 2: The critical 19th century
Conceptualizing the link between the thyroid and myxoedema

Stefan Slater
R Soc Med 2011; 104: 59–63. http://dx.doi.org:/10.1258/jrsm.2010.10k051

Sir William Withey Gull (1816–1890)

Frederik Ruysch, anatomist in Leyden around 1690, adopted, according to Albrecht von Haller in 1766, the opinion that a peculiar fluid was elaborated in the gland and poured into the veins’. The 19th century thus began with thyroidology at best in embryo; but during that century endocrinology was born and the thyroid was its standard bearer. In 1836, Thomas Wilkinson King of Guys Hospital, regarded by some as the ‘Father of Endocrinology’, anticipated on the basis of observation and experiment the internal secretion of the thyroid. In a meticulous paper on its anatomy: he wrote of the thyroid gland that ‘its absorbent vessels carry its peculiar secretion to the great veins of the body’. This language is almost identical to that of Ruysch and Haller more than a century earlier. The idea was prompted by the thyroid’s disproportionately large vascular supply in the absence of any evident mechanical or other local function and also at what he described as its ‘peculiar’ fluid. King notes that his view ‘has been indirectly surmised by Morgagni [probably in 1761] and others’.
In 1850, at a meeting of the Royal Medical and Chirurgical Society of London, chaired by Thomas Addison, Thomas Blizzard Curling, surgeon at the London Hospital, provided a clear clinicopathological correlate in a paper entitled ‘Two cases of absence of the thyroid body and symmetrical swellings of fat tissue at the sides of the neck, connected with defective cerebral development’. Postmortem examination in each revealed no trace of thyroid tissue and that the swellings consisted only of fat. Curling’s important observation was not pursued until 1871 when, at another meeting of the Society, Curling himself then in the chair, Charles Hilton Fagge, a physician at Guy’s Hospital, presented a paper on sporadic cretinism. He described four living cases and noted that none of them had a goiter and that one had been well up to the age of eight and, although now physically cretinous at age 16, she remained very intelligent. He referred to Curling’s paper and reached the same conclusion that the ‘healthy thyroid body is capable of exerting a counteracting influence [on cretinism]’.
Two years later, in 1873, Fagge’s senior colleague at Guy’s, Sir William Withey Gull, presented before the Clinical Society of London two of the five cases he had seen of what he called ‘A Cretinoid State supervening in Adult Life in Women’. He described their cretin-like appearance, drawing particular attention to the broad and thick tongue and the guttural voice and its pronunciation ‘as if the tongue were too large for the mouth’. He acknowledged his remarks were tentative, hence, he said, his use of the word ‘cretinoid’, but he had no doubt this was a ‘substantive’ condition and not one of cardiac or renal origin.
Gull was an interesting personality with apparently a remarkable presence, resembling Napoleon in face, form and manner (Figure). In the 1970s, 80 years after his death in 1890, he was the subject of a theory, quickly discredited, that he had been ‘Jack the Ripper’, the killer in the still unsolved murders and mutilations of at least five Whitechapel prostitutes in 1888. He figured in the 1988 TV film series, Jack the Ripper, starring Michael Caine as the detective. Gull is credited with the first description of hypothyroidism in adults and his paper was important in defining a recognizable clinical syndrome.
Then, in 1877, William Miller Ord, read his paper before the Royal Medical and Chirurgical Society of London and proposed the term ‘myxoedema’ for the adult condition. He described the non-pitting, ‘mucous edema’. He also presented an engaging theory to explain the lethargy, inertia and slow responses associated with the disease. He suggested that these might result from the sheathing and insulation of the body in a ‘jelly-like’, mucin-laden integument that interfered with sensory perceptions and stimulation. Six years later, he chaired the committee set up by the Clinical Society of London to investigate the whole matter. He also later undertook some of the earliest metabolic studies of the effects of treating myxoedema with thyroid extract, showing the rapid weight loss and rise in temperature and in urinary volume and nitrogen excretion that occurred.
The key papers, which advanced these English authors observations, were those of the Swiss surgeons, Jaques-Louis Reverdin in Geneva and Emil Theodor Kocher in Bern, Kocher later receiving the Nobel Prize for his work on the thyroid. How fitting it is that it should be two Swiss doctors whose practices unlocked an understanding of the importance of the thyroid. For they each identified the late effects of total ablation (extirpation) of goiters. they

noted the great similarity of Gull’s and Ord’s myxoedema cases with their affected postoperative patients, referring to the comparison as a ‘rapprochement complet’, clearly making the connection. They acknowledged Gull’s primacy in describing the clinical manifestations and Ord’s ‘christening’ the condition ‘myxoedema’, and proposed that surgical cases be known as ‘myxoedème opératoire’. In light of his findings in 1882, Reverdin thereafter sought to conserve a part of the gland during thyroidectomy for goiter, speculating that its complete removal may have been responsible for these late effects. He had noticed that no such problems followed a just unilateral lobectomy. Kocher called the disease picture in his affected cases ‘cachexia strumipriva’ – literally, a bad condition due to the removal of a struma (goiter) without reference to the earlier work of Reverdin. Halsted noted in his monumental review of goiter surgery: ‘It is interesting to follow the argumentation of a mind so exceptionally keen and sane as Kocher’s in its futile efforts to explain insufficiently illuminated phenomena’. In reading Kocher’s 1909 Nobel Prize Lecture (in English translation), one gets the impression that Kocher was aware in 1883 of Gull’s and Ord’s reports, despite not referring to them, and he dismisses Reverdin’s contribution.
There ensued a competition over the contribution to the thyroid discovery. When post-thyroidectomy myxedema wsas brought to the attention of Kocher, he agreed it was analogous to his cases of cachexia strumipriva. It is also obvious that Kocher, like many surgeons of the time, cannot have engaged in routine postoperative outpatient follow-up, for otherwise the ensuing problems in his goiter-operated patients would have been detected years earlier. In respect of this key moment in the history of the thyroid, Reverdin could be said to hold the intellectual property. The thought has been expressed that perhaps he should have shared the 1909 Nobel Prize with Kocher.
The Emerging Roles of Thyroglobulin

Yuqian Luo, Yuko Ishido, Naoki Hiroi, Norihisa Ishii, and Koichi Suzuki
Adv in Endocr 2014, Article ID 189194, 7 pp http://dx.doi.org/10.1155/2014/189194

Thyroglobulin (Tg), the most important and abundant protein in thyroid follicles, is well known for its essential role in thyroid hormone synthesis. In addition to its conventional role as the precursor of thyroid hormones, we have uncovered a novel function of Tg as an endogenous regulator of follicular function over the past decade. The newly discovered negative feedback effect of Tg on follicular function observed in the rat and human thyroid provides an alternative explanation for the observation of follicle heterogeneity. Given the essential role of the regulatory effects of Tg, we consider that dysregulation of normal Tg function is associated with multiple human thyroid diseases including autoimmune thyroid disease and thyroid cancer. Additionally, extrathyroid Tg may serve a regulatory function in other organs. Further exploration of Tg action, especially at the molecular level, is needed to obtain a better understanding of both the physiological and pathological roles of Tg.

The Surgical Management of Thyroid Cancer

Sara A. Morrison, Hyunsuk Suh, and Richard A. Hodin
Rambam Maimonides Med J 2014; 5(2):e0008. http://dx.doi.org:/10.5041/RMMJ.10142

There are approximately 63,000 reported cases of thyroid carcinoma annually in the United States, representing roughly 4% of all documented malignancies.1 Diagnosis typically stems from work-up of a thyroid nodule. Data from the Framingham study suggests that palpable thyroid nodules are present in 4% of the US population,2 but non-palpable nodules may exist in up to 67% of the population. Such nodules are often found incidentally secondary to the rising use of imaging modalities in medical settings. The large majority of thyroid nodules are benign, with an overall reported risk of malignancy from 5% to 15%.
Thyroid cancer has been increasing in incidence, with the number of reported cases in the US rising by 25% over the last 3 years. With growing technological advances in the field and improved contributions of diagnostics, surgical decision-making and operative planning have taken on new challenges. Herein, we review the current clinical practice recommendations and active areas of surgical controversy, reflective of the most recently published professional consensus guidelines and a systematic review of the literature.
The use of FNA in current clinical practice has resulted in post-surgical pathology findings of malignancy in over 50% of specimens.7 The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) was developed in order to allow pathologists among varying institutions to communicate results to clinical care-takers with widely under-stood descriptors. Results of FNA biopsies are broken down into the following categories with the corresponding risks of malignancy: non-diagnostic or unsatisfactory (1%–4%), benign (0%–3%), atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS; 5%–15%), follicular neoplasm or suspicious for a follicular neoplasm (FN/sFN; 15%–30%), suspicious for malignancy (60%–75%), and malignant (97%–99%).
Mutational Panels.
AsuragenmiR Inform (Austin, TX, USA) mutation analysis assay and Thyroid Cancer Mutation Panel by Quest Diagnostics (Madison, NJ, USA) are the two main commercially available mutational tests which test for known genetic alterations such as BRAF, RAS, RET/PTC, and PAX8/PPAR. These mutational panels are highly specific for malignancy; however, due to the low overall frequency of these mutations in thyroid cancers, negative results do not rule out cancer. Therefore, mutational panel tests are considered a “rule-in” test. If a preoperative mutational test is positive, the nodule should be considered malignant, and total thyroidectomy should be recommended.
Gene Expression Profiling.
The most widely known gene expression profiling test is Afirma Gene Expression Classifier (Veracyte, San Francisco, CA, USA), and, with its recent clinical validation by Alexander et al., Afirma is already being utilized in many clinical settings. The Afirma Gene Expression Classifier (GEC) is an RNA-based assay that utilizes FNA samples to evaluate 167 molecular genes associated with benign nodules based on their proprietary algorithm. Unlike the mutational panel testing, Afirma testing is considered a “rule-out” test since the test has a high negative predictive value in distinguishing benign nodules. However, a positive result reported as “suspicious” carries only 38% risk of malignancy.
In all, these molecular tests should be utilized judiciously and should be considered as a complementary diagnostic tool in the management of thyroid nodules. In the future, molecular testing could become more cost-effective and accurate as a diagnostic tool while providing prognostic and therapeutic information.
Papillary Thyroid Cancer.
Total thyroidectomy is the gold standard for patients with a preoperative diagnosis of papillary thyroid cancer when the nodule is greater than 1 cm in size. Completion thyroidectomy is indicated in patients who have undergone prior lobectomy and are found on final pathology to have papillary thyroid cancer that is larger than 1 cm. The completion thyroidectomy should generally be performed within 6 months of the original procedure in order to minimize the risk of lymph node metastasis.
Involvement of cervical lymph nodes in papillary thyroid cancer is frequent, reported to occur in up to 50% of patients. The role of neck dissection at the time of total thyroidectomy is somewhat controversial, however, since most of the nodal involvement is microscopic and does not affect overall survival. It is generally agreed upon that a therapeutic neck dissection should be pursued in the setting of well-differentiated thyroid cancer patients with clinically positive lymph nodes, whether in the central or lateral neck compartments. Prophylactic neck dissection is not done for follicular thyroid cancer, as the rates of lymph node metastasis are typically less than 10%.
Medullary thyroid cancer (MTC) comprises 4% of all thyroid malignancies. The majority of cases are sporadic in nature; approximately 20%–25% represent familiar/hereditary syndromes. Diagnosis is commonly made by FNA biopsy with specific staining for the presence of calcitonin in the tissue specimen. All patients with a diagnosis of medullary thyroid cancer must be evaluated for multiple endocrine neoplasia (MEN) 2 and be ruled out for the synchronous presence of pheochromocytoma prior to scheduling thyroid surgery.
Effects of Dose Level of Anti-thyroid Drug Carbimazole on Thermoregulation and Blood Constituents in Male Rabbits (Oryctolagus cuniculus)

Intisar H. Saeed, Abdalla M. Abdelatif and Mohamed E. Elnageeb
Adv in Research 2014; 2(3): 129-144. Article no. AIR.2014.002

Carbimazole (CBZ) is an anti-thyroid drug commonly used in the treatment of hyperthyroidism. The objective of this study was to evaluate the effects of dose level of CBZ on thermoregulation and blood constituents in mature male rabbits. Twenty animals were assigned to 4 groups (A, B, C, D) of 5 each. Group A served as control and treated animals in groups B,C,D, received daily orally CBZ doses of 10, 15 and 20 mg/animal for 3 weeks, respectively.
The values of rectal temperature (Tr,), respiration rate (RR) and heart rate (HR) decreased in treated rabbits and the mean values of HR decreased with increase in the dose level of CBZ. The packed cell volume (PCV), Hb concentration and total leukocyte count (TLC) were lower in CBZ treated rabbits. Serum levels of total protein and globulins increased and serum albumin level decreased in treated groups of rabbits. Serum urea level was lower in CBZ treated groups and there was an increase in serum urea level with increase in CBZ dose level. Serum cholesterol level was higher in treated groups and there was an increase in serum cholesterol level with increase in CBZ dose level. Plasma glucose level decreased significantly in CBZ treated groups compared with the control and the mean values decreased with increase in the dose level of CBZ. The results indicate that the responses of basic physiological parameters were almost dose dependent in the range adopted in this study.
Phosphatase Inhibitor Calyculin A Activates TRPC2 Channels in Thyroid FRTL-5 Cells

Pramod Sukumaran, MY Asghar, C Löf, T Viitanen, and Kid Törnquist
Calcium Signaling Jun 2014; 1(2) http://www.researchpub.org/journal/cs/cs.html

We have previously shown that rat thyroid FRTL-5 cells express a calcium entry pathway regulated by a phosphatase. The nature of the calcium entry pathway is presently unknown. We have also shown that FRTL-5 cells express only the TRPC2 channel of the TRPC family of cation channels. In the present investigation we show, using pharmacological inhibitors, the measurement of sodium and calcium entry, stable TRPC2 knock-down cells, and transfection with a non-conducting form of TRPC2, that the calcium entry pathway regulated by a phosphatase is, in fact, the TRPC2 channel. Our data thus point to a novel mechanism by which the TRPC2 channels can be regulated.

Thyroxine Uptake by Perfused Rat Liver
No Evidence for Facilitation by Five Different Thyroxine-binding Proteins

Carl M. Mendel and Richard A. Weisiger
J. Clin. Invest. 1990; 86: 1840-1847

For each of the five protein-hormone complexes studied, the rate of hepatic uptake of T4 (measured under conditions expected to result in dissociation-limited uptake) closely approximated the rate of spontaneous dissociation of the protein-hormone complex within the hepatic sinusoids. These findings indicate an absence of special cellular mechanisms that facilitate the hepatic uptake of T4 from its plasma binding proteins, and support the view that uptake occurs from the free T4 pool after spontaneous dissociation of T4 from its binding proteins.
Thyroxine Transport and Distribution in Nagase Analbuminemic Rats

Carl M. Mendel, RR Cavalieri, LA Gavin, T Pettersson, and M Inoue
J. Clin. Invest. 1989; 83: 143-148

The postulate that thyroxine (T4) in plasma enters tissues by protein-mediated transport or enhanced dissociation from plasma-binding proteins leads to the conclusion that almost all T4 uptake by tissues in the rat occurs via the pool of albumin bound T4 (Pardridge, W. M., B. N. Premachandra, and G. Fierer. 1985. Am. J. Physiol. 248:G545-G550).
To directly test this postulate, and to test more generally whether albumin might play a special role in T4 transport in the rat, we performed in vivo kinetics studies in six Nagase analbuminemic rats and in six control rats, all of whom had similar serum T4 concentrations and percent free T4 values.
Evaluation of the plasma disappearance curves of simultaneously injected ¹²⁵I-T4 and ^I31I-albumin indicated that the flux of T4 from the extracellular compartment into the rapidly exchangeable intracellular compartment was similar in the analbuminemic rats (51±21 ng/min, mean±SD) and in the control rats (54±15 ng/min), as was the size of the rapidly exchangeable intracellular pool of T4 (1.13±0.53 vs. 1.22±036 Mg). This latter finding was confirmed by direct analysis of tissue samples (liver, kidney, and brain). We also performed in vitro kinetics studies using the isolated perfused rat liver. The single-pass fractional extraction by normal rat liver of T4 in pooled analbuminemic rat serum was indistinguishable from that of T4 in pooled control rat serum (10.9±3.3%, n = 3, vs. 11.4±3.4%). When > 98% of the albumin was removed from normal rat serum by chromatography with Affi-Gel blue, the single-pass fractional extraction of T4 (measured by a bolus injection method) did not change (16.3±2.1%, n = 5, vs. 15.2±2.5%). These data provide the first valid experimental test of the enhanced

dissociation hypothesis and indicate that there is no special, substantive role for albumin in T4 transport in the rat.
Influence of thyroid receptors on breast cancer cell proliferation

Conde, R. Paniagua, J. Zamora, M. J. Blanquez, B. Fraile, A. Ruiz & M. I. Arenas
Ann Oncol 2005; http://dx.doi.org:/10.1093/annonc/mdj040

Background: The involvement of thyroid hormones in the development and differentiation of normal breast tissue has been established. However, the association between breast cancer and these hormones is controversial. Therefore, the objective of the present study was to determine the protein expression pattern of thyroid hormone receptors in different human breast pathologies and to evaluate their possible relationship with cellular proliferation.
Patients and methods: The presence of thyroid hormone receptors was evaluated by immunohistochemistry and western blot analysis in 84 breast samples that included 12 cases of benign proliferative diseases, 20 carcinomas in situ and 52 infiltrative carcinomas.
Results: TR-α was detected in the nuclei of epithelial cells from normal breast ducts and acini, while in any pathological type this receptor was located in the cytoplasm. However, TR-b presented a nuclear location in benign proliferative diseases and carcinomas in situ and a cytoplasmatic location in normal breast and infiltrative carcinomas. The highest proliferation index was observed in carcinomas in situ, although in infiltrative carcinomas an inverse correlation between this index and the TR-α expression was encountered.
Conclusions: The results of this study reveal substantial changes in the expression profile of thyroid hormone.
Zebrafish as a model for monocarboxyl transporter 8-deficiency

GD Vatine, D Zada, T Lerer-Goldshtein, A Tovin, G Malkinson, K Yaniv and L Appelbaum
J Biol Chem Nov 2012; Manuscript M112.413831
http://dx.doi.org:/10.1074/jbc.M112.413831

Background: Mutations in the thyroid hormone transporter MCT8 are associated with psychomotor retardation AHDS.
Results: In zebrafish, as in humans, mct8 is expressed primarily in the nervous system. Elimination of MCT8 causes severe neural impairment.
Conclusion: MCT8 is a crucial regulator during zebrafish embryonic development. Significance: Establishment of the first vertebrate model for MCT8-deficiency, which exhibits a neurological phenotype.
Unusual Ratio between Free Thyroxine and Free Triiodothyronine in a Long-Lived Mole-Rat Species with Bimodal Ageing

Yoshiyuki Henning, Christiane Vole, Sabine Begall, Martin Bens, et al.
PlusOne Nov 2014; 9(11),e113698. http://dx.doi.org:/10.1371/journal.pone.0113698

Ansell’s mole-rats (Fukomys anselli) are subterranean, long-lived rodents, which live in eusocial families, where the maximum lifespan of breeders is twice as long as that of non-breeders. Their metabolic rate is significantly lower than expected based on allometry, and their retinae show a high density of S-cone opsins. Both features may indicate naturally low thyroid hormone levels.
In the present study, we sequenced several major components of the thyroid hormone pathways and analyzed free and total thyroxine and triiodothyronine in serum samples of breeding and non-breeding F. anselli to examine whether
a) their thyroid hormone system shows any peculiarities on the genetic level,
b) these animals have lower hormone levels compared to euthyroid rodents (rats and guinea pigs), and
c) reproductive status, lifespan and free hormone levels are correlated.
Genetic analyses confirmed that Ansell’s mole-rats have a conserved thyroid hormone system as known from other mammalian species. Interspecific comparisons revealed that free thyroxine levels of F. anselli were about ten times lower than of guinea pigs and rats, whereas the free triiodothyronine levels, the main biologically active form, did not differ significantly amongst species. The resulting fT4:fT3 ratio is unusual for a mammal and potentially represents a case of natural hypothyroxinemia.
Comparisons with total thyroxine levels suggest that mole-rats seem to possess two distinct mechanisms that work hand in hand to downregulate fT4 levels reliably. We could not find any correlation between free hormone levels and reproductive status, gender or weight. Free thyroxine may slightly increase with age, based on subsignificant evidence. Hence, thyroid hormones do not seem to explain the different ageing rates of breeders and nonbreeders. Further research is required to investigate the regulatory mechanisms responsible for the unusual proportion of free thyroxine and free triiodothyronine.
Transthyretin Regulates Thyroid Hormone Levels in the Choroid Plexus, But Not in the Brain Parenchyma: Study in a Transthyretin-Null Mouse Model

JA Palha, R Fernandes, GM De Escobar, V Episkopou, M Gottesman, and MJ Saraiva
Endocr 2000; 141(9): 3267–3272.

Transthyretin (TTR) is the major T4-binding protein in rodents. Using a TTR-null mouse model we asked the following questions.
1) Do other T4 binding moieties replace TTR in the cerebrospinal fluid (CSF)?
2) Are the low whole brain total T4 levels found in this mouse model associated with hypothyroidism, e.g. increased 59-deiodinase type 2 (D2) activity and RC3-neurogranin messenger RNA levels?
3) Which brain regions account for the decreased total whole brain T4 levels?
4) Are there changes in T3 levels in the brain?
Our results show the following.
1) No other T4-binding protein replaces TTR in the CSF of the TTR-null mice.
2) D2 activity is normal in the cortex, cerebellum, and hippocampus, and total brain RC3-neurogranin messenger RNA levels are not altered.
3) T4 levels measured in the cortex, cerebellum, and hippocampus are normal. However T4 and T3 levels in the choroid plexus are only 14% and 48% of the normal values, respectively.
4) T3 levels are normal in the brain parenchyma.
The data presented here suggest that TTR influences thyroid hormone levels in the choroid plexus, but not in the brain. Interference with the blood-choroid-plexus-CSF-TTR-mediated route of T4 entry into the brain caused by the absence of TTR does not produce measurable features of hypothyroidism. It thus appears that TTR is not required for T4 entry or for maintenance of the euthyroid state in the mouse brain.
Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter

E.C.H. Friesema, S Ganguly, A. Abdalla, J.E.M. Fox, AP. Halestrap, and TJ. Visser
J Biol Chem 2003; Manuscript M300909200
http://dx.doi.org/10.1074/jbc.M300909200

Transport of thyroid hormone across the cell membrane is required for its action and

metabolism. Recently, a T-type amino acid transporter was cloned which transports aromatic amino acids but not iodothyronines. This transporter belongs to the monocarboxylate transporter (MCT) family, and is most homologous with MCT8 (SLC16A2). Therefore, we cloned rat MCT8, and tested it for thyroid hormone transport in Xenopus laevis oocytes. Oocytes were injected with rat MCT8 cRNA, and after 3 days immunofluorescence microscopy demonstrated expression of the protein at the plasma membrane. MCT8 cRNA induced a ~10-fold increase in uptake of 10 nM ¹²⁵I-labeled thyroxine (T4), 3,3′,5-triiodothyronine (T3), 3,3′,5′-triiodothyronine (rT3) and 3,3′-diiodothyronine. Due to the rapid uptake of the ligands, transport was only linear with time for <4 min. MCT8 did not transport Leu, Phe, Trp or Tyr. [¹²⁵I]T4 transport was strongly inhibited by L-T4, D-T4, L-T3, D-T3, 3,3’,5-triiodothyroacetic acid, N-bromoacetyl-T3, and bromosulfophthalein. T3 transport was less affected by these inhibitors. Iodothyronine uptake in uninjected oocytes was reduced by albumin but the stimulation induced by MCT8 was markedly increased. Saturation analysis provided apparent Km values of 2-5 μM for T4, T3 and rT3. Immunohistochemistry showed high expression in liver, kidney, brain and heart. In conclusion, we have identified MCT8 as a very active and specific thyroid hormone transporter.
Thyroid hormones,T3 andT4, in the brain
Amy C. Schroeder and Martin L. Privalsky
Front Endocr Mar 2014; 5 article 40. http://dx.doi.org:/10.3389/fendo.2014.00040

Thyroid hormones (THs) are essential for fetal and post-natal nervous system development and also play an important role in the maintenance of adult brain function. Of the two major THs, T4 (3,5,30,50-tetraiodo-l-thyronine) is classically viewed as an pro-hormone that must be converted toT3 (3,5,30-tri-iodo-l-thyronine) via tissue-level deiodinases for biological activity. THs primarily mediate their effects by binding to thyroid hormone receptor (TR) isoforms, predominantly TRα1 and TRβ1, which are expressed in different tissues and exhibit distinctive roles in endocrinology. Notably, the ability to respond toT4 and toT3 differs for the two TR isoforms, with TRα1 generally more responsive to T4 than TRβ1. TRα1 is also the most abundantly expressed TR isoform in the brain, encompassing 70–80% of all TR expression in this tissue. Conversion of T4 into T3 via deiodinase 2 in astrocytes has been classically viewed as critical for generating local T3 for neurons. However, deiodinase-deficient mice do not exhibit obvious defectives in brain development or function. Considering that TRα1 is well-established as the predominant isoform in brain, and that TRα1 responds to both T3 and T4, we suggest T4 may play a more active role in brain physiology than has been previously accepted.
Thyroid hormone action: astrocyte–neuron communication

Beatriz Morte and Juan Bernal
Front Endocr May 2014; 5, Article 82 http://dx.doi.org:/10.3389/fendo.2014.00082

Thyroid hormone (TH) action is exerted mainly through regulation of gene expression by binding of T3 to the nuclear receptors.T4 plays an important role as a source of intracellular T3 in the central nervous system via the action of the type 2 deiodinase (D2), expressed in the astrocytes. A model of T3 availability to neural cells has been proposed and validated. The model contemplates that brain T3 has a double origin: a fraction is available directly from the circulation, and another is produced locally from T4 in the astrocytes by D2. The fetal brain depends almost entirely on theT3 generated locally. The contribution of systemic T3 increases subsequently during development to account for approximately 50% of total brain T3 in the late postnatal and adult stages. In this article, we review the experimental data in support of this model, and how the factors affectingT3 availability in the brain, such as deiodinases and transporters, play a decisive role in modulating local TH action during development.
The Significance of Thyroid Hormone Transporters in the Brain

Juan Bernal
Endocr Apr 2005; 146(4):1698–1700. http://dx.doi.org:/10.1210/en.2005-0134

The MCT family comprises up to 14 members, some of which are involved in the transport of important substrates for the brain such as lactate and pyruvate. MCT8 has been shown to act as a specific transporter for T4 and T3 and displays slightly higher affinity for T3. Heuer et al. have also studied the regional expression of MCT8 mRNA. In addition to high expression levels in the choroid plexus, they found that MCT8 is expressed in neurons of the neocortex, hippocampus, basal ganglia, amygdala, hypothalamus, and the Purkinje cells of the cerebellum, all regions known to be sensitive to thyroid hormones. Expression of MCT8 in neurons suggests that neuronal uptake of the T3 produced in astrocytes is facilitated by this transporter.
The physiological significance ofMCT8 as a transporter for thyroid hormone is supported by the finding of mutations in humans by Dumitrescu et al. and Friesema et al. The syndrome affects children from an early age and consists of severe developmental delay and neurological damage together with an unusually altered pattern of thyroid hormone levels in blood. The patients presented low total and free T4, high total and free T3, and low rT3. TSH was moderately elevated in two of the patients and normal or slightly elevated in the other five. Inactivating mutations of the MCT8 transporter could result in the altered thyroid hormone levels. In vitro uptake of T4 and T3 by fibroblasts isolated from affected males was strongly reduced, and intracellular D2 was increased 6- to 8-fold. It is thus hypothesized that the resulting increase in intracellularly generated T3 accumulates in blood because of its poor reuptake into cells.
The second trimester is also the period when thyroid hormone receptors increase in concentration in the brain. If MCT8 is needed at this stage of development for T3 entry into neurons, mutations of the transporter could interfere with T3-dependent developmental processes. Knowledge of the ontogenetic patterns of MCT8 in the human fetal brain would certainly be helpful. On the other hand, there is also the possibility that MCT8 mutations interfere with transport of other substrates for brain metabolism that could be even more important than T3 in determining the severity and outcome of the syndrome. Other members of the family transport metabolic substrates such as pyruvate and lactate, but MCT8 so far appears to be specific for iodothyronines

Peripheral markers of thyroid function: The effect of T4 monotherapy versus T4/T3 combination therapy in hypothyroid subjects: A randomized cross-over study

Ulla Schmidt, B Nygaard, EW Jensen, J Kvetny, A Jarløv, and Jens Faber
Endocrine Connections Jan 10, 2013 http://dx.doi.org:/10.1530/EC-12-0

Background: A recent randomized controlled trial suggests that hypothyroid subjects may find L-T4 and L-T3 combination therapy to be

superior to L-T4 monotherapy in terms of quality of life, suggesting that the brain registered increased T3 availability during the

combination therapy.

Hypothesis: Peripheral tissue might also be stimulated during T4/T3 combination therapy compared to T4 monotherapy.
Methods: Serum levels of Sex Hormone-Binding Globulin (SHBG), pro-collagen-1-N-terminal peptide (PINP), and N-terminal pro-brain natriuretic peptide (NT-proBNP) (representing hepatocyte, osteoblast, and cardiomyocyte stimulation, respectively) were measured in 26 hypothyroid subjects in a double blind, randomized, cross-over trial, which compared the replacement therapy with T4/T3 in combination (50 Fg T4 was substituted with 20 Fg T3) to T4 alone (once daily regimens). This was performed to obtain unaltered serum thyroid stimulating hormone (TSH) levels during the trial and between the two treatment groups. Blood sampling was performed 24 hours after the last intake of thyroid hormone medication.
Results: TSH remained unaltered between the groups ((median) 0.83 vs. 1.18 mU/l in T4/T3 combination and T4 mono-therapy, respectively; p=0.534). SHBG increased from (median) 75 nmol/l at baseline to 83 nmol/l in the T4/T3 group (p=0.015), but remained unaltered in the T4 group (67 nmol/l); thus, it was higher in the T4/T3 vs. T4 group (p=0.041). PINP levels were higher in the T4/T3 therapy (48 vs. 40 Fg/l (p<0.001)). NT-proBNP did not differ between the groups. Conclusions: T4/T3 combination therapy in hypothyroidism seems to have more metabolic effects than the T4 monotherapy.
Stimulatory effects of thyroid hormone on brain angiogenesis in vivo and in vitro

Liqun Zhang, CM Cooper-Kuhn, U Nannmark, K Blomgren and HG Kuhn
J Cereb Blood Flow & Metab 2010; 30:323–335. http://dx.doi.org:/10.1038/jcbfm.2009.216

Thyroid hormone is critical for the proper development of the central nervous system. However, the specific role of thyroid hormone on brain angiogenesis remains poorly understood. Treatment of rats from birth to postnatal day 21 (P21) with propylthiouracil (PTU), a reversible blocker of triiodothyronine (T3) synthesis, resulted in decreased brain angiogenesis, as indicated by reduced complexity and density of microvessels. However, when PTU was withdrawn at P22, these parameters were fully recovered by P90. These changes were paralleled by an altered expression of vascular endothelial growth factor A (Vegfa) and basic fibroblast growth factor (Fgf2). Physiologic concentrations of T3 and thyroxine (T4) stimulated proliferation and tubulogenesis of rat brain derived endothelial (RBE4) cells in vitro. Protein and mRNA levels of VEGF-A and FGF-2 increased after T3 stimulation of RBE4 cells. The thyroid hormone receptor blocker NH-3 abolished T3-induced Fgf2 and Vegfα upregulation, indicating a receptor-mediated effect. Thyroid hormone inhibited the apoptosis in RBE4 cells and altered mRNA levels of apoptosis-related genes, namely Bcl2 and Bad. The present results show that thyroid hormone has a substantial impact on vasculature development in the brain. Pathologically altered vascularization could, therefore, be a contributing factor to the neurologic deficits induced by thyroid hormone deficiency.

Molecules important for thyroid hormone

synthesis and action – known facts and future perspectives

Klaudia Brix, Dagmar Führer, Heike Biebermann
Thyroid Research 2011, 4(Suppl 1):S9 http://www.thyroidresearchjournal.com/content/4/S1/S9

Thyroid hormones are of crucial importance for the functioning of nearly every organ. Remarkably, disturbances of thyroid hormone synthesis and function are among the most common endocrine disorders affecting approximately one third of the working German population. Over the last ten years our understanding of biosynthesis and functioning of these hormones has increased tremendously. This includes the identification of proteins involved in thyroid hormone biosynthesis like Thox2 and Dehal where mutations in these genes are responsible for certain degrees of hypothyroidism. One of the most important findings was the identification of a specific transporter for triiodothyronine (T3), the monocarboxylate transporter 8 (MCT8) responsible for directed transport of T3 into target cells and for export of thyroid hormones out of thyroid epithelial cells. Genetic disturbances of MCT8 in patients result in a biochemical constellation of high T3 levels in combination with low or normal TSH and thyroxine levels leading to a new syndrome of severe X-linked mental retardation. Importantly mice lacking MCT8 presented only with a mild phenotype, indicating that compensatory mechanisms exist in mice. Moreover, it has become clear that not only genomic actions of T3 exist. T3 is also capable to activate adhesion receptors and it signals via activation of PI3K and MAPK pathways. Most recently, thyroid hormone derivatives were identified, the thyronamines which are decarboxylated thyroid hormones initiating physiological actions like lowering body temperature and heart rate, thereby acting in opposite direction to the classical thyroid hormones. So far it is believed that thyronamines function via the activation of a G-protein coupled receptor, TAAR1. The objective of this review is to summarize the recent findings in thyroid hormone synthesis and action and to discuss their implications for diagnosis of thyroid disease and for treatment of patients.

Retinoic Acid Induces Expression of the Thyroid Hormone Transporter, Monocarboxylate Transporter 8 (Mct8)

T Kogai, Yan-Yun Liu, LL Richter, K Mody, H Kagechika, and GA Brent
J Biol Chem Jun 2010. Manuscript M110.123158
http://www.jbc.org/cgi/doi/10.1074/jbc.M110.123158

Retinoic acid (RA) and thyroid hormone are critical for differentiation and organogenesis in the embryo. The monocarboxylate transporter-8 (Mct8), expressed predominantly in brain and placenta, mediates thyroid hormone uptake from the circulation and is required for normal neural development. RA induces differentiation of F9 mouse teratocarcinoma cells towards neurons as well as extraembryonal endoderm. We hypothesized that Mct8 is functionally expressed in F9 cells and induced by RA. All trans RA (tRA), and other RA receptor (RAR) agonists, dramatically (> 300-fold) induced Mct8. tRA treatment significantly increased uptake of triiodothyronine and thyroxine (4.1 fold and 4.3 fold, respectively), which was abolished by a selective Mct8 inhibitor, bromosulfophthalein. Sequence inspection of the Mct8 promoter region and
5′-rapid amplification of cDNA ends (5’-RACE) PCR analysis in F9 cells identified
11 transcription start sites and a proximal Sp1 site, but no TATA-box. tRA significantly enhanced Mct8 promoter activity through a consensus RA responsive element located 6.6 kilobases upstream of the coding region. Chromatin immunoprecipitation assay demonstrated binding of RAR and retinoid-X receptor (RXR) to the RA response element. The promotion of thyroid hormone uptake through the transcriptional up-regulation of Mct8 by RAR is likely to be important for extraembryonic endoderm development and neural differentiation. This finding demonstrates crosstalk between RA signaling and thyroid hormone signaling in early development at the level of the thyroid hormone transporter.
Abnormal thyroid hormone metabolism in mice lacking the monocarboxylate transporter 8

Marija Trajkovic, Theo J. Visser, Jens Mittag, Sigrun Horn, et al.
J. Clin. Invest. 2007; 117:627–635. http://dx.doi.org:/10.1172/JCI28253

In humans, inactivating mutations in the gene of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8; SLC16A2) lead to severe forms of psychomotor retardation combined with imbalanced thyroid hormone serum levels. The MCT8-null mice described here, however, developed without overt deficits but also exhibited distorted 3,5,3′-triiodothyronine (T3) and thyroxine (T4) serum levels, resulting in increased hepatic activity of type 1 deiodinase (D1). In the mutants’ brains, entry of T4 was not affected, but uptake of T3 was diminished. Moreover, the T4 and T3 content in the brain of MCT8-null mice was decreased, the activity of D2 was increased, and D3 activity was decreased, indicating the hypothyroid state of this tissue. In the CNS, analysis of T3 target genes revealed that in the mutants, the neuronal T3 uptake was impaired in an area-specific manner, with strongly elevated thyrotropin-releasing hormone transcript levels in the hypothalamic paraventricular nucleus and slightly decreased RC3 mRNA expression in striatal neurons; however, cerebellar Purkinje cells appeared unaffected, since they did not exhibit dendritic outgrowth defects and responded normally to T3 treatment in vitro.
In conclusion, the circulating thyroid hormone levels of MCT8-null mice closely resemble those of humans with MCT8 mutations, yet in the mice, CNS development is only partially affected.
3-Monoiodothyronamine: the rationale for its action as an endogenous adrenergic-blocking neuromodulator

HS Gompf, JH Greenberg, G Aston-Jones, A Ianculescu, TS Scanlan, and MB Dratman
Brain Res. 2010 Sep 10; 1351: 130–140. http://dx.doi.org:/10.1016/j.brainres.2010.06.067

The investigations reported here were designed to gain insights into the role of
3-monoiodothyronamine (T1AM) in the brain, where the amine was originally identified and characterized.
Extensive deiodinase studies indicated that T1AM was derived from the T4 metabolite, reverse triiodothyronine (revT3), while functional studies provided well-confirmed evidence that T1AM has strong adrenergic blocking effects. Because a state of adrenergic overactivity prevails when triiodothyronine (T3) concentrations becomes excessive, the possibility that T3’s metabolic partner, revT3, might give rise to an antagonist of those T3 actions was thought to be reasonable.
All T1AM studies thus far have required use of pharmacological doses.
Therefore we considered that choosing a physiological site of action was a priority and focused on the locus coeruleus (LC), the major noradrenergic control center in the brain. Site-directed injections of T1AM into the LC elicited a significant, dose-dependent neuronal firing rate change in a subset of adrenergic neurons with an EC50=2.7 μM, a dose well within the physiological range. Further evidence for its physiological actions came from autoradiographic images obtained following intravenous carrier-free 125I-labeled T1AM injection. These showed that the amine bound with high affinity to the LC and to other selected brain nuclei, each of which is both an LC target and a known T3 binding site. This new evidence points to a physiological role for T1AM as an endogenous adrenergic-blocking neuromodulator in the central noradrenergic system.

Thyroid hormones are transported through the blood-brain barrier

Thyroid hormones are transported through the blood-brain barrier (OATP) or the blood-CSF barrier (OATP and MCT8). In the astrocytes and tanycytes T4 is converted to T3 which then enters the neurons through MCT8. In the neurons both T4 and T3 are degraded by D3. T3 from the tanycytes may reach the portal vessels in the median eminence. Other transporters may be present on the astrocyte or tanycyte membranes. In most cases the transport could be bidirectional, although only one direction is shown.
Juan Bernal – Instituto de Investigaciones Biomedicas – 28029 Madrid, Spain

the interactions of maternal, placental and fetal thyroid

Old and new concepts of thyroid hormone action.

A: Old concept of thyroid hormone action. In former times it was assumed that thyroid hormones are able to pass the plasma membrane by passive transport. Once in the cytosol T4 is deiodinated to T3 which exerts genomic effects by binding to the thyroid hormone receptor (TR). After hetero-dimerization with other nuclear receptors like retinoic X receptor (RXR), transcriptional regulation is initiated resulting in activation or inactivation of target genes.
B: New concepts of thyroid hormone action. Thyroid hormones enter a target cell via specific transporters, e.g. T3 uses the monocarboxylate transporter MCT8 while T4 entry is mediated by Lat2 or Oatp14. Moreover, T3 can interact with avb3 integrins to induce ERK1/2 signalling. Cytosolic T3 exerts genomic effects but can additionally also act by non-genomic means after TR binding and activation of down-stream PI-3 kinase. Likewise, the naturally occurring iodothyronine T2 is believed to stimulate metabolic rates via mitochondrial pathways, thereby bypassing genomic regulation. Besides thyroid hormones, derivatives like the thyronamines T1AM or T0AM, modulate the action of T3, e.g. counter-acting its effects in certain target cells. Thyronamines (TAMs) bind to and activate G-protein coupled receptors (GPCRs) of the trace amine associated receptor (TAAR) family. So far, it is only known that TAAR1 is activated by TAMs and signals via adenylylcyclase (AC) activation with subsequent rise of cAMP levels. However other GPCRs are likely targets for thyroid hormone derivatives

Brix et al.: Molecules important for thyroid hormone synthesis and action – known facts and future perspectives. Thyroid Research 2011 4(Suppl 1):S9.
http://dx.doi.org:/10.1186/1756-6614-4-S1-S9

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Proteomics

Posted in Amino acids, Ca2+ triggered activation, Cell Biology, Clinical Diagnostics, Coagulation Therapy and Internal Bleeding, Curation, Cytoskeleton, Diagnostics and Lab Tests, Disease Biology, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Hematology, Ionic Transporters Na+, Mass automation of plasma proteins, Myocardial Metabolism, Neurodegenerative Diseases, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Pharmaceutical Discovery, Proteins, Proteomics, Rapid automation of plasma protein pools, Signaling & Cell Circuits, tagged Cancer - General, Cardiovascular disease, cell-cell interactions, Cytoskeleton, Proteomics, receptors on January 31, 2015| Leave a Comment »

Proteomics

Writer and Curator: Larry H. Bernstein, MD, FCAP

The previous discussion concerned genomics, metabolomics, and cancer. The discussion that follows is concerned with the expanding filed of proteomics, which has implication for disease discovery, pharmaceutical targeting, and diagnostics.

The human proteome – a scientific opportunity for transforming diagnostics, therapeutics, and healthcare

Marc Vidal, Daniel W Chan, Mark Gerstein, Matthias Mann, Gilbert S Omenn, et al.
Clinical Proteomics 2012, 9:6 http://www.clinicalproteomicsjournal.com/content/9/1/6

A National Institutes of Health (NIH) workshop was convened in Bethesda, MD on September 26–27, 2011, with representative scientific leaders in the field of proteomics and its applications to clinical settings. The main purpose of this workshop was to articulate ways in which the biomedical research community can capitalize on recent technology advances and synergize with ongoing efforts to advance the field of human proteomics. This executive summary and the following full report describe the main discussions and outcomes of the workshop.

Proteomics Pioneer Award 2013: Professor Amos Bairoch, University of Geneva, Switzerland

Eupa Open Proteomics 2 (2014) 34 http://dx.doi.org/10.1016/j.euprot.2013.12.002

Amos Bairoch has always been fascinated by computer science, genetics and biochemistry. His fi rst project, as a PhD student, was the development of PC/Gene, a MS-DOS based software package for the analysis of protein and nucleotide sequences. While working on this project, he realized that there was no single resource for protein sequences, and started to develop the first annotated protein sequence database, which became Swiss-Prot and was first released in July 1986. In 1988, he created PROSITE, a database of protein families and domains, and a little later ENZYME, an enzyme nomenclature database.

Amos Bairoch led the Swiss-Prot group from its creation in 1988 until 2009. During this period, Swiss-Prot became the primary protein sequence resource in the world and has been a key research instrument for both bioinformaticians and laboratory-based scientists, particularly in the field of proteomics.

Since 2009, Amos Bairoch’s group is developing neXtProt, a knowledgebase
specifically dedicated to human proteins.neXtProt has been chosen as the reference protein database for the HUPO Human Proteome Projects.

For his major contributions in the field of proteomic databases, Amos Bairoch received the Friedrich Miescher Award from the Swiss Society of Biochemistry in 1993, the Helmut Horten Foundation Incentive Award in 1995, the Pehr Edman award and the European Latsis Prize in 2004, the Otto Naegeli prize in 2010, and the HUPO Distinguished Achievement Award in Proteomic Sciences in 2011.

National Heart, Lung, and Blood Institute Clinical Proteomics Working Group Report

CB Granger, JE Van Eyk, SC Mockrin and N. Leigh Anderson
Circulation. 2004;109:1697-1703
http://dx.doi.org:/10.1161/01.CIR.0000121563.47232.2A

The National Heart, Lung, and Blood Institute (NHLBI) Clinical Proteomics Working Group was charged with identifying opportunities and challenges in clinical proteomics and using these as a basis for recommendations aimed at directly improving patient care. The group included representatives of clinical and translational research, proteomic technologies, laboratory medicine, bioinformatics, and 2 of the NHLBI Proteomics Centers, which form part of a program focused on innovative technology development. This report represents the results from a one-and-a-half-day meeting on May 8 and 9, 2003. For the purposes of this report, clinical proteomics is defined as the systematic, comprehensive, large-scale identification of protein patterns (“fingerprints”) of disease and the application of this knowledge to improve patient care and public health through better assessment of disease susceptibility, prevention of disease, selection of therapy for the individual, and monitoring of treatment response.

The -omics era: Proteomics and lipidomics in vascular research

Athanasios Didangelos, Christin Stegemann, Manuel Mayr
Atherosclerosis 221 (2012) 12– 17
http://dx.doi.org:/10.1016/j.atherosclerosis.2011.09.043

The retention of proatherogenic low-density lipoprotein (LDL) particles on the subendothelial extracellular matrix (ECM) is a hallmark of atherosclerosis. Apolipoprotein B (apoB)-containing lipoprotein particles are trapped in the arterial intima by proteoglycans in atherosclerosis-prone areas and eventually become modified, commonly by aggregation and oxidation. The initial accumulation of proatherogenic lipoproteins initiates an inflammatory response, which results in the release of proteolytic enzymes and induces the dedifferentiation of vascular smooth muscle cells (SMCs) resulting in alterations of their matrix producing properties. The precise mechanisms responsible for the accumulation of certain matrix components and subsequent lipoprotein retention on the vessel wall are not fully elucidated. Undoubtedly, ECM remodeling contributes to the formation of atherosclerotic lesions and the lipid composition of apolipoproteins influences their binding properties to the matrix. An unbiased discovery approach, which is not limited to known molecules of presumed importance, will be invaluable for the identification of novel, previously unknown mediators of disease. Although descriptive, the detailed examination of atherosclerotic plaques using advanced proteomics and lipidomics techniques can generate novel insights and form the basis for further mechanistic investigations.

The Revolution in Proteomics Ionization –
CaptiveSpray nanoBooster™
Bruker, LC-MS Source

Bruker CaptiveSpray principle:

Stable and robust nanoflow LC/MS is still a challenge in proteomics analysis. The Bruker CaptiveSpray source is a revolutionary ion source with a patented design that provides provides easy operation just as simple normal flow electrospray.

CaptiveSpray delivers nanospray sensitivity, resists plugging, and provides reproducible uninterrupted flow for even the most complex proteomics samples.

CaptiveSpray nanoBooster brings your MS to the next performance level and provides even higher flexibility.

Boost nanoflow sensitivity
• Push up ID rates
• Enabling Glycoanalysis
• Supercharging capability

CaptiveSpray provides a vortex gas that sweeps around the emitter spray tip to desolvate and to focus the Taylor cone into the MS inlet capillary. The vacuum seal to the MS ion guide draws all of the sample ions into the MS increasing the efficiency of sample transfer from the spray tip into the mass spectrometer. The direct connection to the inlet capillary eliminates the need for any source adjustment making the CaptiveSpray source truly Plug-and-Play.

CaptiveSpray Illustration

Structure elucidation

Tissue Proteomics for the Next Decade? Towards a Molecular Dimension in Histology

R Longuespee, M Fleron, C Pottier, F Quesada-Calvo, Marie-Alice Meuwis, et al.
OMICS A Journal of Integrative Biology 2014; 18(9)
http://dx.doi.org:/10.1089/omi.2014.0033

Currently, sampling methods, biochemical procedures, and MS instrumentations allow scientists to perform ‘‘in depth’’ analysis of the protein content of any type of tissue of interest. This article reviews the salient issues in proteomics analysis of tissues. We first outline technical and analytical considerations for sampling and biochemical processing of tissues and subsequently the instrumental possibilities for proteomics analysis such as shotgun proteomics in an anatomical context. Specific attention concerns formalin fixed and paraffin embedded (FFPE) tissues that are potential ‘‘gold mines’’ for histopathological investigations. In all, the matrix assisted laser desorption/ionization (MALDI) MS imaging, which allows for differential mapping of hundreds of compounds on a tissue section, is currently the most striking evidence of linkage and transition between ‘‘classical’’ and ‘‘molecular’’ histology. Tissue proteomics represents a veritable field of research and investment activity for modern biomarker discovery and development for the next decade.

A transcriptome-proteome integrated network identifies ERp57 as a hub that mediates bone metastasis

N Santana-Codina, R Carretero, R Sanz-Pamplona1, T Cabrera, et al.
The American Society for Biochemistry and Molecular Biology
MCP Apr 26, 2013; Manuscript M112.022772
E-mail: asierra@idibell.cat

Bone metastasis is the most common distant relapse in breast cancer. The identification of key proteins involved in the osteotropic phenotype would represent a major step toward the development of new prognostic markers and therapeutic improvements. The aim of this study was to characterize functional phenotypes that favor bone metastasis in human breast cancer.
We used the human breast cancer cell line MDA-MB-231 and its osteotropic BO2 subclone to identify crucial proteins in bone metastatic growth. We identified 31 proteins, 15 underexpressed and 16 overexpressed, in BO2 cells compared to parental cells. We employed a network-modeling approach in which these 31 candidate proteins were prioritized with respect to their potential in metastasis formation, based on the topology of the protein–protein interaction network and differential expression. The protein–protein interaction network provided a framework to study the functional relationships between biological molecules by attributing functions to genes whose functions had not been characterized.
The combination of expression profiles and protein interactions revealed an endoplasmic reticulum-thiol oxidoreductase, ERp57, functioning as a hub which retained 4 downregulated nodes involved in antigen presentation associated with the human major histocompatibility complex class I molecules, including HLA-A, HLA-B, HLA-E and HLA-F. Further analysis of the interaction network revealed an inverse correlation between ERp57 and vimentin, which influences cytoskeleton reorganization. Moreover, knockdown of ERp57 in BO2 cells confirmed its bone organ-specific prometastatic role. Altogether, ERp57 appears as a multifunctional chaperone that can regulate diverse biological processes to maintain the homeostasis of breast cancer cells and promote the development of bone metastasis.

Tandem-repeat protein domains across the tree of life

Kristin K. Jernigan and Seth R. Bordenstein
PeerJ 3:e732; 2015 http://dx.doi.org:/10.7717/peerj.732

Tandem-repeat protein domains, composed of repeated units of conserved stretches of 20–40 amino acids, are required for a wide array of biological functions. Despite their diverse and fundamental functions, there has been no comprehensive assessment of their taxonomic distribution, incidence, and associations with organismal lifestyle and phylogeny.
In this study, we assess for the first time the abundance of armadillo (ARM) and tetratricopeptide (TPR) repeat domains across all three domains in the tree of life and compare the results to our previous analysis on ankyrin (ANK) repeat domains in this journal. All eukaryotes and a majority of the bacterial and archaeal genomes analyzed have a minimum of one TPR and ARM repeat. In eukaryotes, the fraction of ARM-containing proteins is approximately double that of TPR and ANK-containing proteins, whereas bacteria and archaea are enriched in TPR-containing proteins relative to ARM- and ANK-containing proteins.
We show in bacteria that phylogenetic history, rather than lifestyle or pathogenicity, is a predictor of TPR repeat domain abundance, while neither phylogenetic history nor lifestyle predicts ARM repeat domain abundance. Surprisingly, pathogenic bacteria were not enriched in TPR-containing proteins, which have been associated within virulence factors in certain species. Taken together, this comparative analysis provides a newly appreciated view of the prevalence and diversity of multiple types of tandem-repeat protein domains across the tree of life.
A central finding of this analysis is that tandem repeat domain-containing proteins are prevalent not just in eukaryotes, but also in bacterial and archaeal species.

Detection of colorectal adenoma and cancer based on transthyretin and C3a-desArg serum levels

Anne-Kristin Fentz, Monika Sporl, Jorg Spangenberg, Heinz Joachim List, et al.
Proteomics Clin. Appl. 2007, 1, 536–544
http://dx.doi.org:/10.1002/prca.200600664

Colorectal cancer is the second leading cause of cancer death, and it develops from benign colorectal adenomas in over 95% of patients. Early detection of these cancer precursors by screening tests and their removal can potentially eradicate more than 95% of colorectal cancers before they develop.
To discover sensitive and specific biomarkers for improvement of pre-clinical diagnosis of colorectal adenoma and cancer, we analysed in two independent studies (n = 87 and n = 83 patients) serum samples from colorectal cancer (stage III), colorectal adenoma and control patients using SELDI-TOF-MS. Extensive statistical analysis was performed to establish homogeneous patient groups based on their clinical data.
Two biomarkers that were each able to distinguish control patients from either colorectal adenoma or colorectal cancer patients (p,0.001) were identified as transthyretin (pre-albumin) and C3adesArg by MS/MS and were further validated by antibody-based assays (radial immunodiffusion, ELISA). A combination of both proteins clearly indicated the presence of colorectal adenoma or carcinoma. Using a cut-off of >0.225 g/L for transthyretin and >1974 ng/mL for C3a-desArg, we found a sensitivity and specificity for colorectal adenoma of 96% and 70%, respectively.

The essential biology of the endoplasmic reticulum stress response for structural and computational biologists

Sadao Wakabayashi, Hiderou Yoshida
CSBJ Mar 2013; 6(7), e201303010 http://dx.doi.org/10.5936/csbj.201303010

The endoplasmic reticulum (ER) stress response is a cytoprotective mechanism that maintains homeostasis of the ER by upregulating the capacity of the ER in accordance with cellular demands. If the ER stress response cannot function correctly, because of reasons such as aging, genetic mutation or environmental stress, unfolded proteins accumulate in the ER and cause ER stress-induced apoptosis, resulting in the onset of folding diseases, including Alzheimer’s disease and diabetes mellitus. Although the mechanism of the ER stress response has been analyzed extensively by biochemists, cell biologists and molecular biologists, many aspects remain to be elucidated. For example, it is unclear how sensor molecules detect ER stress, or how cells choose the two opposite cell fates (survival or apoptosis) during the ER stress response. To resolve these critical issues, structural and computational approaches will be indispensable, although the mechanism of the ER stress response is complicated and difficult to understand holistically at a glance. Here, we provide a concise introduction to the mammalian ER stress response for structural and computational biologists.

Sequence co-evolution gives 3D contacts and structures of protein complexes

Thomas A Hopf, Charlotta P I Schärfe, João P G L M Rodrigues, et al.
eLife 2014;3:e03430 http://dx.doi.org:/10.7554/eLife.03430

Protein–protein interactions are fundamental to many biological processes. Experimental screens have identified tens of thousands of interactions, and structural biology has provided detailed functional insight for select 3D protein complexes. An alternative rich source of information about protein interactions is the evolutionary sequence record. Building on earlier work, we show that analysis of correlated evolutionary sequence changes across proteins identifies residues that are close in space with sufficient accuracy to determine the three-dimensional structure of the protein complexes. We evaluate prediction performance in blinded tests on 76 complexes of known 3D structure, predict protein–protein contacts in 32 complexes of unknown structure, and demonstrate how evolutionary couplings can be used to distinguish between interacting and non-interacting protein pairs in a large complex. With the current growth of sequences, we expect that the method can be generalized to genome-wide elucidation of protein–protein interaction networks and used for interaction predictions at residue resolution.
S-Glutathionylation of Cryptic Cysteines Enhances Titin Elasticity by Blocking Protein Folding

Jorge Alegre-Cebollada, P Kosuri, D Giganti, E Eckels, JA Rivas-Pardo, et al.
Cell, Mar 13, 2014; 156: 1235–1246. http://dx.doi.org/10.1016/j.cell.2014.01.056

The giant elastic protein titin is a determinant factor in how much blood fills the left ventricle during diastole and thus in the etiology of heart disease. Titin has been identified as a target of S-glutathionylation, an end product of the nitric-oxide-signaling cascade that increases cardiac muscle elasticity. However, it is unknown how S-glutathionylation may regulate the elasticity of titin and cardiac tissue.
Here, we show that mechanical unfolding of titin immunoglobulin (Ig) domains exposes buried cysteine residues, which then can be S-glutathionylated. S-glutathionylation of cryptic cysteines greatly decreases the mechanical stability of the parent Ig domain as well as its ability to fold. Both effects favor a more extensible state of titin. Furthermore, we demonstrate that S-glutathionylation of cryptic cysteines in titin mediates mechanochemical modulation of the elasticity of human cardiomyocytes.
We propose that posttranslational modification of cryptic residues is a general mechanism to regulate tissue elasticity.
Encounter complexes and dimensionality reduction in protein–protein association

Dima Kozakov, Keyong Li, David R Hall, Dmitri Beglov, Jiefu Zheng, et al.
eLife 2014;3:e01370 http://dx.doi.org:/10.7554/eLife.01370.001

An outstanding challenge has been to understand the mechanism whereby proteins associate. We report here the results of exhaustively sampling the conformational space in protein–protein association using a physics-based energy function. The agreement between experimental intermolecular paramagnetic relaxation enhancement (PRE) data and the PRE profiles calculated from the docked structures shows that the method captures both specific and non-specific encounter complexes. To explore the energy landscape in the vicinity of the native structure, the nonlinear manifold describing the relative orientation of two solid bodies is projected onto a Euclidean space in which the shape of low energy regions is studied by principal component analysis. Results show that the energy surface is canyon-like, with a smooth funnel within a two dimensional subspace capturing over 75% of the total motion. Thus, proteins tend to associate along preferred pathways, similar to sliding of a protein along DNA in the process of protein-DNA recognition.

Cardiovascular Proteomics: Evolution and Potential

Kent Arrell, Irina Neverova and Jennifer E. Van Eyk
Circ Res. 2001;88:763-773 http://dx.doi.org:/doi:/10.1161/hh0801.090193

The development of proteomics is a timely one for cardiovascular research. Analyses at the organ, subcellular, and molecular levels have revealed dynamic, complex, and subtle intracellular processes associated with heart and vascular disease. The power and flexibility of proteomic analyses, which facilitate protein separation, identification, and characterization, should hasten our understanding of these processes at the protein level. Properly applied, proteomics provides researchers with cellular protein “inventories” at specific moments in time, making it ideal for documenting protein modification due to a particular disease, condition, or treatment. This is accomplished through the establishment of species- and tissue-specific protein databases, providing a foundation for subsequent proteomic studies. Evolution of proteomic techniques has permitted more thorough investigation into molecular mechanisms underlying cardiovascular disease, facilitating identification not only of modified proteins but also of the nature of their modification. Continued development should lead to functional proteomic studies, in which identification of protein modification, in conjunction with functional data from established biochemical and physiological methods, has the ability to further our understanding of the interplay between proteome change and cardiovascular disease.

Advances in Proteomic Technologies and Its Contribution to the Field of Cancer

Mehdi Mesri

Advances in Medicine 2014, Article ID 238045, 25 pages http://dx.doi.org/10.1155/2014/238045

Systematic studies of the cancer genome have generated a wealth of knowledge in recent years. These studies have uncovered a number of new cancer genes not previously known to be causal targets in cancer. Genetic markers can be used to determine predisposition to tumor development, but molecularly targeted treatment strategies are not widely available for most cancers. Precision care plans still must be developed by understanding and implementing basic science research into clinical treatment. Proteomics is continuing to make major strides in the discovery of fundamental biological processes as well as more recent transition into an assay platform capable of measuring hundreds of proteins in any biological system. As such, proteomics can translate basic science discoveries into the clinical practice of precision medicine. The proteomic field has progressed at a fast rate over the past five years in technology, breadth and depth of applications in all areas of the bioscience. Some of the previously experimental technical approaches are considered the gold standard today, and the community is now trying to come to terms with the volume and complexity of the data generated. Here I describe contribution of proteomics in general and biological mass spectrometry in particular to cancer research, as well as related major technical and conceptual developments in the field.

Chemoproteomics reveals Toll-like receptor fatty acylation

Nicholas M Chesarino, Jocelyn C Hach, James L Chen, Balyn W Zaro, et al.
BMC Biology 2014, 12:91 http://www.biomedcentral.com/1741-7007/12/91

Background: Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells.
Results: A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands. Conclusions: This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. Spalmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.

Comparative Proteomics and Network Analysis Identify PKC Epsilon Underlying Long-Chain Fatty Acid Signaling

T Yonezawa, R Kurata, A Tajima, X Cui, H Maruta, H Nakaoka, K Nakajima and H Inokio
J Proteomics Bioinform 2014: 7:11 http://dx.doi.org/10.4172/jpb.1000337

Long-chain fatty acid possesses myriad roles in the biological function of the cells, not only as an energy substrate but also as substrates for cell membrane synthesis and as precursors for intracellular signaling molecules. However, little is known about the biological pathways that are stimulated by long-chain fatty acid. In order to identify the pathway of long-chain fatty acid, we performed 2-dimensional gel electrophoresis in the cells treated with or without oleate, and then analyzed 648 protein spots using PDQuest software and narrowed down 22 significant changing spots by statistical criterion. We also tried to determine these spots by MALDI-QIT-TOF-MS and SWISSPROT database query. We identified 11 proteins and predicted the biological network using available data sets from protein-protein interaction database. This prediction indicated that several protein kinase Cs (PKCs) underlie long chain fatty acid signaling. Indeed, oleate stimulated predicted PKC pathways. In expression array, oleate significantly up-regulated only PKC epsilon, but not other PKCs, in transcriptional levels. Collectively, our proteomics and network analysis implicates that PKC epsilon pathway plays an important role in long-chain fatty acid signaling.
Editorial: The art of proteomics translation

Translational Proteomics 2013; 1: 1–2 http://dx.doi.org/10.1016/j.trprot.2013.03.001

Over the years, the difficulties of transferring fundamental proteomics discoveries to clinical applications have caused a lot of frustration to proteomics researchers and clinicians alike, in both academia and industry. One of the reasons for this barrier is the lack of understanding between basic scientists and physicians: they have been trained using opposing concepts. Whilst the former want to control and understand all variables, the latter need rapid actions on patients, rather than absolute certainties. Both disciplines are difficult to con-dense into a single scientist and therefore interdisciplinary associations need to be fostered. Translational research has often been viewed as a two-way street: bedside to bench, and back to bedside. We should perhaps look at it as a roundabout, with the patient and his disease in the center, surrounded by a constant, iterative inter-play between basic, translational and clinical scientists, from both the public and private sectors. Proteomics research needs more than just a translation road bridge from discoveries to cures. Rather, it requires networks of road junctions to fill all the gaps and to allow cross-fertilization and synergies. Translational research and translational proteomics are more than just interesting concepts and hot keywords, they are supposed to improve the quality of people’s lives. With the launch of Translational Proteomics, we want to help the scientific and medical communities overcome the challenges on the long path from discovery to patient care. By focusing on connecting basic proteomics research to its ultimate clinical applications, the Journal will provide a space for publications detailing proteomics experiments, from early discovery to validation and the bedside.

Structural Basis of Diverse Membrane Target Recognitions by Ankyrins

C Wang, Z Wei, K Chen, F Ye, C Yu, V Bennett, and M Zhang
eLife 2014; http:dx.doi.org:/10.7554/eLife.04353

Ankyrin adaptors together with their spectrin partners coordinate diverse ion channels and cell adhesion molecules within plasma membrane domains and thereby promote physiological activities including fast signaling in the heart and nervous system. Ankyrins specifically bind to numerous membrane targets through their 24 ankyrin repeats (ANK repeats), although the mechanism for the facile and independent evolution of these interactions has not been resolved. Here we report the structures of ANK repeats in complex with an inhibitory segment from the C-terminal regulatory domain and with a sodium channel Nav1.2 peptide, respectively, showing that the extended, extremely conserved inner groove spanning the entire ANK repeat solenoid contains multiple target binding sites capable of accommodating target protein with very diverse sequences via combinatorial usage of these sites. These structures establish a framework for understanding the evolution of ankyrins’ membrane targets, with implications for other proteins containing extended ANK repeat domains.

Fusion of Protein Aggregates Facilitates Asymmetric Damage Segregation

Miguel Coelho, Steven J. Lade, Simon Alberti, Thilo Gross, Iva M. Tolic
PLOS Biology June 2014; 12(6):e1001886
http://dx.doi.org:/10.1371/journal.pbio.1001886

Asymmetric segregation of damaged proteins at cell division generates a cell that retains damage and a clean cell that supports population survival. In cells that divide asymmetrically, such as Saccharomyces cerevisiae, segregation of damaged proteins is achieved by retention and active transport. We have previously shown that in the symmetrically dividing Schizosaccharomyces pombe there is a transition between symmetric and asymmetric segregation of damaged proteins. Yet how this transition and generation of damage-free cells are achieved remained unknown. Here, by combining in vivo imaging of Hsp104-associated aggregates, a form of damage, with mathematical modeling, we find that fusion of protein aggregates facilitates asymmetric segregation. Our model predicts that, after stress, the increased number of aggregates fuse into a single large unit, which is inherited asymmetrically by one daughter cell, whereas the other one is born clean. We experimentally confirmed that fusion increases segregation asymmetry, for a range of stresses, and identified Hsp16 as a fusion factor. Our work shows that fusion of protein aggregates promotes the formation of damage-free cells. Fusion of cellular factors may represent a general mechanism for their asymmetric segregation at division.

Symmetric exchange of multi-protein building blocks between stationary focal adhesions and the cytosol

Jan-Erik Hoffmann, Y Fermin, R LO Stricker, K Ickstadt, E Zamir
eLife 2014;3:e02257. http://dx.doi.org:/10.7554/eLife.02257.001

How can the integrin adhesome get self-assembled locally, rapidly, and correctly as diverse cell-matrix adhesion sites? Here, we investigate this question by exploring the cytosolic state of integrin-adhesome components and their dynamic exchange between adhesion sites and cytosol. Using fluorescence cross-correlation spectroscopy (FCCS) and fluorescence recovery after photo-bleaching (FRAP) we found that the integrin adhesome is extensively pre-assembled already in the cytosol as multi-protein building blocks for adhesion sites. Stationary focal adhesions release symmetrically the same types of protein complexes that they recruit, thereby keeping the cytosolic pool of building blocks spatiotemporally uniform. We conclude a model in which multi-protein building blocks enable rapid and modular self-assembly of adhesion sites and symmetric exchange of these building blocks preserves their specifications and thus the assembly logic of the system.

Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress

Jie Wang, Kristeen A Pareja, Chris A Kaiser, Carolyn S Sevier
eLife 2014;3:e03496. http://dx.doi.org:/10.7554/eLife.03496

Oxidative protein folding in the endoplasmic reticulum (ER) has emerged as a potentially significant source of cellular reactive oxygen species (ROS). Recent studies suggest that levels of ROS generated as a byproduct of oxidative folding rival those produced by mitochondrial respiration. Mechanisms that protect cells against oxidant accumulation within the ER have begun to be elucidated yet many questions still remain regarding how cells prevent oxidant-induced damage from ER folding events. Here we report a new role for a central well-characterized player in ER homeostasis as a direct sensor of ER redox imbalance. Specifically we show that a conserved cysteine in the lumenal chaperone BiP is susceptible to oxidation by peroxide, and we demonstrate that oxidation of this conserved cysteine disrupts BiP’s ATPase cycle. We propose that alteration of BiP activity upon oxidation helps cells cope with disruption to oxidative folding within the ER during oxidative stress.

Current perspectives on cadherin-cytoskeleton interactions and dynamics

Xuan Liang, Guillermo A Gomez, Alpha S Yap
Cell Health and Cytoskeleton 2015:7 11–24
http://dx.doi.org/10.2147/CHC.S76107

Cells are linked together dynamically by adhesion molecules, such as the classical cadherins. E-cadherin, which mediates epithelial cell–cell interactions, plays fundamental roles in tissue organization and is often perturbed in diseases such as cancer. It has long been recognized that the biology of E-cadherin arises from cooperation between adhesion and the actin cytoskeleton. A major feature is the generation of contractile forces at junctions, yielding patterns of tension that contribute to tissue integrity and patterning. Here we discuss recent developments in understanding how cadherin junctions integrate signaling and cytoskeletal dynamics to sense and generate force.

N-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct β-catenin- and γ-catenin-containing AJs

Basem T Jamal, M Nita-Lazar, Z Gao, B Amin, J Walker, MA Kukuruzinska
Cell Health and Cytoskeleton 2009:1 67–80

N-glycosylation of E-cadherin has been shown to inhibit cell–cell adhesion. Specifically, our recent studies have provided evidence that the reduction of E-cadherin N-glycosylation promoted the recruitment of stabilizing components, vinculin and serine/threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs) and enhanced the association of AJs with the actin cytoskeleton. Here, we examined the details of how N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions. Using the hypoglycosylated E-cadherin variant, V13, we show that V13/β-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein. This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that increased association of PP2A with V13-containing AJs promoted their tethering to microtubules. On the other hand, V13/γ-catenin complexes associated more with vinculin, suggesting that they mediated the interaction of AJs with the actin cytoskeleton. N-glycosylation driven changes in the molecular organization of AJs were physiologically significant because transfection of V13 into A253 cancer cells, lacking both mature AJs and tight junctions (TJs), promoted the formation of stable AJs and enhanced the function of TJs to a greater extent than wild-type E-cadherin. These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through the assembly of distinct β-catenin- and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components.

Mapping the dynamics of force transduction at cell-cell 4 junctions of epithelial clusters

Mei Rosa Ng, Achim Besser, Joan S. Brugge, Gaudenz Danuser
eLife 2014;10.7554/eLife.03282
http://dx.doi.org/10.7554/eLife.03282

Force transduction at cell-cell adhesions regulates tissue development, maintenance and adaptation. We developed computational and experimental approaches to quantify, with both subcellular and multi-cellular resolution, the dynamics of force transmission in cell clusters. Applying this technology to spontaneously-forming adherent epithelial cell clusters, we found that basal force fluctuations were coupled to E-cadherin localization at the level of individual cell-cell junctions. At the multi-cellular scale, cell-cell force exchange depended on the cell position within a cluster, and was adaptive to reconfigurations due to cell divisions or positional rearrangements. Importantly, force transmission through a cell required coordinated modulation of cell-matrix adhesion and actomyosin contractility in the cell and its neighbors. These data provide insights into mechanisms that could control mechanical stress homeostasis in dynamic epithelial tissues, and highlight our methods as a resource for the study of mechanotransduction in cell-cell adhesions.

G-protein-coupled receptor signaling and polarized actin dynamics drive cell-in-cell invasion

Vladimir Purvanov, Manuel Holst, Jameel Khan, Christian Baarlink, Robert Grosse
eLife 2014;3:e02786. http://dx.doi.org:/10.7554/eLife.02786

Homotypic or entotic cell-in-cell invasion is an integrin-independent process observed in carcinoma cells exposed during conditions of low adhesion such as in exudates of malignant disease. Although active cell-in-cell invasion depends on RhoA and actin, the precise mechanism as well as the underlying actin structures and assembly factors driving the process are unknown. Furthermore, whether specific cell surface receptors trigger entotic invasion in a signal-dependent fashion has not been investigated. In this study, we identify the G-protein-coupled LPA receptor 2 (LPAR2) as a signal transducer specifically required for the actively invading cell during entosis. We find that G12/13 and PDZ-RhoGEF are required for entotic invasion, which is driven by blebbing and a uropod-like actin structure at the rear of the invading cell. Finally, we provide evidence for an involvement of the RhoA-regulated formin Dia1 for entosis downstream of LPAR2. Thus, we delineate a signaling process that regulates actin dynamics during cell-in-cell invasion.

Cytoskeletal Basis of Ion Channel Function in Cardiac Muscle

Matteo Vatta, and Georgine Faulkner
Future Cardiol. 2006 Jul 1; 2(4): 467–476. http://dx.doi.org:/10.2217/14796678.2.4.467

The heart is a force-generating organ that responds to self-generated electrical stimuli from specialized cardiomyocytes. This function is modulated by sympathetic and parasympathetic activity.

In order to contract and accommodate the repetitive morphological changes induced by the cardiac cycle, cardiomyocytes depend on their highly evolved and specialized cytoskeletal apparatus. Defects in components of the cytoskeleton, in the long term, affect the ability of the cell to compensate at both functional and structural levels. In addition to the structural remodeling, the myocardium becomes increasingly susceptible to altered electrical activity leading to arrhythmogenesis. The development of arrhythmias secondary to structural remodeling defects has been noted, although the detailed molecular mechanisms are still elusive. Here I will review the current knowledge of the molecular and functional relationships between the cytoskeleton and ion channels and, I will discuss the future impact of new data on molecular cardiology research and clinical practice.

Structure and transport mechanism of the sodium/proton 2 antiporter MjNhaP1

Cristina Paulino, D Wöhlert , E Kapotova, Ö Yildiz & W Kühlbrandt
eLife 2014; http://dx.doi.org/10.7554/eLife.03583

Sodium/proton antiporters are essential for sodium and pH homeostasis and play a major role in human health and disease. We determined the structures of the archaeal sodium/proton antiporter MjNhaP1 in two complementary states. The inward-open state was obtained by x-ray crystallography in the presence of sodium at pH8, where the transporter is highly active. The outward-open state was obtained by electron crystallography without sodium at pH4, where MjNhaP1 is inactive. Comparison of both structures reveals a 7° tilt of the 6-helix bundle. Na+ uptake measurements indicate non-cooperative transport with an activity maximum at pH7.5. We conclude that binding of a Na+ ion from the outside induces helix movements that close the extracellular cavity, open the cytoplasmic funnel, and result in a ~5 Å vertical relocation of the ion binding site to release the substrate ion into the cytoplasm.

Integrated control of transporter endocytosis and recycling by the arrestin-related protein Rod1 and the ubiquitin ligase Rsp5

Michel Becuwe, Sébastien Léon
eLife 2014; http://dx.doi.org/10.7554/eLife.03307

After endocytosis, membrane proteins can recycle to the cell membrane or be degraded in lysosomes. Cargo ubiquitylation favors their lysosomal targeting and can be regulated by external signals, but the mechanism is ill-defined. Here, we studied the post-endocytic trafficking of Jen1, a yeast monocarboxylate transporter, using microfluidics-assisted live cell imaging. We show that the ubiquitin ligase Rsp5 and the glucose-regulated arrestin related (ART) protein Rod1, involved in the glucose-induced internalization of Jen1, are also required for the post-endocytic sorting of Jen1 to the yeast lysosome. This new step takes place at the trans-Golgi network (TGN), where Rod1 localizes dynamically upon triggering endocytosis. Indeed, transporter trafficking to the TGN after internalization is required for their degradation. Glucose removal promotes Rod1 relocalization to the cytosol and Jen1 deubiquitylation, allowing transporter recycling when the signal is only transient. Therefore, nutrient availability regulates transporter fate through the localization of the ART/Rsp5 ubiquitylation complex at the TGN.

McKenney, W Huynh, ME. Tanenbaum, G Bhabha, and RD. Vale
Science Express 19 June 2014 /10.1126/science.1254198
http://www.sciencemag.org/content/early/recent/10.1126/science.1254198

Cytoplasmic dynein is a molecular motor that transports a large variety of cargoes (e.g., organelles, mRNAs, and viruses) along microtubules over long intracellular distances. The dynactin protein complex is important for dynein activity in vivo, but its precise role has been unclear. Here, we found that purified mammalian dynein did not move processively on microtubules in vitro. However, when dynein formed a complex with dynactin and one of four different cargo-specific adapter proteins, the motor became ultra-processive, moving for distances similar to those of native cargoes in living cells. Thus, we propose that dynein is largely inactive in the cytoplasm and that a variety of adapter proteins activate processive motility by linking dynactin to dynein only when the motor is bound to its proper cargo.

Removal of surface charge–charge interactions from ubiquitin leaves the protein folded and very stable

Vakhtang V. Loladze And George I. Makhatadze
Protein Science (2002), 11:174–177
http://www.proteinscience.org/cgi/doi/10.1101/ps.29902.

The contribution of solvent-exposed charged residues to protein stability was evaluated using ubiquitin as a model protein. We combined site-directed mutagenesis and specific chemical modifications to first replace all Arg residues with Lys, followed by carbomylation of Lys- amino groups. Under the conditions in which all carboxylic groups are protonated (at pH 2), the chemically modified protein is folded and very stable (dG= 18 kJ/mol). These results indicate that surface charge–charge interactions are not an essential fundamental force for protein folding and stability.

Phase Transitions of Multivalent Proteins Can Promote Clustering of Membrane Receptors

Sudeep Banjade and Michael K. Rosen
eLife 2014; http://dx.doi.org/10.7554/eLife.04123

Clustering of proteins into micrometer-sized structures at membranes is observed in many signaling pathways. Most models of clustering are specific to particular systems, and relationships between physical properties of the clusters and their molecular components are not well understood. We report biochemical reconstitution on supported lipid bilayers of protein clusters containing the adhesion receptor Nephrin, and its cytoplasmic partners, Nck and N-WASP. With Nephrin attached to the bilayer, multivalent interactions enable these proteins to polymerize on the membrane surface and undergo two-dimensional phase separation, producing micrometer-sized clusters. Dynamics and thermodynamics of the clusters are modulated by the valencies and affinities of the interacting species. In the presence of the Arp2/3 complex, the clusters assemble actin filaments, suggesting that clustering of regulatory factors could promote local actin assembly at membranes. Interactions between multivalent proteins could be a general mechanism for cytoplasmic adaptor proteins to organize membrane receptors into micrometer-scale signaling zones.

The quantitative architecture of centromeric chromatin

Dani L Bodor, João F Mata, Mikhail Sergeev, Ana Filipa David, et al.
eLife 2014;3:e02137. http://dx.doi.org:/10.7554/eLife.02137

The centromere, responsible for chromosome segregation during mitosis, is epigenetically defined by CENP-A containing chromatin. The amount of centromeric CENP-A has direct implications for both the architecture and epigenetic inheritance of centromeres. Using complementary strategies, we determined that typical human centromeres contain ∼400 molecules of CENP-A, which is controlled by a mass-action mechanism. This number, despite representing only ∼4% of all centromeric nucleosomes, forms a ∼50-fold enrichment to the overall genome. In addition, although pre-assembled CENP-A is randomly segregated during cell division, this amount of CENP-A is sufficient to prevent stochastic loss of centromere function and identity. Finally, we produced a statistical map of CENP-A occupancy at a human neocentromere and identified nucleosome positions that feature CENP-A in a majority of cells. In summary, we present a quantitative view of the centromere that provides a mechanistic framework for both robust epigenetic inheritance of centromeres and the paucity of neocentromere formation.

Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion

Jiajie Diao, Patricia Grob, Daniel J Cipriano, Minjoung Kyoung
eLife 2012;1:e00109. http://dx.doi.org:/10.7554/eLife.00109

The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle–vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca2+-injection at 250–500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca2+-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca2+-triggered immediate fusion started from a membrane–membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca2+-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca2+-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.

Cytoskeleton, cytoskeletal interactions, and vascular endothelial function

Jingli Wang, Michael E Widlansky
Cell Health and Cytoskeleton 2012:4 119–127
http://dx.doi.org/10.2147/CHC.S21823

Far from being inert, the vascular endothelium is a critical regulator of vascular function. While the endothelium participates in autocrine, paracrine, and endocrine signaling, it also transduces mechanical signals from the cell surface involving key cell structural elements. In this review, we discuss the structure of the vascular endothelium and its relationship to traditional cardiovascular risk factors and clinical cardiovascular events. Further, we review the emerging evidence that cell structural elements, including the glycocalyx, intercellular junctions, and cytoskeleton elements, help the endothelium to communicate with its environment to regulate vascular function, including vessel permeability and signal transduction via nitric oxide bioavailability. Further work is necessary to better delineate the regulatory relationships between known key regulators of vascular function and endothelial cell structural elements.

Cellular prion protein is required for neuritogenesis: fine-tuning of multiple signaling pathways involved in focal adhesions and actin cytoskeleton dynamics

Aurélie Alleaume-Butaux, C Dakowski, M Pietri, S Mouillet-Richard, et al.
Cell Health and Cytoskeleton 2013:5 1–12
http://dx.doi.org/10.2147/CHC.S28081

Neuritogenesis is a dynamic phenomenon associated with neuronal differentiation that allows a rather spherical neuronal stem cell to develop dendrites and axon, a prerequisite for the integration and transmission of signals. The acquisition of neuronal polarity occurs in three steps: (1) neurite sprouting, which consists of the formation of buds emerging from the postmitotic neuronal soma; (2) neurite outgrowth, which represents the conversion of buds into neurites, their elongation and evolution into axon or dendrites; and (3) the stability and plasticity of neuronal polarity. In neuronal stem cells, remodeling and activation of focal adhesions (FAs) associated with deep modifications of the actin cytoskeleton is a prerequisite for neurite sprouting and subsequent neurite outgrowth. A multiple set of growth factors and interactors located in the extracellular matrix and the plasma membrane orchestrate neuritogenesis by acting on intracellular signaling effectors, notably small G proteins such as RhoA, Rac, and Cdc42, which are involved in actin turnover and the dynamics of FAs. The cellular prion protein (PrPC), a glycosylphosphatidylinositol (GPI)-anchored membrane protein mainly known for its role in a group of fatal neurodegenerative diseases, has emerged as a central player in neuritogenesis. Here, we review the contribution of PrPC to neuronal polarization and detail the current knowledge on the signaling pathways fine-tuned by PrPC to promote neurite sprouting, outgrowth, and maintenance. We emphasize that PrPC-dependent neurite sprouting is a process in which PrPC governs the dynamics of FAs and the actin cytoskeleton via β1 integrin signaling. The presence of PrPC is necessary to render neuronal stem cells competent to respond to neuronal inducers and to develop neurites. In differentiating neurons, PrPC exerts a facilitator role towards neurite elongation. This function relies on the interaction of PrPC with a set of diverse partners such as elements of the extracellular matrix, plasma membrane receptors, adhesion molecules, and soluble factors that control actin cytoskeleton turnover through Rho-GTPase signaling. Once neurons have reached their terminal stage of differentiation and acquired their polarized morphology, PrPC also takes part in the maintenance of neurites. By acting on tissue nonspecific alkaline phosphatase, or matrix metalloproteinase type 9, PrPC stabilizes interactions between neurites and the extracellular matrix.

Broader implications: biological and clinical significance of microtubule acetylation

Sharon M Rymut, Thomas J Kelley
Cell Health and Cytoskeleton 2015:7 71–82
http://dx.doi.org/10.2147/CHC.S77040

Microtubule acetylation is a key posttranslational modification that enhances organelle transport, drives cell signaling, and regulates cell cycle regulation. The optimal level of microtubule acetylation is regulated by the acetyltransferase alpha-tubulin-N-acetyltransferase 1and two deacetylases, histone deacetylase 6 and sirtuin-2. Alterations in microtubule acetylation levels have been associated with the pathophysiology of a number of diseases, including various forms of neurodegenerative conditions, cancer, and even cystic fibrosis. In this review, we will highlight the biological and clinical significance of microtubule acetylation and the potential of targeting this pathway for therapeutics.

Inositol-1,4,5-trisphosphate 1 (IP3)-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake

Deak, S. Blass, M. J. Khan, L. N. Groschner, M. Waldeck-Weiermair, et al.
Journal of Cell Science 2014 advanced print

Mitochondria contribute to cell signaling by controlling store-operated Ca2+ entry (SOCE). SOCE is activated by Ca2+ release from the endoplasmic reticulum (ER), whereupon the stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER-plasma membrane junctions, and opens plasma membrane Ca2+ channels. Mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study we used a shRNA approach to investigate the direct involvement of mitochondrial Ca2+ buffering in SOCE. We demonstrate that knock-down of two proteins that are essential for mitochondrial Ca2+ uptake, either the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca2+-buffering or ER Ca2+ depletion by sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca2+ uptake. However, MCU-dependent mitochondrial sequestration of Ca2+ entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus specific contribution of mitochondrial Ca2+ uptake to the SOCE machinery likely by shaping cytosolic Ca2+ micro-domains.

Role of forkhead box protein A3 in age-associated metabolic decline

Xinran Ma, Lingyan Xu, Oksana Gavrilov, and Elisabetta Mueller
PNAS | September 30, 2014 | vol. 111 | no. 39 | 14289–14294
www.pnas.org/cgi/doi/10.1073/pnas.1407640111

Aging is associated with increased adiposity and diminished thermogenesis, but the critical transcription factors influencing these metabolic changes late in life are poorly understood. We recently demonstrated that the winged helix factor forkhead box protein A3 (Foxa3) regulates the expansion of visceral adipose tissue in high-fat diet regimens; however, whether Foxa3 also contributes to the increase in adiposity and the decrease in brown fat activity observed during the normal aging process is currently unknown.
Here we report that during aging, levels of Foxa3 are significantlyand selectively up-regulated in brown and inguinal white fat depots, and that midage Foxa3-null mice have increased white fat browning and thermogenic capacity, decreased adipose tissue expansion, improved insulin sensitivity, and increased longevity. Foxa3 gain-of-function and loss-of-function studies in inguinal adipose depots demonstrated a cell-autonomous function for Foxa3 in white fat tissue browning. Furthermore, our analysis revealed that the mechanisms of Foxa3 modulation of brown fat gene programs involve the suppression of peroxisome proliferator activated receptor γ coactivtor 1 α (PGC1α) levels through interference with cAMP responsive element binding protein 1-mediated transcriptional regulation of the PGC1α promoter. Overall, our data demonstrate a role for Foxa3 in energy expenditure and in age-associated metabolic disorders.

Prediction of enzyme function by combining sequence similarity and protein interactions

Jordi Espadaler, Narayanan Eswa, Enrique Querol, Francesc X Avilés, et al.
BMC Bioinformatics 2008, 9:249 http://dx.doi.org:/10.1186/1471-2105-9-249

Background: A number of studies have used protein interaction data alone for protein function prediction. Here, we introduce a computational approach for annotation of enzymes, based on the observation that similar protein sequences are more likely to perform the same function if they share similar interacting partners.
Results: The method has been tested against the PSI-BLAST program using a set of 3,890 protein sequences from which interaction data was available. For protein sequences that align with at least 40% sequence identity to a known enzyme, the specificity of our method in predicting the first three EC digits increased from 80% to 90% at 80% coverage when compared to PSI-BLAST.
Conclusion: Our method can also be used in proteins for which homologous sequences with known interacting partners can be detected. Thus, our method could increase 10% the specificity of genome-wide enzyme predictions based on sequence matching by PSI-BLAST alone.

Plasma Transthyretin Indicates the Direction of both Nitrogen Balance and Retinoid Status in Health and Disease

Ingenbleek Yves and Bienvenu Jacques
The Open Clinical Chemistry Journal, 2008, 1, 1-12

Whatever the nutritional status and the disease condition, the actual transthyretin (TTR) plasma level is determined by opposing influences between anabolic and catabolic alterations. Rising TTR values indicate that synthetic processes prevail over tissue breakdown with a nitrogen balance (NB) turning positive as a result of efficient nutritional support and / or anti-inflammatory therapy. Declining TTR values point to the failure of sustaining NB as an effect of maladjusted dietetic management and / or further worsening of the morbid condition. Serial measurement of TTR thus appears as a dynamic index defining the direction of NB in acute and chronic disorders, serving as a guide to alert the physician on the validity of his therapeutic strategy. The level of TTR production by the liver also works as a limiting factor for the cellular bioavailability of retinol and retinoid derivatives which play major roles in the brain ageing process. Optimal protein nutritional status, as assessed by TTR values within the normal range, prevents the occurrence of vascular and cerebral damages while maintaining the retinoid-mediated memory, cognitive and behavioral activities of elderly persons.

Prof. Dr. Volker Haucke
Institut für Chemie-Biochemie
Takustrasse 6
http://userpage.chemie.fu-berlin.de/biochemie/aghaucke/teaching.html

Eukaryotic cells contain three major types of cytoskeletal filaments

major types of cytoskeletal filaments

Intermediate Filaments support the nuclear membrane and connect cells at cell junctions

microtubules (MTs; green) radiate from MTOCs (yellow) towards the cell periphery

Actin polymerization in vitro reveals a critical dependence of filament assembly on G-actin concentration via a 3-step nucleation mechanism

Binding-proteins and receptors

Motor, visual and emotional deficits in mice after closed-head mild traumatic brain injury are alleviated by the novel CB2 inverse agonist SMM-189
Reiner, A., Heldt, S.A., Presley, C.S., (…), Gurley, S.N., Moore, B.M.
2015 International Journal of Molecular Sciences 16 (1), pp. 758-787

We have developed a focal blast model of closed-head mild traumatic brain injury (TBI) in mice. As true for individuals that have experienced mild TBI, mice subjected to 50-60 psi blast show motor, visual and emotional deficits, diffuse axonal injury and microglial activation, but no overt neuron
loss. Because microglial activation can worsen brain damage after a concussive event and because microglia can be
modulated by their cannabinoid type 2 receptors (CB2), we evaluated the effectiveness of the novel CB2 receptor inverse agonist SMM-189 in altering microglial activation and mitigating deficits after mild TBI. In vitro analysis indicated that SMM-189 converted human microglia from the pro-inflammatory M1 phenotype to the pro-healing M2 phenotype. Studies in mice showed that daily administration of SMM-189 for two weeks beginning shortly after blast greatly reduced the motor, visual, and emotional deficits otherwise evident after 50-60 psi blasts, and prevented brain injury that may contribute to these deficits. Our results suggest that treatment with the CB2 inverse agonist SMM-189 after a mild TBI event can reduce its adverse consequences by beneficially modulating microglial activation. These
findings recommend further evaluation of CB2 inverse agonists as a novel therapeutic approach for treating mild TBI.

The novel small leucine-rich protein chondroadherin-like (CHADL) is expressed in cartilage and modulates chondrocyte differentiation
Tillgren, V., Ho, J.C.S., Önnerfjord, P., Kalamajski, S.
2015 Journal of Biological Chemistry 290 (2), pp. 918-925

The constitution and biophysical properties of extracellular matrices can dramatically influence cellular phenotype during development, homeostasis, or pathogenesis. These effects can be signaled through a differentially regulated assembly of collagen fibrils, orchestrated by a family of collagen-associated small leucine-rich proteins (SLRPs). In this report, we describe the tissue-specific expression and function of a previously uncharacterized SLRP, chondroadherin-like (CHADL). We developed antibodies against CHADL and, by immunohistochemistry, detected CHADL expression mainly in skeletal tissues, particularly in fetal cartilage and in the pericellular space of adult chondrocytes. In situ hybridizations and immunoblots on tissue lysates confirmed this tissue-specific expression pattern. Recombinant CHADL bound collagen in cell culture and inhibited in vitro collagen fibrillogenesis. After Chadl shRNA knockdown, chondrogenic ATDC5 cells increased their differentiation, indicated by increased transcript levels of Sox9, Ihh, Col2a1, and Col10a1. The knockdown increased collagen II and aggrecan deposition in the cell layers.

Microarray analysis of the knockdown samples suggested collagen receptor-related changes, although other upstream effects could not be excluded. Together, our data indicate that the novel SLRP CHADL is expressed in cartilaginous tissues, influences collagen fibrillogenesis, and modulates chondrocyte differentiation. CHADL appears to have a negative regulatory role, possibly ensuring the formation of a stable extracellular matrix.

P53 protein-mediated Up-regulation of MAP kinase phosphatase 3 (MKP-3) contributes to the establishment of the cellular senescent phenotype through dephosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2)
Zhang, H., Chi, Y., Gao, K., Zhang, X., Yao, J.
2015 Source of the DocumentJournal of Biological Chemistry 290 (2), pp. 1129-1140

Growth arrest is one of the essential features of cellular senescence. At present, the precise mechanisms responsible for the establishment of the senescence-associated arrested phenotype are still incompletely understood. Given that ERK1/2 is one of the major kinases controlling cell growth and proliferation, we examined the possible implication of ERK1/2. Exposure of normal rat epithelial cells to etoposide caused cellular senescence, as manifested by enlarged cell size, a flattened cell body, reduced cell proliferation, enhanced ?-galactosidase activity, and elevated p53 and p21. Senescent cells displayed a blunted response to growth factor-induced cell proliferation, which was preceded by impaired ERK1/2 activation. Further analysis revealed that senescent cells expressed a significantly higher level of mitogenactivated protein phosphatase 3 (MKP-3, a cytosolic ERK1/2-targeted phosphatase), which was suppressed by blocking the transcriptional activity of the tumor suppressor p53 with pifithrin-?. Inhibition of MKP-3 activity with a specific inhibitor or siRNA enhanced basal ERK1/2 phosphorylation and promoted cell proliferation. Apart from its role in growth arrest, impairment of ERK1/2 also contributed to the resistance of senescent cells to oxidant-elicited cell injury. These results therefore indicate that p53-mediated up-regulation of MKP-3 contributes to the establishment of the senescent cellular phenotype through dephosphorylating ERK1/2. Impairment of ERK1/2 activation could be an important mechanism by which p53 controls cellular senescence.

Dynamics and interaction of Interleukin-4 receptor subunits in living cells
Gandhi, H., Worch, R., Kurgonaite, K., (…), Bökel, C., Weidemann, T.
2015 Biophysical Journal 107 (11), pp. 2515-2527

It has long been established that dimerization of Interleukin-4 receptor (IL-4R) subunits is a pivotal step for JAK/STAT signal transduction. However, ligand-induced complex formation at the surface of living cells has been challenging to observe. Here we report an experimental assay employing trisNTA dyes for orthogonal, external labeling of eGFP-tagged receptor constructs that allows the quantification of receptor heterodimerization by dual-color fluorescence cross-correlation spectroscopy. Fluorescence cross-correlation spectroscopy analysis at the plasma membrane shows that IL-4R subunit dimerization is indeed a strictly ligand-induced process.

Under conditions of saturating cytokine occupancy, we determined intramembrane dissociation constants (Kd,2D) of 180 and 480 receptors per ?m2 for the type-2 complexes IL-4:IL-4R?/IL-13R?1 and IL-13:IL-13R?1/IL-4R?, respectively. For the lower affinity type-1 complex IL-4:IL-4R?/IL-2R?, we estimated a Kd,2D of ?1000 receptors per ?m2. The receptor densities required for effective dimerization thus exceed the typical, average expression levels by several orders of magnitude. In addition, we find that all three receptor subunits accumulate rapidly within a subpopulation of early sorting and recycling endosomes stably anchored just beneath the plasma membrane (cortical endosomes, CEs). The receptors, as well as labeled IL-4 and trisNTA ligands are specifically trafficked into CEs by a constitutive internalization mechanism. This may compensate for the inherent weak affinities that govern ligand-induced receptor dimerization at the plasma membrane. Consistently, activated receptors are also concentrated at the CEs. Our observations thus suggest that receptor trafficking may play an important role for the regulation of IL-4R-mediated JAK/STAT signaling.

Role of mitochondria in nonalcoholic fatty liver disease
Nassir, F., Ibdah, J.A.
2015 International Journal of Molecular Sciences 15 (5), pp. 8713-8742

Nonalcoholic fatty liver disease (NAFLD) affects about 30% of the general population in the United States and includes a spectrum of disease that includes simple steatosis, non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Significant insight has been gained into our understanding of the pathogenesis of NALFD; however the key metabolic aberrations underlying lipid accumulation in hepatocytes and the progression of NAFLD remain to be elucidated. Accumulating and emerging evidence indicate that hepatic mitochondria play a critical role in the development and pathogenesis of steatosis and NAFLD. Here, we review studies that document a link between the pathogenesis of NAFLD and hepatic mitochondrial dysfunction with particular focus on new insights into the role of impaired fatty acid oxidation, the transcription factor peroxisome proliferator-activated receptor-? coactivator-1? (PGC-1?), and sirtuins in development and progression of NAFLD.

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The Colors of Life Function

Posted in Amino acids, Cell Biology, Curation, Discovery process, Evolutionary cognition, Experimental validation, Explanatory, Historical relevance, Metabolism, Metabolomics, Proteins, Proteomics, Signaling, tagged Amino acids, catalysts, conformation, energy metabolism, fluorescence correlation spectroscopy (FCS), ligand-binding, metalloenzymes, Near-Infrared Spectroscopy, oxidation, Proteins, Raman spectroscopy on January 20, 2015| Leave a Comment »

The Colors of Life Function

Writer and Curator: Larry H. Bernstein, MD, FCAP

2.5.1 Type 1 Copper Proteins

The Cu(II) state of this category has an intense blue color due to a thiolate ligand
to Cu(II) charge transfer, and unusual EPR properties arising from the asymmetrical
Cu site (distorted trigonal-pyramidal). The proteins all have a low molecular
mass and have, so far, rather arbitrarily been divided into sub-groups, such as
azurins, plastocyanins, pseudoazurins, amicyanins and various other blue
proteins. Of these the azurins, amicyanins, pseudo-azurins and plastocyanins
apparently have similar copper coordination by two histidine, one cysteine and
one methionine residue. Where the function of Type I copper proteins is known,
it is invariably electron transfer. As yet the names for these proteins are all trivial
and are often derived from source, function or color. The different classes are
usually discerned on the basis of their primary and tertiary structure.

The first bacterial blue proteins to be described were called azurins. Rusticyanin is
another example of a bacterial protein. It has unusual properties with a reduction
potential of 680 mV, and is functional at pH 2. The azurins have well-defined electron
-transfer functions.

The so-called pseudo-azurins differ from the azurins in the N-terminal amino acid
sequence and the optical spectra, which resemble those of plastocyanins.

The blue proteins known as plastocyanins occur in plants, blue-green and green
algae. Their electron transfer role is well defined, i.e. from the bc1 complex
(EC 1.10.2.2) to the photooxidized P-700.

Amicyanins are electron carriers between methylamine dehydrogenase and
cytochrome c, with a characteristic amino acid sequence.

Of the remaining blue proteins stellacyanin is a well- known example. Umecyanin,
plantacyanin and mavicyanin are also considered to belong to this group.
Although these proteins undergo redox reactions in vitro, their true biological
function remains unknown. Most of these proteins exhibit an unusual EPR signal
in which the copper hyperfine splitting pattern is poorly resolved. There is good
evidence that at least for stellacyanin, methionine does not function as a ligand
for copper.

2.5.2 Type 2 Copper Proteins

The copper centres in these proteins are spectroscopically consistent with square
planar or pyramidal coordination, containing oxygen and/or nitrogen ligation.
The Cu(II) is EPR active, with a ‘normal’ signal. There is no intense blue color.
This group includes the copper/zinc superoxide dismutase (EC 1.15.1.1),
dopamine b-monooxygenase (EC 1.14.17.1), galactose oxidase (EC 1.1.3.9)
and the various copper-containing amine oxidases. Some members of this last
group may also contain an organic prosthetic group, such as PQQ
(see section 10), or a modified amino-acid residue.

2.5.3 Type 3 Copper Proteins

In this group a pair of copper atoms comprise a dinuclear centre, with no EPR
activity as for single Cu’s. The best known example of an enzyme containing a
single Type 3 centre is tyrosinase (catechol oxidase, EC 1.10.3.1). This protein
contains a metal center which is a structural analogue of the dinuclear copper
center in hemocyanin (ref 31).

2.5.4 Multi-Copper Oxidases

In addition to the above, there are several proteins with catalytic activity that
contain Types 1, 2 and 3 centres in various stoichiometric ratios. These
include L-ascorbate oxidase (EC 1.10.3.3), laccase (EC 1.10.3.2) and
ceruloplasmin (ferro-oxidase, EC 1.16.3.1), the latter two having aromatic diamine
and diphenol oxidase activity. There is growing evidence that in these proteins
the Type 2 and Type 3 copper centres are juxtaposed. Recently it has been
shown that in L-ascorbate oxidase, a trinuclear copper site is present, consisting
of a type 3 copper site, very close (3.9 Å) and possibly bridged to a type 2 copper
site (ref 32). There is a view that ceruloplasmin functions as a ferro-oxidase
and the Fe(III) produced in this reaction can then oxidize the same substrates
as laccase.

2.5.5 Copper Centres in Cytochrome Oxidase

There are two copper centres that appear to be unique. Both are present in
cytochrome-c oxidase (EC 1.9.3.1). The first appears to be an isolated metal ion
and has been referred to as Cud and CuA. The second appears to be part
of a dinuclear centre with cytochrome a3. It has been referred to as Cuu,
Cua3 and CuB. At the moment the ascriptions CuA and CuB are most frequently
used; however, the recent discovery (ref 33) of a cytochrome oxidase in which
cytochrome a has been replaced by cytochrome b, leads to the recommendation
that CuB shall be referred to as Cua3.

There is a striking similarity between two of the Cu centres of N2O reductase
and CuA (ref 34, 35).

2.5.6 Molybdenum enzymes (general)

Molybdenum enzymes contain molybdenum at the catalytic center responsible
for reaction with substrate. They may be divided into those that contain
the iron-molybdenum cofactor and those that contain the pterin-molybdenum
cofactor.

2.5.7 Additional centers

If a molybdenum enzyme contains flavin, it may be called either a molybdenum
flavoprotein or a flavomolybdenum protein, as indicated above. Other centers
should be treated similarly, e.g. an iron-sulfur molybdenum protein.

2.5.8 Molybdenum enzymes containing the iron-molybdenum cofactor

The only enzymes at present known to belong to this group are the nitrogenases
(EC 1.18.6.1; and EC 1.19.6.1): see pp 89-116 in (ref 36) and pp 91-100 in (ref 37).

2.5.9 Molybdenum enzymes containing the pterin-molybdenum cofactor

These enzymes [see pp 411-415 in (ref 36) and (ref 38)] may be divided
into those in which the molybdenum bears a cyanide-labile sulfido (or thio
– see Note 1) ligand i.e. containing the S2- ligand as Mo=S) and those
lacking this ligand. The former group includes xanthine oxidase (EC 1.1.3.22),
xanthine dehydrogenase (EC 1.1.1.204), aldehyde oxidase (EC 1.2.3.1) and
purine hydroxylase (EC: see Note 2 and 3). These may be called ‘molybdenum-
containing hydroxylase’ as is widely done. Molybdenum enzymes lacking the
sulfide (thio) ligand include sulfite oxidase (EC 1.8.3.1), NAD(P)+-independent
aldehyde dehydrogenase and nitrate reductases (assimilatory and dissimilatory)
(EC 1.6.6.1-3).

2.5.10 Molybdenum enzymes containing the pterin-molybdenum cofactor

These enzymes [see pp 411-415 in (ref 36) and (ref 38)] may be divided into those
in which the molybdenum bears a cyanide-labile sulfido (or thio – see Note 1)
ligand i.e. containing the S2- ligand as Mo=S) and those lacking this ligand. The
former group includes xanthine oxidase (EC 1.1.3.22), xanthine dehydrogenase
(EC 1.1.1.204), aldehyde oxidase (EC 1.2.3.1) and purine hydroxylase. These
may be called ‘molybdenum-containing hydroxylase’ as is widely done.
Molybdenum enzymes lacking the sulfide (thio) ligand include sulfite oxidase
(EC 1.8.3.1), NAD(P)+-independent aldehyde dehydrogenase and nitrate
reductases (assimilatory and dissimilatory) (EC 1.6.6.1-3).

2.5.11 Metal-Substituted Metalloproteins

Scientists from several areas, dealing with spectroscopy and electron-transfer
mechanisms, often use metalloproteins in which a metal at the active site has
been substituted by another metal ion, like Co, Zn, Hg, Cd. Examples are zinc-
substituted cytochromes and cobalt-substituted ferredoxins.

The names for such modified proteins are easily given by using indications
like: ‘zinc-substituted ….’. In case of multi-metal proteins, where ambiguity might
arise about which metal has been substituted, one could easily add in parentheses
the name of the metal that has been replaced, such as: cobalt- substituted [Fe]
nitrogenase.

In formulae fragments or short names one could use the following notation:
[3Fe1Co-4S]2+, cytochrome c'[Fe[arrow right]CoFe], plastocyanin[Cu
[arrow right]Hg].

Ambler, R.P. (1980) in From Cyclotrons to Cytochromes (Kaplan, N.O. &
Robinson, A., eds) Academic Press, New York

Moore, G. & Pettigrew, F.(1987) Cytochromes c, Springer-Verlag, Berlin

Bartsch, R.G. (1963) in Bacterial Photosynthesis (Gest, H., San Pietro, A. &
Vernon, L.P., ed.) p. 315, Antioch Press, Yellow Springs, Ohio.

Stiefel, E.I. & Cramer, S.P. (1985) in Molybdenum Enzymes (Spiro, T.G., ed.),
Wiley-Interscience, New York, 89-116.

Smith B.E. et al. (1988), in Nitrogen Fixation Hundred Years After (Bothe,
H., de Bruijn, F.J. & Newton, W.E., ed.), Gustav Fischer, Stuttgart, New York,
91-100

Type-2 copper-containing enzymes.
MacPherson IS1, Murphy ME.
Cell Mol Life Sci. 2007 Nov;64(22):2887-99.

Type-2 Cu sites are found in all the major branches of life and are often
involved in the catalysis of oxygen species. Four type-2 Cu protein
families are selected as model systems for review: amine oxidases,
Cu monooxygenases, nitrite reductase/multicopper oxidase, and
CuZn superoxide dismutase. For each model protein, the availability
of multiple crystal structures and detailed enzymological studies provides
a detailed molecular view of the type-2 Cu site and delineation of the
mechanistic role of the Cu in biological function. Comparison of these
model proteins leads to the identification of common properties of the
Cu sites and insight into the evolution of the trinuclear active site found
in multicopper oxidases.

Copper proteins and copper enzymes.
Cass AE, Hill HA.
Ciba Found Symp. 1980;79:71-91.
http://www.chm.bris.ac.uk/motm/caeruloplasmin/copper_proteins/t1.htm

The copper proteins that function in homeostasis, electron transport, dioxygen
transport and oxidation are discussed. Particular emphasis is placed on the
role of the ligands, their type and disposition which, in conjunction with other
residues in the active site, determine the role of the copper ion. It is proposed that
copper proteins can be considered in four groups. Those in Group I contain a
single copper ion in an approximately tetrahedral environment with nitrogen and
sulphur-containing ligands. Group II proteins have a single copper ion in a square-
planar-like arrangement. Group III proteins have two copper ions in close
proximity. Group IV consists of multi-opper proteins, composed of sites
representative of the other three groups.

Such centers owe their name to the intense blue coloration of the corresponding
Cu(II) proteins. The color is particularly distinctive since the metal centers are
so optically diluted in these metalloenzymes that only intense absorption in the
visible region, resulting from symmetry allowed electronic transitions, can give
rise to conspicuous colors. In contrast, the comparatively pale blue color of normal
Cu(II)) is the result of forbidden electronic transitions between d-orbitals of
different symmetry; in Cu2+(aq) this gives a molar extinction coefficient of
10 M-1cm-1 from a broad absorption between 10,000 cm-1 and 15,000 cm-1
compared to about 3000 M-1cm-1 observed for blue Cu(II) centers. For the
T1 centers the intense absorption is attributed to a ligand-to-metal charge
transfer between the Cu2+ and a bonded cysteinate ligand. Typically, as in
azurin or plastocyanin this occurs around 16,000 cm-1. Ceruloplasmin has
three T1 centers, and the blue absorption is at 16,400 cm-1 (610nm).

Plastocyanine geometry

around the copper Crystal structures show a very irregular ‘tetrahedral’ coordination
with two sulphurs from methionine and cysteinate, and two histidine nitrogens.
However a comparison of azurin with plastocyanin shows that the geometry
is in some ways closer to a trigonal bipyramid, with and without one extra apical
ligand, so that azurin has a weakly bound glutamine oxygen, and plastocyanine
does not. The T1 coppers in caruloplasmin are in plastocyanine-type domains.
Each of these are coordinated to two histidines and a cysteine, in two of the T1
domains there is also a methionine residue, the third T1 domain has a leucine
residue which may only have a van der Waals type contact with the copper.

T1 copper centers are functional in the reversible electron transfer:

Cu2+ + e- = Cu+

The strongly distorted geometry represents a compromise (entactic-state
situation) between d10 Cu(I), with its preferred tetrahedral or trigonal
coordination through soft sulfur ligands, and d9 Cu(II) with preferential
square planar or square pyramidal geometry and nitrogen ligand
coordination. This irregular, high energy arrangement at the metal
center resembles the transition-state geometry between the tetrahedral
and square planar equilibrium configurations of the two oxidation states
involved and permits enhanced rates of electron transfer. The potential
range for proteins with T1 copper centers runs from 180 mV in
stellacyanin to 680 mV in rusticyanin.

Zinc proteins: enzymes, storage proteins, transcription factors, and replication
proteins.
Coleman JE.
Annu Rev Biochem. 1992;61:897-946.

In the past five years there has been a great expansion in our knowledge of
the role of zinc in the structure and function of proteins. Not only is zinc
required for essential catalytic functions in enzymes (more than 300 are known
at present), but also it stabilizes and even induces the folding of protein
subdomains. The latter functions have been most dramatically illustrated
by the discovery of the essential role of zinc in the folding of the DNA-binding
domains of eukaryotic transcription factors, including the zinc
finger transcription factors, the large family of hormone receptor proteins,
and the zinc cluster transcription factors from yeasts. Similar functions are
highly probable for the zinc found in the RNA polymerases and the zinc-
containing accessory proteins involved in nucleic acid replication. The rapid
increase in the number and nature of the proteins in which zinc functions
is not unexpected since zinc is the second most abundant trace metal found in
eukaryotic organisms, second only to iron. If one subtracts the amount of iron
found in hemoglobin, zinc becomes the most abundant trace metal found
in the human body.

Zinc Coordination Spheres in Protein Structures
ACS ChemWorx
Mikko Laitaoja , Jarkko Valjakka , and Janne Jänis
Inorg. Chem., 2013, 52 (19), pp 10983–10991
http://dx.doi.org:/10.1021/ic401072d
Sept 23, 2013

Synopsis
A statistical analysis in terms of zinc coordinating amino acids, metal-to-ligand
bond lengths, coordination number, and structural classification was performed,
revealing coordination spheres from classical tetrahedral cysteine/histidine binding
sites to more complex binuclear sites with carboxylated lysine residues. According
to the results, coordination spheres of hundreds of crystal structures in the PDB
could be misinterpreted due to symmetry-related molecules or missing electron
densities for ligands.

Protein-folding location can regulate manganese-binding versus copper- or
zinc-binding.
Tottey S, Waldron KJ, Firbank SJ, Reale B, Bessant C, Sato K, Cheek TR, et al.
Nature. 2008 Oct 23;455(7216):1138-42. http://dx.doi.org:/10.1038/nature07340

Metals are needed by at least one-quarter of all proteins. Although metallo-
chaperones insert the correct metal into some proteins, they have not been
found for the vast majority, and the view is that most metalloproteins acquire
their metals directly from cellular pools. However, some metals form more
stable complexes with proteins than do others. For instance, as described
in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable
complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage
metal acquisition by most nascent proteins. To investigate this question, we
identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the
most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of
the cyanobacterium Synechocystis PCC 6803. Each of these newly identified
proteins binds its respective metal via identical ligands within a cupin fold.
Consistent with the Irving-Williams series, MncA only binds Mn(2+) after
folding in solutions containing at least a 10(4) times molar excess of Mn(2+)
over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal
does not exchange with Cu(2+). MncA and CucA have signal peptides for
different export pathways into the periplasm, Tat and Sec respectively. Export
by the Tat pathway allows MncA to fold in the cytoplasm, which contains only
tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In
contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal
a mechanism whereby the compartment in which a protein folds overrides its
binding preference to control its metal content. They explain why the cytoplasm
must contain only tightly bound and buffered copper and Zn(2+).

Predicting copper-, iron-, and zinc-binding proteins in pathogenic species of the
Paracoccidioides genus
GB Tristão, L do Prado Assunção, LPA dos Santos, CL Borges, MG Silva-Bailão,
CM de Almeida Soares, G Cavallaro and AM Bailão*
Front. Microbiol., 9 Jan 2015 http://dx.doi.org:/10.3389/fmicb.2014.00761

Approximately one-third of all proteins have been estimated to contain at least
one metal cofactor, and these proteins are referred to as metalloproteins. These
represent one of the most diverse classes of proteins, containing metal ions that
bind to specific sites to perform catalytic, regulatory and structural functions.
Bioinformatic tools have been developed to predict metalloproteins encoded by
an organism based only on its genome sequence. Its function and the type of
metal binder can also be predicted via a bioinformatics approach. Paracoccidioides
complex includes termodimorphic pathogenic fungi that are found as saprobic
mycelia in the environment and as yeast, the parasitic form, in host tissues. They
are the etiologic agents of Paracoccidioidomycosis, a prevalent systemic mycosis
in Latin America. Many metalloproteins are important for the virulence of several
pathogenic microorganisms. Accordingly, the present work aimed to predict the
copper, iron and zinc proteins encoded by the genomes of three phylogenetic species
of Paracoccidioides (Pb01, Pb03, andPb18). The metalloproteins were identified
using bioinformatics approaches based on structure, annotation and domains. Cu-,
Fe-, and Zn-binding proteins represent 7% of the total proteins encoded by
Paracoccidioides spp. genomes. Zinc proteins were the most abundant metallo-
proteins, representing 5.7% of the fungus proteome, whereas copper and iron
proteins represent 0.3 and 1.2%, respectively. Functional classification revealed that
metalloproteins are related to many cellular processes. Furthermore, it was observed
that many of these metalloproteins serve as virulence factors in the biology of the
fungus. Thus, it is concluded that the Cu, Fe, and Zn metalloproteomes of the
Paracoccidioides spp. are of the utmost importance for the biology and virulence
of these particular human pathogens.

Zinc finger proteins: new insights into structural and functional diversity
John H Laity, Brian M Lee, Peter E Wright
Current Opinion in Structural Biology Feb 2001; 11(1): 39–46
http://epigenie.com/key-epigenetic-players/chromatin-modifying-and-dna-
binding-proteins/zinc-finger-proteins/

Zinc finger proteins are among the most abundant proteins in eukaryotic genomes.
Their functions are extraordinarily diverse and include DNA recognition, RNA
packaging, transcriptional activation, regulation of apoptosis, protein folding
and assembly, and lipid binding. Zinc finger structures are as diverse as their
functions. Structures have recently been reported for many new zinc finger
domains with novel topologies, providing important insights into structure/function
relationships. In addition, new structural studies of proteins containing the
classical Cys2His2 zinc finger motif have led to novel insights into mechanisms
of DNA binding and to a better understanding of their broader functions in
transcriptional regulation.

Zinc Finger Proteins

Zinc finger (ZnF) proteins are a massive, diverse family of proteins that serve a
wide variety of biological functions. Due to their diversity, it is difficult to come up
with a simple definition of what unites all ZnF proteins; however, the most common
approach is to define them as all small, functional domains that require coordination
by at least one zinc ion (Laity et al., 2001). The zinc ion serves to stabilize the
integration of the protein itself, and is generally not involved in binding targets.
The “finger” refers to the secondary structures (α-helix and β-sheet) that are
held together by the Zn ion. Zinc finger containing domains typically serve
as interactors, binding DNA, RNA, proteins or small molecules (Laity et al., 2001).

ZnF Protein Families

Cys2His2 was the first domain discovered (also known as Krüppel-type). It was
initially discovered as a repeating domain in the IIIA transcription factor in
Xenopus laevis (Brown et al., 1985; Miller et al., 1985). IIIA has nine repeats
of the 30 amino acids that make up the Cys2His2 domain. Each domain forms
a left-handed ββα secondary structure, and coordinates a Zn ion between
two cysteines on the β-sheet hairpin and two histidines in the α-helix, hence
the name Cys2His2 (Lee et al., 1989). These resides are highly conserved,
as well as a general hydrophobic core that allows the helix to form. The other
residues can show great sequence diversity (Michael et al., 1992). Cys2His2
zinc fingers that bind DNA tend to have 2-4 tandem domains as part of a
larger protein. The residues of the alpha helices form specific contacts with a
specific DNA sequence motif by “reading” the nucleotides in major groove
of DNA (Elrod-Erickson et al., 1996; Pavletich and Pabo, 1991). Cys2His2
proteins are the biggest group of transcription factors in most species. Non-
DNA binding proteins can have much more flexible tertiary structure.
Examples of Cys2His2 proteins include the Inhibitor of Apoptosis (IAP) family
of proteins and the CTFC transcription factor.

Treble clef fingers are a very diverse group of ZnF protiens both in terms of
structure and function. What makes them a family is a shared fold at their core
that looks a little like a musical treble clef, especially if you squint (Grishin,
2001). Most treble clef finger motifs have a β hairpin, a variable loop region,
a β hairpin, and an α helix. The “knuckle” of the β hairpin and the α helix contain
the Cys-x-x-Cys sequence necessary to coordinate the Zn ion. Treble clef
fingers often form the core of protein structures, for example the L24E and
S14 ribosomal proteins and the RING finger family.

Zinc ribbons are a little less structurally complex than the other two major groups.
Zinc ribbons contain two zinc knuckles, often β hairpins, coordinating a zinc ion via
a two Cys residures separated by 2-4 other residues on one knuckle, and a Cys-x-x-
Cys on the other (Hahn and Roberts, 2000). Examples of zinc ribbon-containing
proteins include the basal transcription factors TFIIS and TFIIB that for a complex
with RNAPII to bind DNA, and the Npl4 nuclear core protein that uses a zinc ribbon
to bind ubiquitin (Alam et al., 2004). Cys2His2, treble clef fingers, and zinc ribbons
form the majority of zinc fingers, but there are several other smaller groups that
don’t fit neatly into these three. Green fluorescent protein as a marker for gene
expression.

Metallothionein proteins expression, copper and zinc concentrations, and lipid
peroxidation level in a rodent model for amyotrophic lateral sclerosis
E Tokuda, Shin-Ichi Ono, K Ishige, A Naganuma, Y Ito, T Suzuki
Toxicology Jan 2007; 229(1–2): 33–41

It has been hypothesized that copper-mediated oxidative stress contributes to the
pathogenesis of familial amyotrophic lateral sclerosis (ALS), a fatal motor neuron
disease in humans. To verify this hypothesis, we examined the copper and zinc
concentrations and the amounts of lipid peroxides, together with that of the
expression of metallothionein (MT) isoforms in a mouse model [superoxide
dismutase1 transgenic (SOD1 Tg) mouse] of ALS. The expression of MT-I and
MT-II (MT-I/II) isoforms were measured together with Western blotting, copper
level, and lipid peroxides amounts increased in an age-dependent manner in the
spinal cord, the region responsible for motor paralysis. A significant increase was
already seen as early as 8-week-old SOD1 Tg mice, at which time the mice had not
yet exhibited motor paralysis, and showed a further increase at 16 weeks of age,
when paralysis was evident. Inversely, the spinal zinc level had significantly
decreased at both 8 and 16 weeks of age. The third isoform, the MT-III level,
remained at the same level as an 8-week-old wild-type mouse, finally increasing
to a significant level at 16 weeks of age. It has been believed that a mutant SOD1
protein, encoded by a mutant SOD1, gains a novel cytotoxic function while
maintaining its original enzymatic activity, and causes motor neuron death
(gain-of-toxic function). Copper-mediated oxidative stress seems to be a probable
underlying pathogenesis of gain-of-toxic function. Taking the above current
concepts and the classic functions of MT into account, MTs could have a disease
modifying property: the MT-I/II isoform for attenuating the gain-of-toxic function
at the early stage of the disease, and the MT-III isoform at an advanced stage.

Prion protein expression level alters regional copper, iron and zinc content in
the mouse brain
MJ Pushie, IJ Pickering, GR Martin, S Tsutsui, FR Jirik and GN George
Metallomics, 2011,3, 206-214 http://dx.doi.org:/10.1039/C0MT00037J

The central role of the prion protein (PrP) in a family of fatal neurodegenerate
diseases has garnered considerable research interest over the past two decades.
Moreover, the role of PrP in neuronal development, as well as its apparent role
in metal homeostasis, is increasingly of interest. The host-encoded form of the
prion protein (PrP^C) binds multiple copper atoms via its N-terminal domain
and can influence brain copper and iron levels. The importance of PrP^C to the
regulation of brain metal homeostasis and metal distribution, however, is not
fully understood. We therefore employed synchrotron-based X-ray fluorescence
imaging to map the level and distributions of several key metals in the brains of
mice that express different levels of PrP^C. Brain sections from wild-type, prion
gene knockout (Prnp^−/−) and PrP^C over-expressing mice revealed striking
variation in the levels of iron, copper, and even zinc in specific brain regions as
a function of PrP^C expression. Our results indicate that one important function
of PrP^C may be to regulate the amount and distribution of specific metals within
the central nervous system. This raises the possibility that PrP^C levels, or its
activity, might regulate the progression of diseases in which altered metal
homeostasis is thought to play a pathogenic role such as Alzheimer’s,
Parkinson’s and Wilson’s diseases and disorders such as hemochromatosis.

Zinc & Copper Imbalances: Immense Biochemical Implications
Mar 27, 2013 by Michael McEvoy
http://metabolichealing.com/zinc-copper-imbalances-immense-biochemical-
implications/

The status of zinc and copper levels may have profound implications for
many people. Much has been written about the significance of these two
trace elements for many, many years. Many health conditions may be
directly caused by abnormal zinc and copper levels.

With all of the recent attention given to methylation status, gene mutations,
MTHFR, and the associated neurological and mental/behavioral disorders
that may ensue, zinc and copper status remains a pivotal ratio in these regards.

While zinc toxicity and copper deficiency are possible, the subject of this
article is on the more common imbalance: copper toxicity and zinc deficiency.

The Physiological Roles Of Zinc & Copper

Zinc and copper are antagonists. The balance between these two trace
elements is an example of the effects of biological dualism. While zinc
toxicity is possible, far more common is zinc deficiency and copper toxicity.
Both zinc and copper play essential roles in the body, and there can be a
number of causes for why imbalances ensue.

It may be easier to identify the roles that zinc doesn’t play in the body,
than the roles it does play. Zinc is an essential trace element that activates
several hundred enzymatic reactions. These reactions are fundamental
to life and biological activity. Some of the activities that zinc are involved in:

DNA & RNA synthesis
Gene expression
Nervous system function
Immune function & immune signaling such as cell
apoptosis
Neuronal transmission
Brain function
Zinc possesses powerful anabolic activities in the cells
Formation of zinc proteins known as “zinc fingers”
Zinc is essential for blood clotting and platelet formation
Zinc is involved in Vitamin A synthesis
Folate is made available through zinc enzyme reactions
Along with copper, Zinc makes up the antioxidant
enzyme
system, ZnCu superoxide dismutase
Steroidal hormone synthesis
Growth & development of children
Testosterone and semen formation
The highest concentration of zinc is found in the
male prostate gland

Copper is an essential trace element serving many important functions
as well. However, copper is well documented to induce several toxic effects
in the body, when elevated. Because copper is a pro-oxidant when free and
unbound, it can quickly generate free radicals.

The major sources for copper toxicity are: exposure to industrial forms
of copper such as copper pipes, copper cookware, birth control, exposure
to copper-based fungicides. Diets high in copper and low in zinc may play
a role in copper toxicity. Pyrrole disorder, which causes depletion of zinc,
may result in elevated levels of copper.

Some of the essential roles copper plays in the body:

Connective tissue formation
ATP synthesis
Iron metabolism
Brain health via neurotransmitter synthesis
Gene transcription
Synthesis of the antioxidant superoxide dismutase
Skin pigmentation
Nerve tissue: myelin sheath formation
Copper tends to rise when estrogen is dominant

Perhaps one of the first reports that zinc and copper imbalances play
a role in human health and disease was their detection in mental
disorders made by Carl Pfeiffer, MD, PhD. Dr. Pfeiffer identified a
condition known as pyrrole disorder, sometimes referred to as
pyrroluria or “mauve factor”.

As it turns out, pyrrole disorder is a major biochemical imbalance
in many people with chronic illnesses such as chronic Lyme disease,
autism, schizophrenia, depression, bi-polar, and chronic fatigue
syndrome. Pyrroles are a byproduct of hemoglobin synthesis.
Apparently, some individuals are more predisposed towards producing
higher amounts of pyrroles. When pyrroles are excessive, they irreversibly
bind to zinc and vitamin B6, causing their excretion. Consequently,
it is common that once zinc levels become depleted, copper levels tend to rise.

Copper Toxicity

Problems associated with copper toxicity include: pyrrole disorder,
estrogen dominance, schizophrenia, depression, anxiety disorder,
chronic fatigue, migraines, liver toxicity, thyroid conditions, chronic
candida yeast infections, PMS, to name a few. Some research has
even implicated copper toxicity with Alzheimer’s Disease and with
cardiovascular disease. Perhaps one of the primary mechanisms
through which copper toxicity can damage tissues is through its
initiation of oxidative stress and free radical formation. Free copper
ions that are not bound to copper proteins such as ceruloplasmin,
are pro-oxidants, and are highly reactive.

Empirical research from clinicians, indicates that there are different
types of copper imbalances. For example, if there is a lot of free,
unbound copper present, this may cause a situation of nutritive
copper deficiency. Another copper imbalance is when high pyrroles
depress zinc levels, and copper levels concomintantly rise. If high
pyrroles are present, B6 will also be lost in high amounts. In a general
but very real sense, all forms of copper excess will affect zinc status,
due to the dualistic nature of zinc and copper.

Copper & Estrogen

It has been known for many years that copper can cause a rise in
estrogen, and conversely estrogen may raise copper. Estrogen
dominance has been extensively studied in its role in breast
cancer development. One possible, critical role that can cause
estrogen to become carcinogenic, is through its oxidation induced by
copper. Once oxidized, estrogen forms volatile hydroxyl radicals and
the associated DNA damage and “mutagenesis”.

Zinc Deficiency

As mentioned previously, pyrrole disorder will directly depress
zinc status, causing high levels of its excretion. When zinc is
lost, copper rises. Because of their essential roles in neuro-
transmitter synthesis, low zinc and high copper levels can
directly effect cognition, behavior and thought processes.
Zinc has been studied in biochemical reactions involving
calcium-driven, synaptic neurotransmission, as well as in
glutamate/GABA balance and with limbic brain function.

Zinc & Reproduction

Zinc is essential for steroidal hormone synthesis, and is a
well known catalyst for testosterone synthesis, as well as
leutinizing hormone. Zinc has demonstrated its ability to
prevent miscarriage and toxicity during pregnancy. The male
prostate gland reportedly contains the highest concentration
of zinc in the body.

Zinc & Brain Function

Much attention has been given to excitotoxicity, such as the
effects induced by MSG (monosodium glutamtate). Excess
stimulation of the excitatory neurotransmitter glutamate,
may cause severe physical and psychological reactions in
certain individuals. Zinc has been studied for its ability to
enhance GABA (glutamate’s antagonistic neurotransmitter)
activity and to suppress excess glutamate.

Studies on mice demonstrated that when depleted of zinc
for two weeks, the mice developed seizures, most likely due
to GABA deficiencies and glutamate excess.

There is an emerging body of evidence that demonstrates
that Alzheimer’s disease may involve copper toxicity and
zinc deficiency. Not only can excess copper cause zinc
depletion, but so can excess lead.

The hippocampus, a major part of the limbic brain, records
memories and is responsible for processing meaningful
experiences. Numerous studies site that if hippocampal
cells are deprived of zinc, the hippocampal cells die. In
addition to hippocampus cell death induced by zinc
deprivation, the amygdala, the other major limbic gland
experiences cell death as well, when deprived of zinc.

Green Fluorescent Protein

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC.
Science. 1994 Feb 11;263(5148):802-5.
http://www.ncbi.nlm.nih.gov/pubmed/8303295

A complementary DNA for the Aequorea victoria green fluorescent protein (GFP)
produces a fluorescent product when expressed in prokaryotic (Escherichia coli)
or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and
cofactors are not required for this fluorescence, GFP expression can be used
to monitor gene expression and protein localization in living organisms.

http://en.wikipedia.org/wiki/Green_fluorescent_protein

The green fluorescent protein (GFP) is a protein composed of 238 amino acid
residues (26.9 kDa) that exhibits bright green fluorescence when exposed
to light in the blue to ultraviolet range. Although many other marine organisms
have similar green fluorescent proteins, GFP traditionally refers to the protein
first isolated from the jellyfish Aequorea victoria. The GFP from A. victoria
has a major excitation peak at a wavelength of 395 nm and a minor one at
475 nm. Its emission peak is at 509 nm, which is in the lower green portion
of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79.
The GFP from the sea pansy (Renilla reniformis) has a single major excitation
peak at 498 nm.

In cell and molecular biology, the GFP gene is frequently used as a reporter of
expression. In modified forms it has been used to make biosensors, and many
animals have been created that express GFP as a proof-of-concept that a gene
can be expressed throughout a given organism. The GFP gene can be introduced
into organisms and maintained in their genome through breeding, injection with a
viral vector, or cell transformation. To date, the GFP gene has been introduced
and expressed in many Bacteria, Yeast and other Fungi, fish (such as zebrafish),
plant, fly, and mammalian cells, including human. Martin Chalfie, Osamu Shimomura,
and Roger Y. Tsien were awarded the 2008 Nobel Prize in Chemistry on 10 October
2008 for their discovery and development of the green fluorescent protein.

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm

In Aequorea victoria a protein called aequorin releases blue light upon binding
with calcium. This blue light is then totally absorbed by the GFP, which in turn
gives off the green light as in the animation below.

In 1994 GFP was cloned. Now GFP is found in laboratories all over the world where
it is used in every conceivable plant and animal. Flatworms, algae, E. coli and pigs
have all been made to fluoresce with GFP.

The importance of GFP was recognized in 2008 when the Nobel Committee awarded
Osamu Shimomura, Marty Chalfie and Roger Tsien the Chemistry Nobel Prize ”
for the discovery and development of the green fluorescent protein, GFP.”

Why is it so popular? Well, I like to think of GFP as the microscope of the twenty-
first century. Using GFP we can see when proteins are made, and where they can go.
This is done by joining the GFP gene to the gene of the protein of interest so that
when the protein is made it will have GFP hanging off it. Since GFP fluoresces, one
can shine light at the cell and wait for the distinctive green fluorescence associated
with GFP to appear.

A variant of yellow fluorescent protein with fast and efficient maturation for
cell-biological applications
T Nagai, K Ibata, E Sun Park, M Kubota, K Mikoshiba & A Miyawaki
Nature Biotechnology 20, 87 – 90 (2002) http://dx.doi.org:/10.1038/nbt0102-87

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria
has provided a myriad of applications for biological systems. Over the last
several years, mutagenesis studies have improved folding properties of GFP.
However, slow maturation is still a big obstacle to the use of GFP variants for
visualization. These problems are exacerbated when GFP variants are expressed
at 37°C and/or targeted to certain organelles. Thus, obtaining GFP variants that
mature more efficiently is crucial for the development of expanded research
applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs)
are relatively acid-sensitive,and uniquely quenched by chloride ion (Cl−)3. For
YFP to be fully and stably fluorescent, mutations that decrease the sensitivity
to both pH and Cl− are desired. Here we describe the development of an
improved version of YFP named “Venus”. Venus contains a novel mutation,
F46L, which at 37°C greatly accelerates oxidation of the chromophore, the rate-
limiting step of maturation. As a result of other mutations, F64L/M153T/
V163A/S175G, Venus folds well and is relatively tolerant of exposure
to acidosis and Cl−. We succeeded in efficiently targeting a neuropeptide
Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion
was readily monitored by measuring release of fluorescence into the medium.
The use of Venus as an acceptor allowed early detection of reliable signals of
fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain
slices. With the improved speed and efficiency of maturation and the increased
resistance to environment, Venus will enable fluorescent labelings that were not
possible before.

Rhodopsin-like Protein from the Purple Membrane of Halobacterium halobium
DIETER OESTERHELT & WALTHER STOECKENIUS
Nature New Biology 29 Sep 1971; 233, 149-152 | http://dx.doi.org:/10.1038/
newbio233149a0

HALOPHILIC bacteria require high concentrations of sodium chloride and lower
concentrations of KCl and MgCl2 for growth. The cell membrane dissociates into
fragments of varying size when the salt is removed1. One characteristic fragment—
termed the “purple membrane” because of its characteristic deep purple colour—
has been isolated in relatively pure form from Halobacterium halobium. We can
now show that the purple colour is due to retinal bound to an opsin-like protein,
the only protein present in this membrane fragment.

References

Stoeckenius, W. , and Rowen, R. , J. Cell Biol., 34, 365 (1967).

Stoeckenius, W. , and Kunau, W. H. , J. Cell Biol., 38, 337 (1968).

Blaurock, A. E. , and Stoeckenius, W. , Nature New Biology, 233, 152 (1971).

Sehgal, S. N. , and Gibbons, N. E. , Canad. J. Microbiol., 6, 165 (1960).

Kelly, M. , Norgård, S. , and Liaach-Jensen, S. , Acta Chem. Scand., 2A, 2169 (1970).

Shapiro, A. L. , Vinnela, E. , and Maizel, jun., J. V. , Biochem. Biophys. Res.
Commun., 28, 815 (1967).

The monomerization of the Purple protein, a member of the GFP-family
Corning, Brooke

Green fluorescent protein (GFP) has been used extensively since its discovery
in the 1960s to report and visualize gene expression. For years it has been the only
known naturally occurring fluorescent pigment that is encoded by a single gene,
making it extremely useful in various fields of biology, because the expression of
this gene directly leads to the appearance of the fluorescent green color. Recently,
however, many more proteins with similar properties to GFP, and available in a
variety of colors, have been isolated from the class of marine organisms called
Anthozoa, which includes the corals. This increase in the availability of colored
proteins in GFP family in turn has expanded the number of available biotech-
nology applications. However, some of these newly discovered GFP-like
proteins do not have wild-type forms that readily allow for the creation of
fusion proteins, particularly because of oligomerization. It is widely accepted
that almost all members of the GFP-family form dimers or tetramers in their
functional forms.

This study investigates a GFP-ike protein, Purple, isolated from two species,
Galaxea fascicularis and Montipora efflorescens. Purple protein forms oligomers
when expressed, which would then interfere with the normal expression of a protein
to be tagged in gene fusion experiments. We selectively mutated 3 amino acids,
which we believed were responsible for oligomerization in Purple. These 3
residues were chosen based on sequence similarities to a very similar protein,
a mutant form of the Rtms5 chromoprotein from Montipora efflorescens. While
we had hoped that the resulting triple-mutant Purple protein would form
monomers in vivo while retaining its purple coloration, this turned out to
be incorrect. The resulting mutants had lost their ability to turn purple. However,
we also determined that we had successfully changed the oligomerization
state of Purple by examining the relative molecular mass of one our
mutant proteins, which turned out to be half the size of the original
purple protein. It is possible that by adding additional mutations in
the future, the original spectral properties could be recovered. If
successful, this would further expand the utility of the GFP family.

Rhodopsin, also known as visual purple, from Ancient Greek ῥόδον
(rhódon, “rose”), due to its pinkish color, and ὄψις (ópsis, “sight”), is
a light-sensitive receptor protein. It is a biological pigment in photo-
receptor cells of the retina. Rhodopsin is the primary pigment found
in rod photoreceptors. Rhodopsins belong to the G-protein-coupled
receptor (GPCR) family. They are extremely sensitive to light, enabling
vision in low-light conditions. Exposed to light, the pigment
immediately photobleaches, and it takes about 45 minutes to regenerate
fully in humans. Its discovery was reported by German physiologist
Franz Christian Boll in 1876.

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Evolution of Myoglobin and Hemoglobin

Posted in Amino acids, Cell Biology, Curation, Evolutionary cognition, Experimental validation, Explanatory, Gene Regulation and Evolution, Genome Biology, Hematology, Historical relevance, Metabolism, Nitric Oxide in Health and Disease, Proteins, Theoretic convergence, tagged Evolutionary Biology, globin chains, heme, Hemoglobin, iron, myoglobin, oxygen affinity, oxygen transport on January 19, 2015| Leave a Comment »

Evolution of Myoglobin and Hemoglobin

Author and Curator: Larry H. Bernstein, MD, FCAP

Nitric oxide dioxygenase function and mechanism of flavohemoglobin, hemoglobin, myoglobin and their associated reductases

Paul R. Gardner
Journal of Inorganic Biochemistry Jan 2005; 99(1): 247–266
http://dx.doi.org:/10.1016/j.jinorgbio.2004.10.003

Microbial flavohemoglobins (flavoHbs) and hemoglobins (Hbs) show large radical dotNO dioxygenation rate constants ranging from 745 to 2900 μM−1 s−1 suggesting a primal radical dotNO dioxygenase (NOD) (EC 1.14.12.17) function for the ancient Hb superfamily. Indeed, modern O2-transporting and storing mammalian red blood cell Hb and related muscle myoglobin (Mb) show vestigial radical dotNO dioxygenation activity with rate constants of 34–89 μM−1 s−1. In support of a NOD function, microbial flavoHbs and Hbs catalyze O2-dependent cellular radical dotNO metabolism, protect cells from radical dotNO poisoning, and are induced by radical dotNO exposures. Red blood cell Hb, myocyte Mb, and flavoHb-like activities metabolize radical dotNO in the vascular lumen, muscle, and other mammalian cells, respectively, decreasing radical dotNO signalling and toxicity. HbFe(III)–OOradical dot, HbFe(III)–OONO and protein-caged [HbFe(III)–Oradical dotradical dotNO2] are proposed intermediates in a reaction mechanism that combines both O-atoms of O2 with radical dotNO to form nitrate and HbFe(III). A conserved Hb heme pocket structure facilitates the dioxygenation reaction and efficient turnover is achieved through the univalent reduction of HbFe(III) by associated reductases. High affinity flavoHb and Hb heme ligands, and other inhibitors, may find application as antibiotics and antitumor agents that enhance the toxicity of immune cell-derived radical dotNO or as vasorelaxants that increase radical dotNO signaling.

http://ars.els-cdn.com/content/image/1-s2.0-S016201340400296X-gr1.sml

http://ars.els-cdn.com/content/image/1-s2.0-S016201340400296X-gr2.sml

http://ars.els-cdn.com/content/image/1-s2.0-S016201340400296X-gr3.sml

The evolution of the globin family genes: Concordance of stochastic and augmented maximum parsimony genetic distances for α hemoglobin, β hemoglobin and myoglobin phylogenies
R Holmquist, TH Jukes, H Moise, M Goodman, GW Moore
Journal of Molecular Biology Jul 1976; 105(1): 39–74
http://dx.doi.org:/10.1016/0022-2836(76)90194-7

We compare the amino acid sequences of 70 globing, representing the following families: (a) α hemoglobin chains; (b) β hemoglobin chains; (c) myoglobins; (d) two lamprey, a mollusc, and two plant globins. The comparisons show a convergence of maximal and minimal estimates of genetic differences as calculated respectively by the stochastic and maximum parsimony procedures, thus demonstrating for the first time the logical consistency and complementarity of the two procedures. Evolutionary rates are non-constant, varying over a range of 1 to 75 nucleotide replacements per 100 codons per 108 years. These rate differentials are resolved into two components (a) due to change in the number of codon sites free to fix mutations during the period of divergence of the species involved; (b) due to change in fixation intensity at each site. These two components also show non-uniformity along different lineages. Positive Darwinian natural selection can bring about an increase in either component, and negative or stabilizing selection in protein evolution can lead to decreases. Accelerated rates of globin evolution were found in lineages of cold-blooded vertebrates, some marsupials, and early placental mammals, while slower rates were found in warm-blooded vertebrates, especially higher primates. One manifestation of negative selection in the globins is that minimal 3-base type amino acid replacements occur less frequently than would be expected if base replacements had occurred and were accepted at random. The selection against these replacements is not due to atypical behavior with respect to the change in electrical charge involved in the replacements. Interestingly, the globins from the lamprey, sea hare and the legumes are as distant from one another as are α-hemoglobin and β-hemoglobin from myoglobin.

Hemoglobin Orthologs
http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2005/Heiner/ortholog.html

Orthologs are sequences of genes that evolved from a common ancestor and can be traced evolutionarily through different species. By comparing the ortholog sequences of a specific gene between many species, the amino acid sequences which are conserved can be determined. These highly conserved sequences are important, because they provide information on which amino acids are essential to the protein structure and function.

Evolution of Hemoglobin

Hemoglobin is derived from the myoglobin protein, and ancestral species just had myoglobin for oxygen transport. 500 million years ago the myoglobin gene duplicated and part of the gene became hemoglobin. Lampreys are the most ancestral animal to have hemoglobin, and the ancestral version was composed of dimers instead of tetramers and was only weakly cooperative. 100 million years later, the hemoglobin gene duplicated again forming alpha and beta subunits. This form of derived hemoglobin is found in bony fish, reptiles, and mammals, which all have both alpha and beta subunits to form a tetramer (Mathews et al., 2000).

Conserved Sequences

When the amino acid sequences of myoglobin, the hemoglobin alpha subunit, and the hemoglobin beta subunit are compared, there are several amino acids that remain conserved between all three globins (Mathews et al., 2000). These amino acid sequences are considered truly essential, because they have remained unchanged throughout evolution, and therefore are fundamental to the function of the protein. These essential amino acids can be seen in Figure 1, which compares myoglobin, and the alpha and beta subunits of hemoglobin. The histidines in helix F8 and helix E7 are highly conserved. These histidines are located proximally and distally to the heme molecule and keep the heme molecule in place within the hemoglobin protein as seen in Figure 2 (Mathews et al., 2000). This shows that the position of the heme molecule within the globin protein is essential to its function. Likewise, the amino acids in the FG region are also highly conserved. This region of the protein is essential to the conformational change between the T to R states (Mathews et al., 2000). Additionally, the amino acids at the alpha-beta subunit interfaces are highly conserved, because they also affect the conformational change between the subunits, which regulates oxygen affinity and cooperativity. In general, the most highly conserved sequences are located within the interior of the hemoglobin protein where the subunits contact each other (Gribaldo et al., 2003).

http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2005/Heiner/figure2.jpg

Figure 2: A cartoon drawing of the structure of hemoglobin around heme molecule. The histadines in helix F8 and E7 interact directly with the heme molecule. http://www.aw-bc.com/mathews/ch07/fi7p5.htm (permission pending).

Figure 1: The amino acid sequences of myoglobin, alpha subunit of hemoglobin, and beta subunit of hemoglobin. The amino acid sequences highlighted in tan are conserved between all three globins and the amino acid sequences highlighted in gray are conserved between alpha and beta hemoglobin. http://www.aw-bc.com/mathews/ch07/fi7p11.htm (permission pending).

http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2005/Heiner/figure1.jpg

Figure 2: A cartoon drawing of the structure of hemoglobin around heme molecule. The histidines in helix F8 and E7 interact directly with the heme molecule. http://www.aw-bc.com/mathews/ch07/fi7p11.htm (permission pending).

Alpha Subunit of Hemoglobin

The alpha subunit of hemoglobin has several amino acid sequences that are conserved across many species and are essential to its function. The alpha subunit of hemoglobin is encoded by the 2 genes HBA1 and HBA2 both located on chromosome 16 (GeneCard, 2005). Click here to see the gene card for HBA1. To determine which amino acid sequences are conserved, I compared the orthologs of HBA1 in Homo sapiens (humans) to 5 additional species including, Xenopus tropicalis (African clawed frog), Danio rerio (Zebra fish), Gallus gallus (Red jungle fowl), Mus musculus (mouse), and Rattus norvegicus (rat) using the Ensembl program. Figure 3 shows the 6 orthologs aligned and the important conserved regions highlighted. The stars indicate amino acids that are conserved between all of the species. As a general observation, the mouse ortholog of HBA is the most similar to human HBA, because it is the most evolutionarily related. The amino acid sequences that are conserved in all globin proteins (highlighted in blue) can be seen in Figure 3. There are also several conserved amino acids that are specifically important to HBA structure (highlighted in red) including: the phenylalanine (F) at position 44, which is in direct contact with the heme group; tyrosine (Y) at position 142, which stabilizes the hemoglobin molecule by forming hydrogen bonds between two of the helices; and glycine (G) at position 26, which is small and therefore allows two of the helices to approach each other, which is important to the structure of hemoglobin (Natzke, 1998). Additionally, there are several proteins found in the alpha subunit that are involved in the movement of the alpha and beta subunits (also highlighted in red) including: the tyrosine (Y) at position 43, which interacts with the beta subunit during the R state, and the arginine (N) at position 143, which interacts with the beta subunit during the T state (Gribaldo et al., 2003).

Mutations

Looking at the effects mutated portions of a gene is also a good way to determine the function of highly conserved sequences. In hemoglobin, deleterious mutations are most common in the heme pockets of the protein and in the alpha and beta subunit interfaces (Mathews et al., 2000). There are several key mutations in highly conserved portions of HBA (highlighted in yellow) including: the substitution of histidine (H) at position 88 to tyrosine (Y), which disrupts the heme molecule leading to decreased oxygen affinity; the substitution of arginine (N) at position 143 to histidine (H), which eliminates a bond in the T state and therefore favors the R state, resulting in increased oxygen affinity; the substitution of proline (P) at position 97 to arginine (N), which alters the alpha-beta contact region and results in the disassociation of the hemoglobin complex; and the substitution of leucine (L) at position 138 for proline (P), which interrupts the helix formation and also results in the disassociation of the hemoglobin complex (Mathews et al., 2000).

Bar-headed Goose Hemoglobin

As mentioned on the previous page, the bar-headed goose has hemoglobin that is specifically adapted to high altitudes. The bar-headed goose hemoglobin has an increased oxygen affinity which allows it to live in low oxygen pressure environments (Liang et al., 2001). This increased oxygen affinity is the result of a mutation at position 121 in the alpha subunit, which is highly conserved in other species, from proline to alanine, as seen in Figure 4 (Liang et al., 2001). This substitution leaves a two-carbon gap between the alpha-beta dimer, which relaxes the T structure and allows it to bind oxygen more readily under lower pressures (Jessen et al. 1991). Thus, comparing orthologs can also be used to explain differences in the oxygen binding capabilities of hemoglobin in different species.

References

Ensembl. Ensembl Genome Browser. http://www.ensembl.org/. Accessed March 2005.

GeneCard. 2005. GeneCard for HBA1. http://genome-www.stanford.edu/cgi-bin/genecards/carddisp?HBA1&search=HBA&suff=txt. Accessed March 2005.

Gribaldo, Simonetta, Didier Casane, Philippe Lopez and Herve Philippe. 2003. Functional Divergence Prediction from Evolutionary Analysis: A Case Study of Vertebrate Hemoglobin. Molecular Biology and Evolution 20 (11): 1754-1759.

Jessen, Timm H et al. 1991. Adaptation of bird hemoglobins to high altitudes: Demonstration of molecular mechanism by protein engineering. Evolution 88: 6519-6522.

Liang, Yuhe et al. 2001. The Crystal Structure of Bar-headed Goose Hemoglobin in Deoxy Form: The Alloseteric Mechanism of a Hemoglobin Species with High Oxygen Affinity. Journal of Molecular Biology 313: 123-137.

Mathews, Christopher, Kensal Van Holde and Kevin Ahern. 2000. Biochemistry 3 rd edition. http://www.aw-bc.com/mathews/ch07/c07emhp.htm . Accessed March 2005.

Natzke, Lisa. 1998. Hemoglobin. http://biology.kenyon.edu/BMB/Chime/Lisa/FRAMES/hemetext.htm. Accessed March 2005.

Divergence pattern and selective mode in protein evolution: the example of vertebrate myoglobins and hemoglobin chains.
Otsuka J1, Miyazaki K, Horimoto K.
J Mol Evol. 1993 Feb; 36(2):153-81.

The evolutionary relation of vertebrate myoglobin and the hemoglobin chains including the agnathan hemoglobin chain is investigated on the basis of a new view of amino acid changes that is developed by canonical discriminant analysis of amino acid residues at individual sites. In contrast to the clear discrimination of amino acid residues between myoglobin, hemoglobin alpha chain, and hemoglobin beta chain in warm-blood vertebrates, the three types of globins in the lower class of vertebrates show so much variation that they are not well discriminated. This is seen particularly at the sites that are ascertained in mammals to carry the amino acid residues participating in stabilizing the monomeric structure in myoglobin and the residues forming the subunit contacts in hemoglobin. At these sites, agnathan hemoglobin chains are evaluated to be intermediate between the myoglobin and hemoglobin chains of gnathostomes. The variation in the phylogenetically lower class of globins is also seen in the internal region; there the amino acid residues of myoglobin and hemoglobin chains in the phylogenetically higher class exhibit an example of parallel evolution at the molecular level. New quantities, the distance of sequence property between discriminated groups and the variation within each group, are derived from the values of discriminant functions along the peptide chain, and this set of quantities simply describes an overall feature of globins such that the distinction between the three types of globins has been clearer as the vertebrates have evolved to become jawed, landed, and warm-blooded. This result strongly suggests that the functional constraint on the amino acid sequence of a protein is changed by living conditions and that severe conditions constitute a driving force that creates a distinctive protein from a less-constrained protein.

The globin gene repertoire of lampreys: Convergent evolution of hemoglobin and myoglobin in jawed and jawless vertebrates
K Schwarze, KL Campbell, T Hankeln, JF Storz, FG Hoffmann and T Burmester
Mol Biol Evol (2014). http://dx.doi.org:/10.1093/molbev/msu216

Agnathans (jawless vertebrates) occupy a key phylogenetic position for illuminating the evolution of vertebrate anatomy and physiology. Evaluation of the agnathan globin gene repertoire can thus aid efforts to reconstruct the origin and evolution of the globin genes of vertebrates, a superfamily that includes the well-known model proteins hemoglobin and myoglobin. Here we report a comprehensive analysis of the genome of the sea lamprey (Petromyzon marinus) which revealed 23 intact globin genes and two hemoglobin pseudogenes. Analyses of the genome of the Arctic lamprey (Lethenteron camtschaticum) identified 18 full length and five partial globin gene sequences. The majority of the globin genes in both lamprey species correspond to the known agnathan hemoglobins. Both genomes harbor two copies of globin X, an ancient globin gene that has a broad phylogenetic distribution in the animal kingdom. Surprisingly, we found no evidence for an ortholog of neuroglobin in the lamprey genomes. Expression and phylogenetic analyses identified an ortholog of cytoglobin in the lampreys; in fact, our results indicate that cytoglobin is the only orthologous vertebrate-specific globin that has been retained in both gnathostomes and agnathans. Notably, we also found two globins that are highly expressed in the heart of P. marinus, thus representing functional myoglobins. Both genes have orthologs in L. camtschaticum. Phylogenetic analyses indicate that these heart-expressed globins are not orthologous to the myoglobins of jawed vertebrates (Gnathostomata), but originated independently within the agnathans. The agnathan myoglobin and hemoglobin proteins form a monophyletic group to the exclusion of functionally analogous myoglobins and hemoglobins of gnathostomes, indicating that specialized respiratory proteins for O2 transport in the blood and O2 storage in the striated muscles evolved independently in both lineages. This dual convergence of O2-transport and O2-storage proteins in agnathans and gnathostomes involved the convergent co-option of different precursor proteins in the ancestral globin repertoire of vertebrates.

Globin evolution
Kent Holsinger
http://darwin.eeb.uconn.edu/eeb348/lecturenotes/molevol-multigene/node2.html

I’ve just pointed out the distinction between myoglobin and hemoglobin. You may also remember that hemoglobin is a multimeric protein consisting of four subunits, 2 α\alpha subunits and 2 β\beta subunits. What you may not know is that in humans there are actually two types of α\alpha hemoglobin and four types of β\beta hemoglobin, each coded by a different genetic locus (see Table 1). The five α\alpha -globin loci (α\alpha_1, α\alpha_2, ς\zeta, and two non-functional pseudogenes) are found in a cluster on chromosome 16. The six β\beta-globin loci (ε\epsilon, ϒ\gamma_G, ϒ\gamma_A, δ\delta, β\beta, and a pseudogene) are found in a cluster on chromosome 11. The myoglobin locus is on chromosome 22.

Table 1: Human hemoglobins arranged in developmental sequence. Adult hemoglobins composed of 2 and 2 subunits typically account for less than 3% of hemoglobins in adults (http://sickle.bwh.harvard.edu/hbsynthesis.html).

Not only do we have all of these different types of globin genes in our bodies, they’re all related to one another. Comparative sequence analysis has shown that vertebrate myoglobin and hemoglobins diverged from one another about 450 million years ago. Figure 1 shows a phylogenetic analysis of globin genes from humans, mice, and a variety of Archaea. Focus your attention on the part of the tree that has human and mouse sequences. You’ll notice two interesting things:

Human and mouse neuroglobins (Ngb) are more closely related to one another than they are to other globins, even those from the same species. The same holds true for cytoglobins (Cyg) and myoglobins (Mb).

Within the hemoglobins, only mouse β\beta-globin (Mouse HbB) is misplaced. All other α\alpha- and β\beta-globins group with the corresponding mouse and human loci.

This pattern is exactly what we expect as a result of duplication and divergence. Up to the time that a gene becomes duplicated, its evolutionary history matches the evolutionary history of the organisms containing it. Once there are duplicate copies, each follows an independent evolutionary history. Each traces the history of speciation and divergence. And over long periods duplicate copies of the same gene share more recent common ancestry with copies of the same gene in a different species than they do with duplicate genes in the same genome.

Figure 1: Evolution of globin genes in Archaea and mammals (from [2]).

http://darwin.eeb.uconn.edu/eeb348/lecturenotes/molevol-multigene/img11.png

Evolution of globin genes in Archaea and mammals

A history of duplication and divergence in multigene families makes it important to distinguish between two classes of related loci: those that represent the same locus in different species and between which divergence is a result of species divergence are orthologs. Those that represent different loci and between which divergence occurred after duplication of an ancestral gene are paralogs. The β\beta-globin loci of humans and chickens are orthologous. The α\alpha $- and $\beta $-globin loci of any pair of taxa are paralogous.

As multigene families go, the globin family is relatively simple and easy to understand. There are only about a dozen loci involved, one isolated locus (myoglobin) and two clusters of loci ($\alpha- and β\beta-globins). You’ll find a diagram of the β\beta-globin cluster in Figure 2. As you can see the β\beta-globins are not only evolutionarily related to one another they occur relatively close to one another on chromosome 11 in humans.

Figure 2: Structure of the human β\beta-globin gene cluster. % identity refers to similarity to the mouse β\beta-globin sequence. From http://globin.cse.psu.edu/html/pip/betaglobin/iplot.ps (retrieved 28 Nov 2006).

Other families are far more complex. Class I and class II MHC loci, for example are part of the same multigene family. Moreover, immunoglobulins, T-cell receptors, and, and MHC loci are part of a larger superfamily of genes, i.e., all are ultimately derived from a common ancestral gene by duplication and divergence. Table 2 lists a few examples of multigene families and superfamilies in the human genome and the number of proteins produced.

Table 2: A few gene families from the human genome (adapted from [5,6]).

Distribution and conservation of sequence

https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcRHcfUpQ09ufj8cleSgnhDfQVUEHsTvnYGNxKaPa5wxMqNFzFU6

Distribution and conservation of sequence motifs throughout mammalian beta-globin gene clusters.A detailed map of the gene cluster is shown on the numbered line

evolutionary history of three hypothetical living species (C, D, and E)

the evolutionary history of three hypothetical living species (C, D, and E), inferred by comparing amino-acid differences in their myoglobin molecules.

http://media-1.web.britannica.com/eb-media/98/52998-004-A8682A5B.jpg

oxyhemoglobin dissociation curve

much higher affinity for oxygen than haemoglobin.

http://i.stack.imgur.com/WQJe9.jpg

myoglobin hs much higher oxygen affinity than Hb

Evolution of Myoglobin / Hemoglobin Proteins

Primitive Globin – Very primitive animals had only a myoglobin-like, single-chain ancestral globin for oxygen storage and were so small that they did not require a transport protein. Roughly 500 million years ago the ancestral myoglobin gene was duplicated. One copy became the ancestor of the myoglobin genes of all higher organisms. The other copy evolved into the gene for an oxygen transport protein and gave rise to the hemoglobins.

Most Primitive Hemoglobin – The most primitive animals to possess hemoglobin are the lampreys. Lamprey hemoglobin can form dimers but not tetramers and is only weakly cooperative. It represents a first step toward allosteric binding. Subsequently a second gene duplication must have occurred, giving rise to the ancestors of the present-day and hemoglobin chain families. This must have happened about 400 million years ago, at about the time of divergence of the sharks and bony fish. The evolutionary line of the bony fish led to the reptiles and eventually to the mammals, all carrying genes for both and globins and capable of forming tetrameric 22 hemoglobins. Further gene duplications have occurred in the hemoglobin line, leading to the embryonic forms and , the fetal form, , and the infant form (Figure 7.22).

Conserved Amino Acid Sequences – During the long evolution of the myoglobin/hemoglobin family of proteins, only a few amino acid residues have remained invariant (Figure 7.11). They include the histidines proximal and distal to the heme iron (F8 and E7- see Figure 7.5b) and Val FG5, which has been implicated in the hemoglobin deoxy/oxy conformation change. These may mark the truly essential positions in the molecule. Other regions highly conserved in hemoglobins are those near the 1 – 2 and 2 – 1 contacts. These parts of the molecule are most directly involved in the allosteric conformational change.

http://web.squ.edu.om/med-lib/med_cd/e_cds/Electronic%20Study%20Guide%20of%20Biochemistry/ch07/c07emhp.htm

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More Complexity in Protein Evolution

Posted in Alzheimer's Disease, Amino acids, Autism Spectrum Disorders, Biological Networks, Biomarkers & Medical Diagnostics, Ca2+ triggered activation, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Clinical Diagnostics, Curation, Disease Biology, Endocrine Diseases, Enzymes and isoenzymes, Etiology, Evolutionary cognition, Experimental validation, Explanatory, Gene Regulation and Evolution, Metabolism, Metabolomics, Mutant Gene Expression, Neurodegenerative Diseases, Neurohumoral Transmission, Neurological Diseases, Pharmaceutical Drug Discovery, Pharmacotherapy and Cell Activity, Proteins, Pyridine nucleotides, Signaling & Cell Circuits, Social Development, Translational Science, tagged Amyloid, amyloidogenesis, crystallins, LDH isoenzymes, lens and retina, oxytocin, oxytocin receptor, social behavior, Transthyretin on January 18, 2015| Leave a Comment »

More Complexity in Protein Evolution

Author and Curator: Larry H. Bernstein, MD, FCAP

Lactate dehydrogenase like crystallin: a potentially protective shield for Indian spiny-tailed lizard (Uromastix ltardwickit) lens against environmental stress?
A Atta, A Ilyas, Z Hashim, A Ahmed and S Zarina
The Protein Journal 2014; 33(2), p. 128-34.
http://dx.doi.org/10.1007/s10930-014-9543-4

Taxon specific lens crystallins in ve1iebrates are either similar or identical with various metabolic enzymes. These bifunctional crystallins serve as structural protein in lens along with their catalytic role. In the present study, we have partially purified and characterized lens crystallin from Indian spiny-tailed lizard (Uroma stix hardwickii). We have found lactate dehydrogenase (LDH) activity in lens indicating presence of an enzyme crystallin with dual functions. Taxon specific lens crystallins are product of gene sharing or gene duplication phenomenon where a pre-existing enzyme is recruited as lens crystallin in addition to structural role. In lens, same gene adopts refractive role in lens without modification or loss of pre-existing function during gene sharing phenomenon. Apart from conventional role of structural protein, LDH activity containing crystallin in Uromastix hardwickii lens is likely to have adaptive characteristics to offer protection against toxic effects of oxidative stress and ultraviolet light, hence justifying its recruitment. Taxon specific crystallins may serve as good models to understand structure-function relationship of these proteins.

αB-Crystallin and 27-kd Heat Shock Protein Are Regulated by Stress Conditions in the Central Nervous System and Accumulate in Rosenthal Fibers
T Iwaki, A Iwaki, J Tateishi, Y Sakaki, and JE Goldmant
Ameri J Pathol 1993; 143(2):487-495.

To understand the significance of the accumulation of αB-crystallin in Rosenthal fibers within astrocytes, the expression and metabolism of αB-crystallin in glioma cell lines were examined under the conditions of heat and oxidative stress. αB-crystallin mRNA was increased after both stresses, and αB-crystallin protein moved from a detergent-soluble to a detergent-insoluble form. In addition, Western blotting of Alexander’s disease brain homogenates revealed that the 27-kd heat shock protein (HSP27), which is related to αB-crystallin, accumulates along with αB-crystallin. The presence of HSP27 in Rosenthal fibers was directly demonstrated by immunohistochemistry. Our results suggest that astrocytes in Alexander’s disease may be involved in an as yet unknown kind of stress reaction that causes the accumulation of αB-ccystallin and HSP27 and results in Rosenthal fiber formation.

α-Crystallin can function as a molecular chaperone
Joseph Horwitz
Proc. Nadl. Acad. Sci. USA Nov 1992; 89: 10449-10453. Biochemistry

The α-crystallins (αA and αB) are major lens structural proteins of the vertebrate eye that are related to the small heat shock protein family. In addition, crystallins (especially αB) are found in many cells organs outside the lens, and aα is overexpressed in several neurological disorders and in cell lines under stress conditions. Here I show that α-crystallin can function as a molecular chaperone. Stoichiometric amounts of αA and αB suppress thermally induced aggregation of various enzymes. In particular, α-crystalln is very efficient in suppressing the thermally induced aggregation of β- and y-crystallins, the two other major mammalian stuctural lens proteins. α-Crystallin was also effective in preventing aggregation and in refolding guanidine hydrochloride-denatured y-crystallin, as judged by circular dichroism spectroscopy. My results thus indicate that α-crystallin refracts light and protects proteins from aggregation in the transparent eye lens and that in nonlens cells α-crystallin may have other functions in addition to its capacity to suppress aggregation of proteins.

Gene sharing by δ-crystallin and argininosuccinate Iyase
J Piatigorsky, WE O’Brient, BL Norman, K Kalumuckt, GJ Wistow, T Borras, et al.
Proc. Natl. Acad. Sci. USA May 1988; 85: 3479-3483. Evolution.

The lens structural protein δ-crystallin and the metabolic enzyme argininosuccinate lyase (ASL; Largininosuccinate argine-lyase, EC 4.3.2.1) have striking sequence similarity. We have demonstrated that duck δ-crystallin has enormously high ASL activity, while chicken δ-crystallin has lower but significant activity. The lenses of these birds had much greater ASL activity than other tissues, suggesting that ASL is being expressed at unusually high levels as a structural component. In Southern blots of human genomic DNA, chicken δ1-crystallin cDNA hybridized only to the human ASL gene; moreover, the two chicken δ-crystallin genes accounted for all the sequences in the chicken genome able to cross-hybridize with a human ASL cDNA, with preferential hybridization to the δ2 gene. Correlations of enzymatic activity and recent data on mRNA levels in the chicken lens suggest that ASL activity depends on expression of the δ2-crystallin gene. The data indicate that the same gene, at least in ducks, encodes two different functions, an enzyme (ASL) and a structural protein (δ-crystallin), although in chickens specialization and separation of functions may have occurred.

Gecko i-crystallin: How cellular retinol-binding protein became an eye lens ultraviolet filter
PJ L Werten, Beate Roll, DMF van Aalten, and WW de Jong
PNAS Mar 2000; 97(7): 3282–3287 http://pnas.org/cgi/doi/10.1073ypnas.050500597

Eye lenses of various diurnal geckos contain up to 12% i-crystallin. This protein is related to cellular retinol-binding protein type I (CRBP I) but has 3,4-didehydroretinol, rather than retinol, as a ligand. The 3,4-didehydroretinol gives the lens a yellow color, thus protecting the retina by absorbing short-wave radiation. i-Crystallin could be either the gecko’s housekeeping CRBP I, recruited for an additional function in the lens, or the specialized product of a duplicated CRBP I gene. The finding of the same CRBP I-like sequence in lens and liver cDNA of the gecko Lygodactylus picturatus now supports the former option. Comparison with i-crystallin of a distantly related gecko, Gonatodes vittatus, and with mammalian CRBP I, suggests that acquiring the additional lens function is associated with increased amino acid changes. Compared with the rat CRBP I structure, the i-crystallin model shows reduced negative surface charge, which might facilitate the required tight protein packing in the lens. Other changes may provide increased stability, advantageous for a long-living lens protein, without frustrating its role as retinol transporter outside the lens. Despite a number of replacements in the ligand pocket, recombinant i-crystallin binds 3,4-didehydroretinol and retinol with similar and high affinity (1.6 nM). Availability of ligand thus determines whether it binds 3,4-didehydroretinol, as in the lens, or retinol, in other tissues. i-Crystallin presents a striking example of exploiting the potential of an existing gene without prior duplication.

Expression of βA3/A1-crystallin in the developing and adult rat eye
G Parthasarathy, Bo Ma, C Zhang, C Gongora, JS Zigler, MK Duncan, D Sinha
J Molec Histol 2011; 42(1): 59-69. http://dx.doi.org:/10.1007/s10735-010-9307-1

Crystallins are very abundant structural proteins of the lens and are also expressed in other tissues. We have previously reported a spontaneous mutation in the rat βA3/A1-crystallin gene, termed Nuc1, which has a novel, complex, ocular phenotype. The current study was undertaken to compare the expression pattern of this gene during eye development in wild type and Nuc1 rats by in situ hybridization (ISH) and immunohistochemistry (IHC).
βA3/A1-crystallin expression was first detected in the eyes of both wild type and Nuc1 rats at embryonic (E) day 12.5 in the posterior portion of the lens vesicle, and remained limited to the lens fibers throughout fetal life.
After birth, βA3/A1-crystallin expression was also detected in the neural retina (specifically in the astrocytes and ganglion cells) and in the retinal pigmented epithelium (RPE).
This suggested that βA3/A1-crystallin is not only a structural protein of the lens, but has cellular function(s) in other ocular tissues.
In summary, expression of βA3/A1-crystallin is controlled differentially in various eye tissues with lens being the site of greatest expression.
Similar staining patterns, detected by ISH and IHC, in wild type and Nuc1 animals suggest that functional differences in the protein, rather than changes in mRNA/protein level of expression likely account for developmental abnormalities in Nuc1.

βA3/A1Crystallin controls anoikis-mediated cell death in astrocytes by modulating PI3K/AKT/mTOR and ERK survival pathways through the PKD/Bit1-signaling axis
B Ma, T Sen, L Asnaghi, M Valapala, F Yang, S Hose, D S McLeod, Y Lu, et la.
Cell Death and Disease 2011; 2(10). http://dx.doi.org:/10.1038/cddis.2011.100

During eye development, apoptosis is vital to the maturation of highly specialized structures such as the lens and retina. Several forms of apoptosis have been described, including anoikis, a form of apoptosis triggered by inadequate or inappropriate cell–matrix contacts. The anoikis regulators, Bit1 (Bcl-2 inhibitor of transcription-1) and protein kinase-D (PKD), are expressed in developing lens when the organelles are present in lens fibers, but are downregulated as active denucleation is initiated.
We have previously shown that in rats with a spontaneous mutation in the Cryba1 gene, coding for βA3/A1-crystallin, normal denucleation of lens fibers is inhibited. In rats with this mutation (Nuc1), both Bit1 and PKD remain abnormally high in lens fiber cells. To determine whether βA3/A1-crystallin has a role in anoikis, we induced anoikis in vitro and conducted mechanistic studies on astrocytes, cells known to express βA3/A1-crystallin.
The expression pattern of Bit1 in retina correlates temporally with the development of astrocytes. Our data also indicate that loss of βA3/A1-crystallin in astrocytes results in a failure of Bit1 to be trafficked to the Golgi, thereby suppressing anoikis. This loss of βA3/A1-crystallin also induces insulin-like growth factor-II, which increases cell survival and growth by modulating the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR and extracellular signal-regulated kinase pathways. We propose that βA3/A1-crystallin is a novel regulator of both life and death decisions in ocular astrocytes.

βA3/A1-crystallin in astroglial cells regulates retinal vascular remodeling during development
D Sinha, A Klise, Y Sergeev, S Hose, IA Bhutto, L Hackler Jr., T Malpic-llanos, et al.
Molec Cell Neurosci 2008; 37(1): 85-95.

http://dx.doi.org:/10.1016/j.mcn.2007.08.016

Vascular remodeling is a complex process critical to development of the mature vascular system. Astrocytes are known to be indispensable for initial formation of the retinal vasculature; our studies with the Nuc1 rat provide novel evidence that these cells are also essential in the retinal vascular remodeling process.
Nuc1 is a spontaneous mutation in the Sprague–Dawley rat originally characterized by nuclear cataracts in the heterozygote and microphthalmia in the homozygote. We report here that the Nuc1 allele results from mutation of the βA3/A1-crystallin gene, which in the neural retina is expressed only in astrocytes. We demonstrate striking structural abnormalities in Nuc1 astrocytes with profound effects on the organization of intermediate filaments. While vessels form in the Nuc1 retina, the subsequent remodeling process required to provide a mature vascular network is deficient. Our data implicate βA3/A1-crystallin as an important regulatory factor mediating vascular patterning and remodeling in the retina.

A developmental defect in astrocytes inhibits programmed regression of the hyaloid vasculature in the mammalian eye
C Zhang, L Asnaghi, C Gongora, B Patek, S Hose, Bo Ma, MA Fard, L Brako, et al.
Eur J Cell Biol 2011; 90(5): 440-448.
http://dx.doi.org:/10.1016/j.ejcb.2011.01.003

Previously we reported the novel observation that astrocytes ensheath the persistent hyaloid artery, both in the Nuc1 spontaneous mutant rat, and in human PFV (persistent fetal vasculature) disease (Developmental Dynamics 234:36–47, 2005). We now show that astrocytes isolated from both the optic nerve and retina of Nuc1 rats migrate faster than wild type astrocytes. Aquaporin 4 (AQP4), the major water channel in astrocytes, has been shown to be important in astrocyte migration. We demonstrate that AQP4 expression is elevated in the astrocytes in PFV conditions, and we hypothesize that this causes the cells to migrate abnormally into the vitreous where they ensheath the hyaloid artery. This abnormal association of astrocytes with the hyaloid artery may impede the normal macrophage-mediated remodeling and regression of the hyaloid system.

βA3/A1-crystallin is required for proper astrocyte template formation and vascular remodeling in the retina.
D Sinha; WJ Stark; M Valapala; IA Bhutto; M Cano; S Hose; GA Lutty; et al. Transgenic research 2012; 21(5):1033-42.

Nuc1 is a spontaneous rat mutant resulting from a mutation in the Cryba1 gene, coding for βA3/A1-crystallin. Our earlier studies with Nuc1 provided novel evidence that astrocytes, which express βA3/A1-crystallin, have a pivotal role in retinal remodeling. The role of astrocytes in the retina is only beginning to be explored. One of the limitations in the field is the lack of appropriate animal models to better investigate the function of astrocytes in retinal health and disease. We have now established transgenic mice that overexpress the Nuc1 mutant form of Cryba1, specifically in astrocytes. Astrocytes in wild type mice show normal compact stellate structure, producing a honeycomb-like network. In contrast, in transgenics over-expressing the mutant (Nuc1) Cryba1 in astrocytes, bundle-like structures with abnormal patterns and morphology were observed. In the nerve fiber layer of the transgenic mice, an additional layer of astrocytes adjacent to the vitreous is evident. This abnormal organization of astrocytes affects both the superficial and deep retinal vascular density and remodeling. Fluorescein angiography showed increased venous dilation and tortuosity of branches in the transgenic retina, as compared to wild type. Moreover, there appear to be fewer interactions between astrocytes and endothelial cells in the transgenic retina than in normal mouse retina. Further, astrocytes overexpressing the mutant βA3/A1-crystallin migrate into the vitreous, and ensheath the hyaloid artery, in a manner similar to that seen in the Nuc1 rat. Together, these data demonstrate that developmental abnormalities of astrocytes can affect the normal remodeling process of both fetal and retinal vessels of the eye and that βA3/A1-crystallin is essential for normal astrocyte function in the retina.

Ontogeny of oxytocin and vasopressin receptor binding in the lateral septum in prairie and montane voles
Z. Wang, L.J. Young
Developmental Brain Research 1997; 104:191–195.

Adult prairie (Microtus ochrogaster). and montane voles (M. montanus). differ in the distribution of oxytocin OT. and vasopressin AVP receptor binding in the brain. The present study examined the ontogenetic pattern of these receptor bindings in the lateral septum in both species to determine whether adult differences in the receptor binding are derived from a common pattern in development. In both species, OT and AVP receptor binding in the lateral septum were detected neonatally, increased during development, and reached the adult level at weaning third week. The progression of OT and AVP receptor differed, as OT receptor binding increased continually until weaning while AVP receptor binding did not change in the first week, increased rapidly in the second week, and was sustained thereafter. For both receptors, the binding increased more rapidly in montane than in prairie voles, resulting in species differences in receptor binding at weaning and in adulthood. Together, these data indicate that OT and AVP could affect the brain during development in a peptide- and species-specific manner in voles.

Evolution of the vasopressin/oxytocin superfamily: Characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis
RE Van Kesteren, AB Smit, RW Dirksi, ND De With, WPM Geraerts, and J Joosse
Proc. Nadl. Acad. Sci. USA May 1992; 89: 4593-4597. Neurobiology

Although the nonapeptide hormones vasopressin, oxytocin, and related peptides from vertebrates and some nonapeptides from invertebrates share similarities in amino acid sequence, their evolutionary relationships are not dear. To investigate this issue, we doned a cDNA encoding a vasopressin-related peptide, Lys-conopressin, produced in the central nervous system of the gastropod mollusc Lymnaea stagnalis. The predicted preproconopressin has the overall architecture of vertebrate preprovasopressins, with a signal peptide, Lys-conopressin, that is flanked at the C terminus by an amidation signal and a pair of basic residues, followed by a neurophysin domain. The Lymnaea neurophysin and the vertebrate neurophysins share high sequence identity, which includes the conservation of all 14 cysteine residues. In addition, the Lymnaea neurophysin possesses unique structural characteristics. It contains a putative N-linked glycosylation site at a position in the vertebrate neurophysins where a strictly conserved tyrosine residue, which plays an essential role in binding of the nonapptide hormones, is found. The C-terminal copeptin homologous extension of the Lymnaea neurophysin has low sequence identity with the vertebrate counterparts and is probably not cleaved from the prohormone, as are the mammalin copeptins. The conopressin gene is expressed in only a few neurons in both pedal ganglia of the central nervous system. The conopressin transcript is present in two sizes, due to alternative use of polyadenylylation signals. The data presented here demonstrate that the typical organization of the prohormones of the vasopressin/oxytocin superfamily must have been present in the common ancestors of vertebrates and invertebrates.

A common allele in the oxytocin receptor gene (OXTR) impacts prosocial temperament and human hypothalamic-limbic structure and function
H Tosta, B Kolachanaa, S Hakimia, H Lemaitrea, BA Verchinskia, et al.
PNAS Aug 3, 2010; 107(31): 13936–13941
http://pnas.org/cgi/doi/10.1073/pnas.1003296107

The evolutionarily highly conserved neuropeptide oxytocin is a key mediator of social and emotional behavior in mammals, including humans. A common variant (rs53576) in the oxytocin receptor gene (OXTR) has been implicated in social-behavioral phenotypes, such as maternal sensitivity and empathy, and with neuropsychiatric disorders associated with social impairment, but the intermediate neural mechanisms are unknown. Here, we used multimodal neuroimaging in a large sample of healthy human subjects to identify structural and functional alterations in OXTR risk allele carriers and their link to temperament. Activation and interregional coupling of the amygdala during the processing of emotionally salient social cues was significantly affected by genotype. In addition, evidence for structural alterations in key oxytocinergic regions emerged, particularly in the hypothalamus. These neural characteristics predicted lower levels of reward dependence, specifically in male risk allele carriers. Our findings identify sex-dependent mechanisms impacting the structure and function of hypothalamic-limbic circuits that are of potential clinical and translational significance.
Test of Association Between 10 SNPs in the Oxytocin Receptor Gene and Conduct Disorder
JT Sakai, TJ Crowley, MC Stallings, M McQueen, JK Hewitt, C Hopfer, et al.
Psychiatr Genet. 2012 Apr; 22(2): 99–102. http://dx.doi.org:/10.1097/YPG.0b013e32834c0cb2

Animal and human studies have implicated oxytocin (OXT) in affiliative and prosocial behaviors. We tested whether genetic variation in the OXT receptor (OXTR) gene is associated with conduct disorder (CD).
Utilizing a family-based sample of adolescent probands recruited from an adolescent substance abuse treatment program, control probands and their families (total sample n=1,750), we conducted three tests of association with CD and 10 SNPs (single nucleotide polymorphisms) in the OXTR gene: (1) family-based comparison utilizing the entire sample; (2) within-Whites, case control comparison of adolescent patients with CD and controls without CD; and (3) within-Whites case-control comparison of parents of patients and parents of controls.
Family-based association tests failed to show significant results (no results p<0.05). While strictly correcting for the number of tests (α=0.002), adolescent patients with CD did not differ significantly from adolescent controls in genotype frequency for the OXTR SNPs tested; similarly, comparison of OXTR genotype frequencies for parents failed to differentiate patient and control family type, except a trend association for rs237889 (p=0.004). In this sample, 10 SNPs in the OXTR gene were not significantly associated with CD.

Leu55Pro transthyretin accelerates subunit exchange and leads to rapid formation of hybrid tetramers
CA Keetch, EHC Bromley, MG McCammon, N Wang, J Christodoulou, CV Robinson
JBC Oct 11, 2005 M508753200. http://jbc.org/cgi/doi/10.1074/jbc.M508753200

Transthyretin is a tetrameric protein associated with the commonest form of

systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry we compared subunit exchange in wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, Leu55Pro. Wild-type subunit exchange occurs via both monomers and dimers , while exchange via dimers is the dominant mechanism for the Leu55Pro variant. Since patients with the Leu55Pro mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and Leu55Pro tetramers . We found that hybrid tetramers containing two or three Leu55Pro subunits dominate in the early stages of the reaction. Surprisingly we also found that in the presence of Leu55Pro transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidos is.

Beyond Genetic Factors in Familial Amyloidotic Polyneuropathy: Protein Glycation and the Loss of Fibrinogen’s Chaperone Activity
G da Costa, RA Gomes, A Guerreiro, E Mateus, E Monteiro, et al.
PLoS ONE 2011; 6(10): e24850. http://dx.doi.org:/10.1371/journal.pone.0024850

Familial amyloidotic polyneuropathy (FAP) is a systemic conformational disease characterized by extracellular amyloid fibril formation from plasma transthyretin (TTR). This is a crippling, fatal disease for which liver transplantation is the only effective therapy. More than 80 TTR point mutations are associated with amyloidotic diseases and the most widely accepted disease model relates TTR tetramer instability with TTR point mutations. However, this model fails to explain two observations. First, native TTR also forms amyloid in systemic senile amyloidosis, a geriatric disease. Second, age at disease onset varies by decades for patients bearing the same mutation and some mutation carrier individuals are asymptomatic throughout their lives. Hence, mutations only accelerate the process and non-genetic factors must play a key role in the molecular mechanisms of disease. One of these factors is protein glycation, previously associated with conformational diseases like Alzheimer’s and Parkinson’s. The glycation hypothesis in FAP is supported by our previous discovery of methylglyoxal-derived glycation of amyloid fibrils in FAP patients. Here we show that plasma proteins are differentially glycated by methylglyoxal in FAP patients and that fibrinogen is the main glycation target. Moreover, we also found that fibrinogen interacts with TTR in plasma. Fibrinogen has chaperone activity which is compromised upon glycation by methylglyoxal. Hence, we propose that methylglyoxal glycation hampers the chaperone activity of fibrinogen, rendering TTR more prone to aggregation, amyloid formation and ultimately, disease.

Aromatic Sulfonyl Fluorides Covalently Kinetically Stabilize Transthyretin to Prevent Amyloidogenesis while Affording a Fluorescent Conjugate
NP Grimster, S Connelly, A Baranczak, J Dong, …, JW Kelly
J Am Chem Soc. 2013 Apr 17; 135(15): 5656–5668. http://dx.doi.org:/10.1021/ja311729d

Molecules that bind selectively to a given protein and then undergo a rapid chemoselective reaction to form a covalent conjugate have utility in drug development. Herein a library of 1,3,4-oxadiazoles substituted at the 2 position with an aryl sulfonyl fluoride and at the 5 position with a substituted aryl known to have high affinity for the inner thyroxine binding subsite of transthyretin (TTR) were conceived of by structure-based design principles and were chemically synthesized. When bound in the thyroxine binding site, most of the aryl sulfonyl fluorides react rapidly and chemoselectively with the pKa-perturbed K15 residue, kinetically stabilizing TTR and thus preventing amyloid fibril formation, known to cause polyneuropathy. Conjugation t50s range from 1 to 4 min, ~ 1400 times faster than the hydrolysis reaction outside the thyroxine binding site. Xray crystallography confirms the anticipated binding orientation and sheds light on the sulfonyl fluoride activation leading to the sulfonamide linkage to TTR. A few of the aryl sulfonyl fluorides efficiently form conjugates with TTR in plasma. A few of the TTR covalent kinetic stabilizers synthesized exhibit fluorescence upon conjugation and therefore could have imaging applications as a consequence of the environment sensitive fluorescence of the chromophore.

Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis
A Lim, T Prokaeva, ME Mccomb, LH Connors, M Skinner, and CE Costello
Protein Science 2003; 12:1775–1786.
http://proteinscience.org/cgi/doi/10.1110/ps.0349703.

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild-type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease. Previously, we have developed a mass spectrometry-based approach, in combination with direct DNA sequence analysis, to fully identify TTR variants. Our methodology uses immunoprecipitation to isolate TTR from serum, and electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry (MS) peptide mapping to identify TTR variants and posttranslational modifications. Unambiguous identification of the amino acid substitution is performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis. The MS and MS/MS analyses also yield information about posttranslational modifications. Using this approach, we have recently identified a novel pathologic TTR variant. This variant has an amino acid substitution (Phe — Cys) at position 33. In addition, like the Cys10 present in the wild type and in this variant, the Cys33 residue was both S-sulfonated and S-thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.

Evolutionary relationships of lactate dehydrogenases (LDHs) from mammals, birds, an amphibian, fish, barley, and bacteria: LDH cDNA sequences from Xenopus, pig, and rat
S Tsuji, MA Qureshi, EW Hou, WM Fitch, and S S.-L. Li
Proc. Natl. Acad. Sci. USA Sep 1994; 91: 9392-9396. Evolution

The nucleotide sequences of the cDNAs encoding LDH (EC 1.1.1.27) subunits LDH-A (muscle), LDH-B (liver), and LDH-C (oocyte) from Xenopus laevis, LDH-A (muscle) and LDH-B (heart) from pig, and LDH-B (heart) and LDH-C (testis) from rat were determined. These seven newly deduced amino acid sequences and 22 other published LDH sequences, and three unpublished fish LDH-A sequences kindly provided by G. N. Somero and D. A. Powers, were used to construct the most parsimonious phylogenetic tree of these 32 LDH subunits from mammals, birds, an amphibian, fish, barley, and bacteria. There have been at least six LDH gene duplications among the vertebrates. The Xenopus LDH-A, LDH-B, and LDH-C subunits are most closely related to each other and then are more closely related to vertebrate LDH-B than LDH-A. Three fish LDH-As, as well as a single LDH of lamprey, also seem to be more related to vertebrate LDH-B than to land vertebrate LDH-A. The mammalian LDH-C (testis) subunit appears to have diverged very early, prior to the divergence of vertebrate LDH-A and LDH-B subunits, as reported previously.

Evidence for neutral and selective processes in the recruitment of enzyme-crystallins in avian lenses
Graeme Wistow, Andrea Anderson, and Joram Piatigorsky
Proc. Natl. Acad. Sci. USA Aug 1990; 87: 6277-6280, Evolution

In apparent contrast to most other tissues, the ocular lenses in vertebrates show striking differences in protein composition between taxa, most notably in the recruitment of different enzymes as major structural proteins. This variability appears to be the result of at least partially neutral evolutionary processes, although there is also evidence for selective modification in molecular structure. Here we describe a bird, the chimney swift (Chaetura pelagica), that lacks δ-crystallin/ argininosuccinate lyase, usually the major crystallin of avian lenses. Clearly, δ-crystallin is not specifically required for a functionally effective avian lens. Furthermore the lens composition of the swift is more similar to that of the related hummingbirds than to that of the barn swallow (Hirundo rustica), suggesting that phylogeny is more important than environmental selection in the recruitment of crystallins. However differences in ε-crystallin/lactate dehydrogenase-B sequence between swift and hummingbird and other avian and reptilian species suggest that selective pressures may also be working at the molecular level. These differences also confirm the close relationship between swifts and hummingbirds.

Enzyme/crystallins and extremely high pyridine nucleotide levels in the eye lens.
Zigler, J. S., Jr.; Rao, P. V.
FASEB J. 1991; 3: 223-225.

Taxon-specific crystallins are proteins present in high abundance in the lens of phylogenetically restricted groups of animals. Recently it has been found that these proteins are actually enzymes which the lens has apparently adopted to serve as structural proteins. Most of these proteins have been shown to be identical to, or related to, oxidoreductases. In guinea pig lens, which contains zeta-crystallin, a protein with an NADPH dependent oxidoreductase activity, the levels of both NADPH and NADP* are extremely high and correlate with the concentration of zeta-crystallin. We report here nucleotide assays on lenses from vertebrates containing other enzyme/crystallins. In each case where the enzyme/crystallin is a pyridine nucleotide-binding protein the level of that particular nucleotide is extremely high in the lens. The presence of an enzyme/crystallin does not affect the lenticular concentrations of those nucleotides which are not specifically bound. The possibility that nucleotide binding may be a factor in the selection of some enzymes to serve as enzyme/crystallins is considered.

Comparison of stability properties of lactate dehydrogenase B4/ε-crystallin from different species
CEM Voorter, LTM Wintjes, PWH Heinstra, H Bloemendal and WW De Jong
Eur. J. Biochem. 1993; 211: 643-648

ε-Crystallin occurs as an abundant lens protein in many birds and in crocodiles and has been identified as heart-type lactate dehydrogenase (LDH-B4). Lens proteins have, due to their longevity and environmental conditions, extraordinary requirements for structural stability. To study lens protein stability, we compared various parameters of LDH-B4/ε-crystallin from lens and/or heart of duck, which has abundant amounts of this enzyme in its lenses, and of chicken and pig, which have no λ-crystallin. Measuring the thermostability of LDH-B4 from the different sources, the t₅₀ values (temperature at which 50% of the enzyme activity remains after a 20-min period) for LDH-B4 from duck heart, duck lens and chicken heart were all found to be around 76°C whereas pig heart LDHB4 was less thermostable, having a t₅₀ value of 625°C. A similar tendency was found with urea inactivation studies. Plotting the first-order rate constants obtained from inactivation kinetic plots against urea concentration, it was clear that LDH-B4 from pig heart was less stable in urea than the homologous enzymes from duck heart, chicken heart and duck lens. The duck and chicken enzymes were also much more resistant against proteolysis than the porcine enzyme. Therefore, it is concluded that avian LDH-B4 is structurally more stable than the homologous enzyme in mammals. This greater stability might make it suitable to function as a ε-crystallin, as in duck, but is not necessarily associated with high lens expression, as in chicken.

Duck lens ε-crystallin and lactate dehydrogenase B4 are identical: A single-copy gene product with two distinct functions
W Hendriks, JWM Mulders, MA Bibby, C Slingsby, H Bloemendal, and WW De Jong
Proc. Natl. Acad. Sci. USA Oct 1988; 85: 7114-7118. Biochemistry

To investigate whether or not duck lens ε-crystaliin and duck heart lactate dehydrogenase (LDH) B4 are the product of the same gene, we have isolated and sequenced cDNA clones of duck ε-crystallin. By using these clones we demonstrate that there is a single-copy Ldh-B gene in duck and in chicken. In the duck lens this gene is overexpressed, and its product is subject to posttranslational modification. Reconstruction of the evolutionary history of the LDH protein family reveals that the mammalian Ldh-C gene most probably originated from an ancestral Ldh-A gene and that the amino acid replacement rate in LDH-C is approximately 4 times the rate in LDH-A. Molecular modeling of LDH-B sequences shows that the increased thermostability of the avian tetramer might be explained by mutations that increase the number of ion pairs. Furthermore, the replacement of bulky side chains by glycines on the corners of the duck protein suggests an adaptation to facilitate close packing in the lens.

Lactate Dehydrogenase A as a Highly Abundant Eye Lens Protein in Platypus (Ornithorhynchus anatinus): Upsilon (υ)-Crystallin
T van Rheede, R Amons, N Stewart, and WW de Jong
Mol. Biol. Evol. 2003; 20(06):994–998. http://dx.doi.org:/10.1093/molbev/msg116

Vertebrate eye lenses mostly contain two abundant types of proteins, the α-crystallins and the β/λ-crystallins. In addition, certain housekeeping enzymes are highly expressed as crystallins in various taxa. We now observed an unusual approximately 41-kd protein that makes up 16% to 18% of the total protein in the platypus eye lens. Its cDNA sequence was determined, which identified the protein as muscle-type lactate dehydrogenase A (LDH-A). It is the first observation of LDH-A as a crystallin, and we designate it upsilon (υ)-crystallin. Interestingly, the related heart-type LDH-B occurs as an abundant lens protein, known as ε-crystallin, in many birds and crocodiles. Thus, two members of the ldh gene family have independently been recruited as crystallins in different higher vertebrate lineages, suggesting that they are particularly suited for this purpose in terms of gene regulatory or protein structural properties. To establish whether platypus LDH-A/υ-crystallin has been under different selective constraints as compared with other vertebrate LDH-A sequences, we reconstructed the vertebrate Ldh-A gene phylogeny. No conspicuous rate deviations or amino acid replacements were observed.

Isozymes, moonlighting proteins and promiscous enzymes
M Nath Gupta, M Kapoor, AB Majumder and V Singh
Current Science Apr 2011; 100(8): 1152-1162.

The structures of isoenzymes differ and yet these catalyse the same type of reaction. These structures evolved to suit the physiological needs and are located in different parts of cells or tissues. Moonlighting proteins represent the same structure performing very different biological functions. Biological promiscuity reveals that the same active sites can catalyse different types of reactions. These three different phenomena, all illustrate similar evolutionary strategies. Viewed together, it emerges that biologists need to take a hard look at the ‘structure–function’ paradigm as well as the notions of biological specificity. Meanwhile, biotechnologists continue to exploit the opportunities which ‘nonspecificity’ offers.

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Summary of Proteomics

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Summary of Proteomics

Author and Curator: Larry H. Bernstein, MD, FCAP

We have completed a series of discussions on proteomics, a scientific endeavor that is essentially 15 years old. It is quite remarkable what has been accomplished in that time. The interest is abetted by the understanding of the limitations of the genomic venture that has preceded it. The thorough, yet incomplete knowledge of the genome, has led to the clarification of its limits. It is the coding for all that lives, but all that lives has evolved to meet a demanding and changing environment with respect to

availability of nutrients
salinity
temperature
radiation exposure
toxicities in the air, water, and food
stresses – both internal and external

We have seen how both transcription and translation of the code results in a protein, lipoprotein, or other complex than the initial transcript that was modeled from tRNA. What you see in the DNA is not what you get in the functioning cell, organ, or organism. There are comparabilities as well as significant differences between plants, prokaryotes, and eukaryotes. There is extensive variation. The variation goes beyond genomic expression, and includes the functioning cell, organ type, and species.

Here, I return to the introductory discussion. Proteomics is a goal directed, sophisticated science that uses a combination of methods to find the answers to biological questions. Graves PR and Haystead TAJ. Molecular Biologist’s Guide to Proteomics.
Microbiol Mol Biol Rev. Mar 2002; 66(1): 39–63. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC120780/

Peptide mass tag searching

Peptide mass tag searching. Shown is a schematic of how information from an unknown peptide (top) is matched to a peptide sequence in a database (bottom) for protein identification. The partial amino acid sequence or “tag” obtained by MS/MS is combined with the peptide mass (parent mass), the mass of the peptide at the start of the sequence (mass tag 1), and the mass of the peptide at the end of the sequence (mass tag 2). The specificity of the protease used (trypsin is shown) can also be included in the search.

ICAT method for measuring differential protein expression

The ICAT method for measuring differential protein expression. (A) Structure of the ICAT reagent. ICAT consists of a biotin affinity group, a linker region that can incorporate heavy (deuterium) or light (hydrogen) atoms, and a thiol-reactive end group for linkage to cysteines. (B) ICAT strategy. Proteins are harvested from two different cell states and labeled on cysteine residues with either the light or heavy form of the ICAT reagent. Following labeling, the two protein samples are mixed and digested with a protease such as trypsin. Peptides labeled with the ICAT reagent can be purified by virtue of the biotin tag by using avidin chromatography. Following purification, ICAT-labeled peptides can be analyzed by MS to quantitate the peak ratios and proteins can be identified by sequencing the peptides with MS/MS.

Strategies for determination of phosphorylation sites in proteins

Strategies for determination of phosphorylation sites in proteins. Proteins phosphorylated in vitro or in vivo can be isolated by protein electrophoresis and analyzed by MS. (A) Identification of phosphopeptides by peptide mass fingerprinting. In this method, phosphopeptides are identified by comparing the mass spectrum of an untreated sample to that of a sample treated with phosphatase. In the phosphatase-treated sample, potential phosphopeptides are identified by a decrease in mass due to loss of a phosphate group (80 Da). (B) Phosphorylation sites can be identified by peptide sequencing using MS/MS. (C) Edman degradation can be used to monitor the release of inorganic ³²P to provide information about phosphorylation sites in peptides.

protein mining strategy

Proteome-mining strategy. Proteins are isolated on affinity column arrays from a cell line, organ, or animal source and purified to remove nonspecific adherents. Then, compound libraries are passed over the array and the proteins eluted are analyzed by protein electrophoresis. Protein information obtained by MS or Edman degradation is then used to search DNA and protein databases. If a relevant target is identified, a sublibrary of compounds can be evaluated to refine the lead. From this method a protein target and a drug lead can be simultaneously identified.

Although the technology for the analysis of proteins is rapidly progressing, it is still not feasible to study proteins on a scale equivalent to that of the nucleic acids. Most of proteomics relies on methods, such as protein purification or PAGE, that are not high-throughput methods. Even performing MS can require considerable time in either data acquisition or analysis. Although hundreds of proteins can be analyzed quickly and in an automated fashion by a MALDI-TOF mass spectrometer, the quality of data is sacrificed and many proteins cannot be identified. Much higher quality data can be obtained for protein identification by MS/MS, but this method requires considerable time in data interpretation. In our opinion, new computer algorithms are needed to allow more accurate interpretation of mass spectra without operator intervention. In addition, to access unannotated DNA databases across species, these algorithms should be error tolerant to allow for sequencing errors, polymorphisms, and conservative substitutions. New technologies will have to emerge before protein analysis on a large-scale (such as mapping the human proteome) becomes a reality.

Another major challenge for proteomics is the study of low-abundance proteins. In some eukaryotic cells, the amounts of the most abundant proteins can be 10⁶-fold greater than those of the low-abundance proteins. Many important classes of proteins (that may be important drug targets) such as transcription factors, protein kinases, and regulatory proteins are low-copy proteins. These low-copy proteins will not be observed in the analysis of crude cell lysates without some purification. Therefore, new methods must be devised for subproteome isolation.

Tissue Proteomics for the Next Decade? Towards a Molecular Dimension in Histology

R Longuespe´e, M Fle´ron, C Pottier, F Quesada-Calvo, Marie-Alice Meuwis, et al.
OMICS A Journal of Integrative Biology 2014; 18: 9. http://dx.doi.org:/10.1089/omi.2014.0033

The concept of tissues appeared more than 200 years ago, since textures and attendant differences were described within the whole organism components. Instrumental developments in optics and biochemistry subsequently paved the way to transition from classical to molecular histology in order to decipher the molecular contexts associated with physiological or pathological development or function of a tissue. In 1941, Coons and colleagues performed the first systematic integrated examination of classical histology and biochemistry when his team localized pneumonia antigens in infected tissue sections. Most recently, in the early 21st century, mass spectrometry (MS) has progressively become one of the most valuable tools to analyze biomolecular compounds. Currently, sampling methods, biochemical procedures, and MS instrumentations
allow scientists to perform ‘‘in depth’’ analysis of the protein content of any type of tissue of interest. This article reviews the salient issues in proteomics analysis of tissues. We first outline technical and analytical considerations for sampling and biochemical processing of tissues and subsequently the instrumental possibilities for proteomics analysis such as shotgun proteomics in an anatomical context. Specific attention concerns formalin fixed and paraffin embedded (FFPE) tissues that are potential ‘‘gold mines’’ for histopathological investigations. In all, the matrix assisted laser desorption/ionization (MALDI) MS imaging, which allows for differential mapping of hundreds of compounds on a tissue section, is currently the most striking evidence of linkage and transition between ‘‘classical’’ and ‘‘molecular’’ histology. Tissue proteomics represents a veritable field of research and investment activity for modern biomarker discovery and development for the next decade.

Progressively, tissue analyses evolved towards the description of the whole molecular content of a given sample. Currently, mass spectrometry (MS) is the most versatile
analytical tool for protein identification and has proven its great potential for biological and clinical applications. ‘‘Omics’’ fields, and especially proteomics, are of particular
interest since they allow the analysis of a biomolecular picture associated with a given physiological or pathological state. Biochemical techniques were then adapted for an optimal extraction of several biocompounds classes from tissues of different natures.

Laser capture microdissection (LCM) is used to select and isolate tissue areas of interest for further analysis. The developments of MS instrumentations have then definitively transformed the scientific scene, pushing back more and more detection and identification limits. Since a few decades, new approaches of analyses appeared, involving the use of tissue sections dropped on glass slides as starting material. Two types of analyses can then be applied on tissue sections: shotgun proteomics and the very promising MS imaging (MSI) using Matrix Assisted Laser Desorption/Ionization (MALDI) sources. Also known as ‘‘molecular histology,’’ MSI is the most striking hyphen between histology and molecular analysis. In practice, this method allows visualization of the spatial distribution of proteins, peptides, drugs, or others analytes directly on tissue sections. This technique paved new ways of research, especially in the field of histopathology, since this approach appeared to be complementary to conventional histology.

Tissue processing workflows for molecular analyses

Tissue processing workflows for molecular analyses. Tissues can either be processed in solution or directly on tissue sections. In solution, processing involves protein
extraction from tissue pieces in order to perform 2D gel separation and identification of proteins, shotgun proteomics, or MALDI analyses. Extracts can also be obtained from
tissues area selection and protein extraction after laser micro dissection or on-tissue processing. Imaging techniques are dedicated to the morphological characterization or molecular mapping of tissue sections. Histology can either be conducted by hematoxylin/eosin staining or by molecular mapping using antibodies with IHC. Finally, mass spectrometry imaging allows the cartography of numerous compounds in a single analysis. This approach is a modern form of ‘‘molecular histology’’ as it grafts, with the use of mathematical calculations, a molecular dimension to classical histology. (AR, antigen retrieval; FFPE, formalin fixed and paraffin embedded; fr/fr, fresh frozen; IHC, immunohistochemistry; LCM, laser capture microdissection; MALDI, matrix assisted laser desorption/ionization; MSI, mass spectrometry imaging; PTM, post translational modification.)

Analysis of tissue proteomes has greatly evolved with separation methods and mass spectrometry instrumentation. The choice of the workflow strongly depends on whether a bottom-up or a top-down analysis has to be performed downstream. In-gel or off-gel proteomics principally differentiates proteomic workflows. The almost simultaneous discoveries of the MS ionization sources (Nobel Prize awarded) MALDI (Hillenkamp and Karas, 1990; Tanaka et al., 1988) and electrospray ionization (ESI) (Fenn et al., 1989) have paved the way for analysis of intact proteins and peptides. Separation methods such as two-dimension electrophoresis (2DE) (Fey and Larsen, 2001) and nanoscale reverse phase liquid chromatography (nanoRP-LC) (Deterding et al., 1991) lead to efficient preparation of proteins for respectively topdown and bottom-up strategies. A huge panel of developments was then achieved mostly for LC-MS based proteomics in order to improve ion fragmentation approaches and peptide
identification throughput relying on database interrogation. Moreover, approaches were developed to analyze post translational modifications (PTM) such as phosphorylations (Ficarro et al., 2002; Oda et al., 2001; Zhou et al., 2001) or glycosylations (Zhang et al., 2003), proposing as well different quantification procedures. Regarding instrumentation, the most cutting edge improvements are the gain of mass accuracy for an optimal detection of the eluted peptides during LC-MS runs (Mann and Kelleher, 2008; Michalski et al., 2011) and the increase in scanning speed, for example with the use of Orbitrap analyzers (Hardman and Makarov, 2003; Makarov et al., 2006; Makarov et al., 2009; Olsen et al., 2009). Ion transfer efficiency was also drastically improved with the conception of ion funnels that homogenize the ion transmission
capacities through m/z ranges (Kelly et al., 2010; Kim et al., 2000; Page et al., 2006; Shaffer et al., 1998) or by performing electrospray ionization within low vacuum (Marginean et al., 2010; Page et al., 2008; Tang et al., 2011). Beside collision induced dissociation (CID) that is proposed for many applications (Li et al., 2009; Wells and McLuckey, 2005), new fragmentation methods were investigated, such as higher-energy collisional dissociation (HCD) especially for phosphoproteomic
applications (Nagaraj et al., 2010), and electron transfer dissociation (ETD) and electron capture dissociation (ECD) that are suited for phospho- and glycoproteomics (An
et al., 2009; Boersema et al., 2009; Wiesner et al., 2008). Methods for data-independent MS2 analysis based on peptide fragmentation in given m/z windows without precursor selection neither information knowledge, also improves identification throughput (Panchaud et al., 2009; Venable et al., 2004), especially with the use of MS instruments with high resolution and high mass accuracy specifications (Panchaud et al., 2011). Gas fractionation methods such as ion mobility (IM) can also be used as a supplementary separation dimension which enable more efficient peptide identifications (Masselon et al., 2000; Shvartsburg et al., 2013; Shvartsburg et al., 2011).

Microdissection relies on a laser ablation principle. The tissue section is dropped on a plastic membrane covering a glass slide. The preparation is then placed into a microscope
equipped with a laser. A highly focused beam will then be guided by the user at the external limit of the area of interest. This area composed by the plastic membrane, and the tissue section will then be ejected from the glass slide and collected into a tube cap for further processing. This mode of microdissection is the most widely used due to its ease of handling and the large panels of devices proposed by constructors. Indeed, Leica microsystem proposed the Leica LMD system (Kolble, 2000), Molecular Machine and Industries, the MMI laser microdissection system Microcut, which was used in combination with IHC (Buckanovich et al., 2006), Applied Biosystems developed the Arcturus
microdissection System, and Carl Zeiss patented P.A.L.M. MicroBeam technology (Braakman et al., 2011; Espina et al., 2006a; Espina et al., 2006b; Liu et al., 2012; Micke
et al., 2005). LCM represents a very adequate link between classical histology and sampling methods for molecular analyses as it is a simple customized microscope. Indeed,
optical lenses of different magnification can be used and the method is compatible with classical IHC (Buckanovich et al., 2006). Only the laser and the tube holder need to be
added to the instrumentation.

After microdissection, the tissue pieces can be processed for analyses using different available MS devices and strategies. The simplest one consists in the direct analysis of the
protein profiles by MALDI-TOF-MS (MALDI-time of flight-MS). The microdissected tissues are dropped on a MALDI target and directly covered by the MALDI matrix (Palmer-Toy et al., 2000; Xu et al., 2002). This approach was already used in order to classify breast cancer tumor types (Sanders et al., 2008), identify intestinal neoplasia protein biomarkers (Xu et al., 2009), and to determine differential profiles in glomerulosclerosis (Xu et al., 2005).

Currently the most common proteomic approach for LCM tissue analysis is LC-MS/MS. Label free LC-MS approaches have been used to study several cancers like head and neck squamous cell carcinomas (Baker et al., 2005), esophageal cancer (Hatakeyama et al., 2006), dysplasic cervical cells (Gu et al., 2007), breast carcinoma tumors (Hill et al., 2011; Johann et al., 2009), tamoxifen-resistant breast cancer cells (Umar et al., 2009), ER + / – breast cancer cells (Rezaul et al., 2010), Barretts esophagus (Stingl et al., 2011), and ovarian endometrioid cancer (Alkhas et al., 2011). Different isotope labeling methods have been used in order to compare proteins expression. ICAT was first used to investigate proteomes of hepatocellular carcinoma (Li et al., 2004; 2008). The O16/O18 isotopic labeling was then used for proteomic analysis of ductal carcinoma of the breast (Zang et al., 2004).

Currently, the lowest amount of collected cells for a relevant single analysis using fr/fr breast cancer tissues was 3000–4000 (Braakman et al., 2012; Liu et al., 2012; Umar et al., 2007). With a Q-Exactive (Thermo, Waltham) mass spectrometer coupled to LC, Braakman was able to identify up to 1800 proteins from 4000 cells. Processing
of FFPE microdissected tissues of limited sizes still remains an issue which is being addressed by our team.

Among direct tissue analyses modes, two categories of investigations can be done. MALDI profiling consists in the study of molecular localization of compounds and can be
combined with parallel shotgun proteomic methods. Imaging methods give less detailed molecular information, but is more focused on the accurate mapping of the detected compounds through tissue area. In 2007, a concept of direct tissue proteomics (DTP) was proposed for high-throughput examination of tissue microarray samples. However, contrary to the classical workflow, tissue section chemical treatment involved a first step of scrapping each FFPE tissue spot with a razor blade from the glass slide. The tissues were then transferred into a tube and processed with RIPA buffer and finally submitted to boiling as an AR step (Hwang et al., 2007). Afterward, several teams proved that it was possible to perform the AR directly on tissue sections. These applications were mainly dedicated to MALDI imaging analyses (Bonnel et al., 2011; Casadonte and Caprioli, 2011; Gustafsson et al., 2010). However, more recently, Longuespe´e used citric acid antigen retrieval (CAAR) before shotgun proteomics associated to global profiling proteomics (Longuespee et al., 2013).

MALDI imaging workflow

MALDI imaging workflow. For MALDI imaging experiments, tissue sections are dropped on conductive glass slides. Sample preparations are then adapted depending on the nature of the tissue sample (FFPE or fr/fr). Then, matrix is uniformly deposited on the tissue section using dedicated devices. A laser beam subsequently irradiates the preparation following a given step length and a MALDI spectrum is acquired for each position. Using adapted software, the different detected ions are then mapped through the tissue section, in function of their differential intensities. The ‘‘molecular maps’’ are called images. (FFPE, formalin fixed and paraffin embedded; fr/fr, fresh frozen; MALDI, matrix assisted laser desorption ionization.)

Proteomics instrumentations, specific biochemical preparations, and sampling methods such as LCM altogether allow for the deep exploration and comparison of different proteomes between regions of interest in tissues with up to 104 detected proteins. MALDI MS imaging that allows for differential mapping of hundreds of compounds on a tissue section is currently the most striking illustration of association between ‘‘classical’’ and ‘‘molecular’’ histology.

Novel serum protein biomarker panel revealed by mass spectrometry and its prognostic value in breast cancer

L Chung, K Moore, L Phillips, FM Boyle, DJ Marsh and RC Baxter* Breast Cancer Research 2014, 16:R63
http://breast-cancer-research.com/content/16/3/R63

Introduction: Serum profiling using proteomic techniques has great potential to detect biomarkers that might improve diagnosis and predict outcome for breast cancer patients (BC). This study used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) to identify differentially expressed proteins in sera from BC and healthy volunteers (HV), with the goal of developing a new prognostic biomarker panel.
Methods: Training set serum samples from 99 BC and 51 HV subjects were applied to four adsorptive chip surfaces (anion-exchange, cation-exchange, hydrophobic, and metal affinity) and analyzed by time-of-flight MS. For validation, 100 independent BC serum samples and 70 HV samples were analyzed similarly. Cluster analysis of protein spectra was performed to identify protein patterns related to BC and HV groups. Univariate and multivariate statistical analyses were used to develop a protein panel to distinguish breast cancer sera from healthy sera, and its prognostic potential was evaluated.
Results: From 51 protein peaks that were significantly up- or downregulated in BC patients by univariate analysis, binary logistic regression yielded five protein peaks that together classified BC and HV with a receiver operating characteristic (ROC) area-under-the-curve value of 0.961. Validation on an independent patient cohort confirmed
the five-protein parameter (ROC value 0.939). The five-protein parameter showed positive association with large tumor size (P = 0.018) and lymph node involvement (P = 0.016). By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, immunoprecipitation and western blotting the proteins were identified as a fragment
of apolipoprotein H (ApoH), ApoCI, complement C3a, transthyretin, and ApoAI. Kaplan-Meier analysis on 181 subjects after median follow-up of >5 years demonstrated that the panel significantly predicted disease-free survival (P = 0.005), its efficacy apparently greater in women with estrogen receptor (ER)-negative tumors (n = 50, P = 0.003) compared to ER-positive (n = 131, P = 0.161), although the influence of ER status needs to be confirmed after longer follow-up.
Conclusions: Protein mass profiling by MS has revealed five serum proteins which, in combination, can distinguish between serum from women with breast cancer and healthy control subjects with high sensitivity and specificity. The five-protein panel significantly predicts recurrence-free survival in women with ER-negative tumors and may have value in the management of these patients.

Cellular prion protein is required for neuritogenesis: fine-tuning of multiple signaling pathways involved in focal adhesions and actin cytoskeleton dynamics

Aurélie Alleaume-Butaux, et al. Cell Health and Cytoskeleton 2013:5 1–12

(1) neurite sprouting, which consists of the formation of buds emerging from the postmitotic neuronal soma;

(2) neurite outgrowth, which represents the conversion of buds into neurites, their elongation and evolution into axon or dendrites; and

(3) the stability and plasticity of neuronal polarity.

In neuronal stem cells, remodeling and activation of focal adhesions (FAs)

associated with deep modifications of the actin cytoskeleton is
a prerequisite for neurite sprouting and subsequent neurite outgrowth.

A multiple set of growth factors and interactors located in

the extracellular matrix and the plasma membrane orchestrate neuritogenesis
by acting on intracellular signaling effectors, notably small G proteins such as RhoA, Rac, and Cdc42,
which are involved in actin turnover and the dynamics of FAs.

The cellular prion protein (PrPC), a glycosylphosphatidylinositol (GPI)-anchored membrane protein

mainly known for its role in a group of fatal neurodegenerative diseases,
has emerged as a central player in neuritogenesis.

Here, we review the contribution of PrPC to neuronal polarization and

detail the current knowledge on the signaling pathways fine-tuned
by PrPC to promote neurite sprouting, outgrowth, and maintenance.

We emphasize that PrPC-dependent neurite sprouting is a process in which

PrPC governs the dynamics of FAs and the actin cytoskeleton via β1 integrin signaling.

The presence of PrPC is necessary to render neuronal stem cells

competent to respond to neuronal inducers and to develop neurites.

In differentiating neurons, PrPC exerts a facilitator role towards neurite elongation.

This function relies on the interaction of PrPC with a set of diverse partners such as

elements of the extracellular matrix,
plasma membrane receptors,
adhesion molecules, and
soluble factors that control actin cytoskeleton turnover

through Rho-GTPase signaling.

Once neurons have reached their terminal stage of differentiation and

acquired their polarized morphology,
PrPC also takes part in the maintenance of neurites.

By acting on tissue nonspecific alkaline phosphatase, or matrix metalloproteinase type 9,

PrPC stabilizes interactions between neurites and the extracellular matrix.

Fusion-pore expansion during syncytium formation is restricted by an actin network

Andrew Chen et al., Journal of Cell Science 121, 3619-3628. http://dx.doi.org:/10.1242/jcs.032169

Cell-cell fusion in animal development and in pathophysiology

involves expansion of nascent fusion pores formed by protein fusogens
to yield an open lumen of cell-size diameter.

Here we explored the enlargement of micron-scale pores in syncytium formation,

which was initiated by a well-characterized fusogen baculovirus gp64.

Radial expansion of a single or, more often, of multiple fusion pores

proceeds without loss of membrane material in the tight contact zone.

Pore growth requires cell metabolism and is

accompanied by a local disassembly of the actin cortex under the pores.

Effects of actin-modifying agents indicate that

the actin cortex slows down pore expansion.

We propose that the growth of the strongly bent fusion-pore rim

is restricted by a dynamic resistance of the actin network and
driven by membrane-bending proteins that are involved in
the generation of highly curved intracellular membrane compartments.

Pak1 Is Required to Maintain Ventricular Ca2+ Homeostasis and Electrophysiological Stability Through SERCA2a Regulation in Mice

Yanwen Wang, et al. Circ Arrhythm Electrophysiol. 2014;7:00-00.

Impaired sarcoplasmic reticular Ca2+ uptake resulting from

decreased sarcoplasmic reticulum Ca2+-ATPase type 2a (SERCA2a) expression or activity
is a characteristic of heart failure with its associated ventricular arrhythmias.

Recent attempts at gene therapy of these conditions explored strategies

enhancing SERCA2a expression and the activity as novel approaches to heart failure management.

We here explore the role of Pak1 in maintaining ventricular Ca2+ homeostasis and electrophysiological stability

under both normal physiological and acute and chronic β-adrenergic stress conditions.

Methods and Results—Mice with a cardiomyocyte-specific Pak1 deletion (Pak1cko), but not controls (Pak1f/f), showed

high incidences of ventricular arrhythmias and electrophysiological instability
during either acute β-adrenergic or chronic β-adrenergic stress leading to hypertrophy,
induced by isoproterenol.

Isolated Pak1cko ventricular myocytes correspondingly showed

aberrant cellular Ca2+ homeostasis.

Pak1cko hearts showed an associated impairment of SERCA2a function and

downregulation of SERCA2a mRNA and protein expression.

Further explorations of the mechanisms underlying the altered transcriptional regulation

demonstrated that exposure to control Ad-shC2 virus infection
increased SERCA2a protein and mRNA levels after
phenylephrine stress in cultured neonatal rat cardiomyocytes.

This was abolished by the

Pak1-knockdown in Ad-shPak1–infected neonatal rat cardiomyocytes and
increased by constitutive overexpression of active Pak1 (Ad-CAPak1).

We then implicated activation of serum response factor, a transcriptional factor well known for

its vital role in the regulation of cardiogenesis genes in the Pak1-dependent regulation of SERCA2a.

Conclusions—These findings indicate that

Pak1 is required to maintain ventricular Ca2+ homeostasis and electrophysiological stability

and implicate Pak1 as a novel regulator of cardiac SERCA2a through
a transcriptional mechanism

fusion in animal development and in pathophysiology involves expansion of nascent fusion pores

formed by protein fusogens to yield an open lumen of cell-size diameter.

Here we explored the enlargement of micron-scale pores in syncytium formation,

which was initiated by a well-characterized fusogen baculovirus gp64.

Radial expansion of a single or, more often, of multiple fusion pores proceeds

without loss of membrane material in the tight contact zone.

Pore growth requires cell metabolism and is accompanied by

a local disassembly of the actin cortex under the pores.

Effects of actin-modifying agents indicate that the actin cortex slows down pore expansion.

We propose that the growth of the strongly bent fusion-pore rim is restricted

by a dynamic resistance of the actin network and driven by
membrane-bending proteins that are involved in the generation of
highly curved intracellular membrane compartments.

Role of forkhead box protein A3 in age-associated metabolic decline

Xinran Maa,1, Lingyan Xua,1, Oksana Gavrilovab, and Elisabetta Muellera,2
PNAS Sep 30, 2014 | 111 | 39 | 14289–14294 http://pnas.org/cgi/doi/10.1073/pnas.1407640111

Significance
This paper reports that the transcription factor forkhead box protein A3 (Foxa3) is

directly involved in the development of age-associated obesity and insulin resistance.

Mice that lack the Foxa3 gene

remodel their fat tissues,
store less fat, and
burn more energy as they age.

These mice also live significantly longer.

We show that Foxa3 suppresses a key metabolic cofactor, PGC1α,

which is involved in the gene programs that turn on energy expenditure in adipose tissues.

Overall, these findings suggest that Foxa3 contributes to the increased adiposity observed during aging,

and that it can be a possible target for the treatment of metabolic disorders.

Aging is associated with increased adiposity and diminished thermogenesis, but

the critical transcription factors influencing these metabolic changes late in life are poorly understood.

We recently demonstrated that the winged helix factor forkhead box protein A3 (Foxa3)

regulates the expansion of visceral adipose tissue in high-fat diet regimens; however,
whether Foxa3 also contributes to the increase in adiposity and the decrease in brown fat activity
observed during the normal aging process is currently unknown.

Here we report that during aging, levels of Foxa3 are significantly and selectively

up-regulated in brown and inguinal white fat depots, and that
midage Foxa3-null mice have increased white fat browning and thermogenic capacity,

decreased adipose tissue expansion,
improved insulin sensitivity, and
increased longevity.

Foxa3 gain-of-function and loss-of-function studies in inguinal adipose depots demonstrated

a cell-autonomous function for Foxa3 in white fat tissue browning.

The mechanisms of Foxa3 modulation of brown fat gene programs involve

the suppression of peroxisome proliferator activated receptor γ coactivtor 1 α (PGC1α) levels
through interference with cAMP responsive element binding protein 1-mediated
transcriptional regulation of the PGC1α promoter.

Our data demonstrate a role for Foxa3 in energy expenditure and in age-associated metabolic disorders.

Control of Mitochondrial pH by Uncoupling Protein 4 in Astrocytes Promotes Neuronal Survival

HP Lambert, M Zenger, G Azarias, Jean-Yves Chatton, PJ. Magistretti,§, S Lengacher
JBC (in press) M114.570879 http://www.jbc.org/cgi/doi/10.1074/jbc.M114.570879

Background: Role of uncoupling proteins (UCP) in the brain is unclear.
Results: UCP, present in astrocytes, mediate the intra-mitochondrial acidification leading to a decrease in mitochondrial ATP production.
Conclusion: Astrocyte pH regulation promotes ATP synthesis by glycolysis whose final product, lactate, increases neuronal survival.
Significance: We describe a new role for a brain uncoupling protein.

Brain activity is energetically costly and requires a steady and

highly regulated flow of energy equivalents between neural cells.

It is believed that a substantial share of cerebral glucose, the major source of energy of the brain,

will preferentially be metabolized in astrocytes via aerobic glycolysis.

The aim of this study was to evaluate whether uncoupling proteins (UCPs),

located in the inner membrane of mitochondria,
play a role in setting up the metabolic response pattern of astrocytes.

UCPs are believed to mediate the transmembrane transfer of protons

resulting in the uncoupling of oxidative phosphorylation from ATP production.

UCPs are therefore potentially important regulators of energy fluxes. The main UCP isoforms

expressed in the brain are UCP2, UCP4, and UCP5.

We examined in particular the role of UCP4 in neuron-astrocyte metabolic coupling

and measured a range of functional metabolic parameters
including mitochondrial electrical potential and pH,

reactive oxygen species production,
NAD/NADH ratio,
ATP/ADP ratio,
CO2 and lactate production, and
oxygen consumption rate (OCR).

In brief, we found that UCP4 regulates the intra-mitochondrial pH of astrocytes

which acidifies as a consequence of glutamate uptake,
with the main consequence of reducing efficiency of mitochondrial ATP production.
the diminished ATP production is effectively compensated by enhancement of glycolysis.
this non-oxidative production of energy is not associated with deleterious H2O2 production.

We show that astrocytes expressing more UCP4 produced more lactate,

used as energy source by neurons, and had the ability to enhance neuronal survival.

Jose Eduardo des Salles Roselino

The problem with genomics was it was set as explanation for everything. In fact, when something is genetic in nature the genomic reasoning works fine. However, this means whenever an inborn error is found and only in this case the genomic knowledge afterwards may indicate what is wrong and not the completely way to put biology upside down by reading everything in the DNA genetic as well as non-genetic problems.

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