Posts Tagged ‘Evolution’

Selye’s Riddle solved

Larry H. Bernstein, mD, FCAP, Curator



Mathematicians Solve 78-year-old Mystery

Mathematicians developed a solution to Selye's riddle which has puzzled scientists for almost 80 years.
Mathematicians developed a solution to Selye’s riddle which has puzzled scientists for almost 80 years.

In previous research, it was suggested that adaptation of an animal to different factors looks like spending of one resource, and that the animal dies when this resource is exhausted. In 1938, Hans Selye introduced “adaptation energy” and found strong experimental arguments in favor of this hypothesis. However, this term has caused much debate because, as it cannot be measured as a physical quantity, adaptation energy is not strictly energy.


Evolution of adaptation mechanisms: Adaptation energy, stress, and oscillating death

Alexander N. Gorbana, , Tatiana A. Tyukinaa, Elena V. Smirnovab, Lyudmila I. Pokidyshevab,


•   We formalize Selye׳s ideas about adaptation energy and dynamics of adaptation.
•   A hierarchy of dynamic models of adaptation is developed.
•   Adaptation energy is considered as an internal coordinate on the ‘dominant path’ in the model of adaptation.
•   The optimal distribution of resources for neutralization of harmful factors is studied.
•   The phenomena of ‘oscillating death’ and ‘oscillating remission’ are predicted.       

In previous research, it was suggested that adaptation of an animal to different factors looks like spending of one resource, and that the animal dies when this resource is exhausted.

In 1938, Selye proposed the notion of adaptation energy and published ‘Experimental evidence supporting the conception of adaptation energy.’ Adaptation of an animal to different factors appears as the spending of one resource. Adaptation energy is a hypothetical extensive quantity spent for adaptation. This term causes much debate when one takes it literally, as a physical quantity, i.e. a sort of energy. The controversial points of view impede the systematic use of the notion of adaptation energy despite experimental evidence. Nevertheless, the response to many harmful factors often has general non-specific form and we suggest that the mechanisms of physiological adaptation admit a very general and nonspecific description.

We aim to demonstrate that Selye׳s adaptation energy is the cornerstone of the top-down approach to modelling of non-specific adaptation processes. We analyze Selye׳s axioms of adaptation energy together with Goldstone׳s modifications and propose a series of models for interpretation of these axioms. Adaptation energy is considered as an internal coordinate on the ‘dominant path’ in the model of adaptation. The phenomena of ‘oscillating death’ and ‘oscillating remission’ are predicted on the base of the dynamical models of adaptation. Natural selection plays a key role in the evolution of mechanisms of physiological adaptation. We use the fitness optimization approach to study of the distribution of resources for neutralization of harmful factors, during adaptation to a multifactor environment, and analyze the optimal strategies for different systems of factors.

In this work, an international team of researchers, led by Professor Alexander N. Gorban from the University of Leicester, have developed a solution to Selye’s riddle, which has puzzled scientists for almost 80 years.

Alexander N. Gorban, Professor of Applied Mathematics in the Department of Mathematics at the University of Leicester, said: “Nobody can measure adaptation energy directly, indeed, but it can be understood by its place already in simple models. In this work, we develop a hierarchy of top-down models following Selye’s findings and further developments. We trust Selye’s intuition and experiments and use the notion of adaptation energy as a cornerstone in a system of models. We provide a ‘thermodynamic-like’ theory of organism resilience that, just like classical thermodynamics, allows for economics metaphors, such as cost and bankruptcy and, more importantly, is largely independent of a detailed mechanistic explanation of what is ‘going on underneath’.”

Adaptation energy is considered as an internal coordinate on the “dominant path” in the model of adaptation. The phenomena of “oscillating death” and “oscillating remission,” which have been observed in clinic for a long time, are predicted on the basis of the dynamical models of adaptation. The models, based on Selye’s idea of adaptation energy, demonstrate that the oscillating remission and oscillating death do not need exogenous reasons. The developed theory of adaptation to various factors gives the instrument for the early anticipation of crises.

Professor Alessandro Giuliani from Istituto Superiore di Sanità in Rome commented on the work, saying: “Gorban and his colleagues dare to make science adopting the thermodynamics style: they look for powerful principles endowed with predictive ability in the real world before knowing the microscopic details. This is, in my opinion, the only possible way out from the actual repeatability crisis of mainstream biology, where a fantastic knowledge of the details totally fails to predict anything outside the test tube.1

Citation: Alexander N. Gorban, Tatiana A. Tyukina, Elena V. Smirnova, Lyudmila I. Pokidysheva. Evolution of adaptation mechanisms: Adaptation energy, stress, and oscillating death. Journal of Theoretical Biology, 2016; DOI:10.1016/j.jtbi.2015.12.017. Voosen P. (2015) Amid a Sea of False Findings NIH tries Reform, The Chronicle of Higher Education.

























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Genomic expression carried over from Neanderthal DNA

Larry H. Bernstein, MD, FCAP, Curator



Neanderthal DNA Shapes Clinical Traits for Modern Humans

GEN  http://www.genengnews.com/gen-news-highlights/neanderthal-dna-shapes-clinical-traits-for-modern-humans/81252363/


Modern humans have inherited many physical traits from the Neanderthals. John Capra, Ph.D., from Vanderbilt University, explains how many of these variants affect a variety of clinical disorders

This graphic shows some of the numerous Neanderthal-influenced traits. [Deborah Brewington, Vanderbilt University]

Today being the 207th birthday celebration of renowned naturalist and evolutionary biologist Charles Darwin, it seemed only appropriate to discuss the recent findings of how Neanderthal DNA has shaped and continues to shape human evolution.

Recent studies have identified that individuals of Eurasian origins inherited somewhere between one and four percent of their DNA from Neanderthals. These findings have led to numerous postulations about how these genetic variants may have affected physical characteristics or the behavior of modern humans, ranging from skin color to heightened allergies to fat metabolism.

Now, a new study from a team of scientists led by researchers at Vanderbilt University has directly compared Neanderthal DNA in the genomes of a large population of adults from European ancestry with their clinical records—confirming that this archaic genetic legacy has a subtle but significant effect on modern human biology.

“Our main finding is that Neanderthal DNA does influence clinical traits in modern humans: We discovered associations between Neanderthal DNA and a wide range of traits, including immunological, dermatological, neurological, psychiatric, and reproductive diseases,” explained senior study author John Capra, Ph.D., assistant professor in the department of biomedical informatics and an investigator in the Center for Human Genetics Research at Vanderbilt University Medical School.

The results of this study were published February 12 in Science through an article entitled “The phenotypic legacy of admixture between modern humans and Neanderthals.

Interestingly, Dr. Capra and his colleagues were able to confirm a few of the previous hypotheses about the influence of Neanderthal DNA on modern Homo sapiens. For instance, investigators found that Neanderthal DNA affects keratinocytes, which help protect the skin from environmental damage such as ultraviolet radiation and pathogens. The new analysis found Neanderthal DNA variants influence skin biology in modern humans, in particular, the risk of developing sun-induced skin lesions called keratosis, which are caused by abnormal keratinocytes.

Surprisingly, the research team found that some regions of Neanderthal DNA were associated with psychiatric and neurological effects. In one example, they found that a specific bit of Neanderthal DNA significantly increased the risk for nicotine addiction, while a separate set of variants influenced the risk for depression (positively and negatively).

“The brain is incredibly complex, so it’s reasonable to expect that introducing changes from a different evolutionary path might have negative consequences,” noted lead author and Vanderbilt doctoral student Corinne Simonti.

In the current study, the authors discussed that the pattern of associations they discovered suggests today’s population retains Neanderthal DNA that may have provided modern humans with some adaptive advantages 40,000 years ago as they migrated into regions outside of Africa with different pathogens and levels of sun exposure.

To study these associations, the scientists used a database containing 28,000 patients whose biological samples have been linked to anonymized versions of their electronic health records. The data came from eMERGE—the Electronic Medical Records and Genomics Network—which links digitized records from Vanderbilt University Medical Center’s BioVU databank and eight other hospitals around the country.

This massive amount of genomic data allowed the researchers to determine if each individual had ever been treated for a particular set of medical conditions, such as heart disease, arthritis, or depression. Subsequently, they analyzed the genomes of each individual to identify the unique set of Neanderthal DNA that each person carried. The comparison of each data set allowed the researchers to test whether each bit of Neanderthal DNA individually and in aggregate influences risk for the traits derived from the medical records.

“Vanderbilt’s BioVU and the network of similar databanks from hospitals across the country were built to enable discoveries about the genetic basis of disease,” Dr. Capra remarked. “We realized that we could use them to answer important questions about human evolution.”

While Dr. Capra and his colleagues were thrilled by their findings—this work establishes a new way to investigate questions about the effects of events in recent human evolution—the researcher team also realized that there is a lot of additional information contained in the medical records, such as lab tests, doctors’ notes, and medical images, that could be used in future analyses to refine their data.

Neanderthals’ Genetic Legacy

Ancient DNA in the genomes of modern humans influences a range of physiological traits.

By Ruth Williams | February 11, 2016


People of Eurasian origin are, genetically speaking, between 1 percent and 4 percent Neanderthal, and new research shows how this archaic DNA in their genomes may be impacting their health. The study, published today (February 11) in Science, utilized the electronic medical records and associated DNA data of more than 28,000 individuals to show that Neanderthal DNA had small but significant effects on the risks of developing—among other things—depression, skin lesions, and excessive blood clotting.

“They’ve looked at huge databases of medical records to see if there are traits that correlate with the presence of particular genes from Neanderthals and have found a number of them,” said anthropologist John Hawks of the University of Wisconsin who was not involved in the study. “The take-away is that these genes that we have from these ancient people have effects on our phenotypes, and that’s pretty cool. They are not just shadows that are not doing anything, they are actually participating in our biology.”

Sequencing of Neanderthal genomes isolated from fragments of bones has revealed that modern humans contain remnants of Neanderthal DNA—a result of interbreeding between the two subspecies. But while certain loci in human genomes have been found to contain an abundance of Neanderthal alleles, it has been unclear whether these alleles have actual functional effects on human traits and, if so, what those are.

Evolutionary and computational geneticist John Capra of Vanderbilt University in Nashville, Tennessee, and colleagues devised an ingenious way to investigate such functional effects on a genome-wide scale. “We realized that we had a great opportunity to answer these questions using large databases of anonymized versions of patient electronic health records linked to their genetic information,” Capra said in a statement.

“A number of previous studies have focused on individual genes,” said evolutionary geneticist Rasmus Neilsen of the University of California, Berkeley, who did not participate in the research. “But this is the first study that really systematically goes through and uses the knowledge we have about genetic variations in humans to answer the question: How much has integration of DNA from Neanderthals affected observable traits in humans?”

Within Neanderthal DNA found in humans, the researchers focused on the most common variants—single nucleotide polymorphisms (SNPs)—and asked, individually and en masse, whether  these variants were associated with any of the medical traits listed for the 28,000 patients.

Investigating the SNPs en masse through a genome-wide complex trait analysis (GCTA), the researchers discovered associations with depression, mood disorders, and a particular type of skin lesion caused by sun exposure. Investigating individual SNPs, on the other hand, the researchers picked out associations tied to tobacco use, urinary problems, and blood hypercoagulation.

Why have such apparently detrimental gene variants been maintained in the human genome? It is important to realize, said Hawks, that “when you look at people’s medical records, you don’t see the good stuff.”

Hawks also noted that “the [observed] associations are really, really small,” meaning that while the links between Neanderthal alleles and certain medical traits were statistically significant, they only represented a tiny percentage of the risk—1 percent to 2 percent in the case of depression, for example.

Further, “many genetic variants, regardless of evolutionary origin and temporal context, are beneficial in some respects but detrimental in others,” Capra added in the statement. For example, while hypercoagulation may increase a person’s thrombosis risk , coagulation is an early innate immune response that protects against injury and infection. As Neanderthals colonized new territories and were exposed to new pathogens, having a souped-up version of this response may therefore have been a favorable defense mechanism.

Capra’s team carried out further experiments to look at whether Neanderthal alleles were associated with classes of traits rather than individual ones, finding neurological and psychiatric traits were both over-represented.

Together with the findings that depression, mood disorders and tobacco use were individually associated with Neanderthal SNPs, this suggested to the researchers that the brains of modern humans have been particularly influenced by Neanderthal DNA. And this might overturn notions of Neanderthals as not-so-bright, said Hawks. “If you had the hypothesis that Neanderthals [died out] because they were stupid,” he said, “you have to explain why their genes are here doing stuff in our brains.”

C.N. Simonti et al., “The phenotypic legacy of admixture between modern humans and Neandertals,” Science, 351:737-41, 2016.


Capra Lab   Evolutionary and Computational Genomics at Vanderbilt University

We use the tools of computer science and statistics to address problems in genetics, evolution, and biomedicine.

Our group is located in the Department of Biological Sciences and affiliated with theVanderbilt Genetics Institute, the Center for Structural Biology, and the Department of Biomedical Informatics at Vanderbilt University.

Humans differ from one another and our closest living relatives, the chimpanzees, in a wide range of traits, including our susceptibility to many diseases. We model the evolutionary processes that have produced these novel traits and develop algorithms that compare genomes to predict the functional relevance of specific genetic differences between individuals and species.

Our research is motivated by several questions:

  • How have evolutionary processes produced the astonishing diversity of form and function present in the natural world?
  • How can better algorithms lead to a deeper understanding of biological systems and networks?
  • How do genomes encode and maintain the information necessary to produce life?
  • How can our increasing knowledge of genomic variation be translated into the treatment and prevention of disease?

We investigate these questions in a number of model systems, but our main focus is on the origins and recent evolution of human populations and their primate relatives.


Borrowing Immunity Through Interbreeding

Neanderthals and Denisovans contributed innate immune genes to modern humans, scientists show.

By Kate Yandell | January 7, 2016


The proportion of Neanderthal-derived toll-like receptors in populations, with Neanderthal alleles in orange and green and non-archaic alleles in blue.DANNEMANN ET AL./AJHG

Modern humans adopted innate immune genes responsible for recognizing invading microbes from Neanderthals and Denisovans, according to two studies published today (January 7) in The American Journal of Human Genetics. The two teams, based in France and Germany, independently concluded that humans picked up some versions of a cluster of toll-like receptors by interbreeding with archaic hominin relatives.

“Once humans came out of Africa and then encountered archaic species, they might also have encountered their pathogens,” said Rasmus Nielsen, an evolutionary biologist at the University of California, Berkeley, who was not involved in the studies. “There might have been pathogens that could affect Neanderthals and Denisovans that also could jump into modern humans.”

“At least partially, Neanderthals may have harbored already adaptive mutations, mutations that rendered them more resistant to infections,” said Lluis Quintana-Murci, an evolutionary geneticist at the Pasteur Institute in Paris and a coauthor of one of the new papers.

Previous studies have shown that modern humans interbred with Neanderthals and Denisovans. For instance, Nielsen and his colleagues showed that humans who migrated to Tibet likely picked up an allelecontrolling blood hemoglobin concentration from local Denisovans, allowing them to adapt to living at high altitudes. Another paper indicated that humans had picked up major histocompatibility genes from Denisovans and Neanderthals.

The authors of the two new studies approached the topic of ancient human evolution from different directions. Quintana-Murci and his colleagues decided to do a broad survey of innate immune genes and their variability among present-day humans around the world, using sequence data gathered through the1,000 Genomes Project. The team demonstrated that innate immune genes have been under stronger-than-average selective pressures. Some innate genes are highly conserved, with little tolerance for variability. Other protein-coding genes have picked up adaptive mutations, mostly occurring within the last 6,000 to 13,000 years after humans transitioned from a hunter-gatherer to agricultural society. The resulting increase in density of human settlements, cohabitation with animals, and increased exposure to sewage may have made humans easier targets for microbial disease, the researchers speculated.

Quintana-Murci and his colleagues also took advantage of a previously published map of areas of the human genome where Neanderthal genes are present, showing that innate immune genes are generally more likely to have been borrowed from Neanderthals than genes coding other types of proteins. Specifically, they noted that 126 innate immune genes in present-day Europeans, Asians, or both groups were among the top 5 percent of genes in the genome of each population most likely to have originated in Neanderthals. The cluster of toll-like receptor genes, encoding TLR 1, TLR 6, and TLR 10, both showed signs of having been borrowed from Neanderthals and having picked up adaptive mutations at various points in history.

Meanwhile, a group led by Janet Kelso of the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, used both the same previously published Neanderthal introgression map that Quintana-Murci used and a second introgression map. The researchers searched for borrowed regions of the genome that were especially long and common in present-day humans, eventually zeroing in TLR6, TLR10, and TLR1. These receptors, which detect conserved microbial proteins such as flagellin, are all encoded along the same segment of DNA on chromosome four.

By looking at 1,000 Genomes Project data, Kelso and her colleagues were able to identify seven distinct versions of the TLR cluster. The researchers were able to match two of these versions to DNA from Neanderthals, and one version to DNA from Denisovans.

“There have been three potentially independent admixtures,” said Kelso. “We suspect it was two different Neanderthals and a Denisovan.”

Kelso and her colleagues then attempted to figure out the functional differences between the Neanderthal and Denisovan versions of the TLR cluster and the versions that likely originated with the modern humans who migrated from Africa to Europe and Asia later than these archaic hominids.

The changes in the Neanderthal and Denisovan TLR clusters do not lead to altered proteins. However, the researchers found that in white blood cells, the Neanderthal and Denisovan TLRs are more highly expressed than the non-borrowed human TLR clusters.

Kelso and her colleagues also did a survey of already-completed genome wide association studies, finding that present-day people who have the borrowed TLR clusters show lower levels of the bacteriumHelicobacter pylori in their bloodstreams than people descended from humans that did not pick up TLR clusters from Neanderthals or Denisovans. People with the borrowed TLR clusters also tend to have elevated allergies to dust and pollen.

Kelso hypothesized that the Denisovan and Neanderthal TLR clusters may have strengthened the human immune systems against novel pathogens they encountered in their new homes in Europe and Asia. This may have yielded an immune system both skilled at fighting off pathogens and slightly oversensitive, leading to the allergies people carrying the archaic TLRs sometimes have today.

But it is less clear exactly how the immune system was strengthened, or what pathogens ancient humans were trying to fight. “What the gene expression results tell us is that there is some kind of a functional effect for introgression,” said Sri Sankararaman, a statistical geneticist at University of California, Los Angeles, who was not involved in the studies but did help make one of the preexisting introgression maps used in the papers. “That’s basically what it has established. Going from there to making a claim about its fitness effect is less obvious.”

And the reduced H. pylori prevalence associated with the borrowed TLR alleles is simply a sign that the variants are associated with altered immunity, not necessarily an indication that breeding with Neanderthals helped humans avoid this particular pathogen. “We may not have the pathogens around today that selection was acting in response to,” said Nielsen.

The studies help confirm that interbreeding between humans, Neanderthals, and Denisovans shaped human evolution, sometimes offering key advantages people of combined lineage. “The things that modern humans took away from the interbreeding with the Neanderthals were regions of the genome involved in adaptation to the environment,” said Kelso.

M. Deschamps et al., “Genomic signatures of selective pressures and introgression from archaic hominins at human innate immunity genes,” The American Journal of Human Genetics, doi:10.1016/j.ajhg.2015.11.014, 2016.

M. Dannemann et al., “Introgression of Neandertal- and Denisovan-like haplotypes contributes to adaptive variation in human toll-like receptors,” The American Journal of Human Genetics,doi:10.1016/j.ajhg.2015.11.015


Genomic Signatures of Selective Pressures and Introgression from Archaic Hominins at Human Innate Immunity Genes

Matthieu Deschamps, Guillaume Laval, Maud Fagny, Yuval Itan, et al.
Am J Human Gen Jan 2016;  98(1):5–21, 7.   http://dx.doi.org/10.1016/j.ajhg.2015.11.014
Human genes governing innate immunity provide a valuable tool for the study of the selective pressure imposed by microorganisms on host genomes. A comprehensive, genome-wide study of how selective constraints and adaptations have driven the evolution of innate immunity genes is missing. Using full-genome sequence variation from the 1000 Genomes Project, we first show that innate immunity genes have globally evolved under stronger purifying selection than the remainder of protein-coding genes. We identify a gene set under the strongest selective constraints, mutations in which are likely to predispose individuals to life-threatening disease, as illustrated by STAT1 and TRAF3. We then evaluate the occurrence of local adaptation and detect 57 high-scoring signals of positive selection at innate immunity genes, variation in which has been associated with susceptibility to common infectious or autoimmune diseases. Furthermore, we show that most adaptations targeting coding variation have occurred in the last 6,000–13,000 years, the period at which populations shifted from hunting and gathering to farming. Finally, we show that innate immunity genes present higher Neandertal introgression than the remainder of the coding genome. Notably, among the genes presenting the highest Neandertal ancestry, we find the TLR6-TLR1-TLR10 cluster, which also contains functional adaptive variation in Europeans. This study identifies highly constrained genes that fulfill essential, non-redundant functions in host survival and reveals others that are more permissive to change—containing variation acquired from archaic hominins or adaptive variants in specific populations—improving our understanding of the relative biological importance of innate immunity pathways in natural conditions.

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Evolutionary Change

Larry H. Bernstein, MD, FCAP, Curator




Change accelerates stalled evolution

biomolbioandco    https://biomolbioandco.wordpress.com/2015/11/17/change-accelerates-stalled-evolution/

Evolution can get stuck. When no mutations are available that can improve the fitness of an organism, evolution cannot proceed. However, using environmental fluctuations that are ubiquitous in nature, evolution can proceed. In the preprint that we posted earlier on the bioRxiv and which has recently been published in PNAS we describe how a transcription factor and its binding site, constraint in the different constant environments, can evolve in fluctuating environments.

Stalled evolution

Concept of fitness landscape. From de Visser & Krug,Nature Reviews Genetics 15, 480–490 (2014)

Concept of fitness landscape. From de Visser & Krug, Nature Reviews Genetics 15, 480–490 (2014)



The accessibility of phenotypes in different environments depends on the wiring of the genotype-phenotype map and the translation of this map into fitness. When sub-optimal genotypes are surrounded by valleys in the adaptive landscape, neighboring genotypes are inaccessible and evolution is unable to proceed by single mutational step-by-step positive Darwinian evolution (Figure 1).

To investigate the connectedness of the fitness increasing genotypes in sequence space we used the lac regulatory system in Escherichia coli. Decades of work have elucidated the physiology of the lac regulatory system. And it is known that a few base pairs in the operator DNA, and a few amino acid residues in the transcription factor are responsible for specificity of binding of these operator-repressor pairs. We here experimentally constructed mutants with mutations in these specific residues (6 residues, yielding 26=64 mutants in total) that together constitute 6!=720 direct trajectories.

Assessing the ability to repress the lac operon in one environment (without a lactose-sugar-derivative), and the ability to express the lac operon in the other environment (with a lactose-sugar-derivative), we find that none of the mutational trajectories allows continuous improvements along the mutational trajectories in one of the constant environments. Interestingly, we find that alternating between these environments doesallow for constant improvements along the mutational trajectories. We find that the shortest route towards the final genotype is of Hamming distance 6, there is thus a direct mutational trajectory from the starting sequence to the final combination of transcription-factor and binding site. With a computational method that describes the mutational and environmental transitions as a Markov process, we can further calculate the crossing rates from the initial to the final genotype for all trajectories in the landscape, allowing mutational detours. We find that the crossing rate is maximal when the environmental switches are on the order of the mutation rate (or the rate at which a sweep can be completed).

Tradeoffs are crucial for crossing the adaptive landscape in fluctuating environments

Cross-environmental tradeoffs are responsible for these continuous improvements. In the most extreme case, suboptimal peaks are translated into valleys, by which constraints are resolved. In the more subtle case, descending slopes are turned into ascending slopes upon environmental change, allowing adaptive trajectories to surf over the slopes with positive selective coefficients. Evolutionary constraints can thus be overcome by the environment-dependent ‘ratcheting’ that allows the crossing of otherwise inaccessible regions in sequence space (Figure 2).




We think that this research not only aids the fundamental understanding of ecological and evolutionary transitions in fluctuating environments, but that it can also help rethinking the evolutionary optimization of certain biotechnological processes (for instance the production of antibodies). And in addition, it cautions against the use of cyclic multi-drug protocols in clinical treatments, as these might potentially increase the speed of adaptation to the drugs, instead of halting it.

Breaking evolutionary constraint with a tradeoff ratchet
M.G.J. de Vos, A. Dawid, V. Sunderlikova, S.J. Tans
PNAS, (2015) | http://dx.doi.org/10.1073/pnas.1510282112


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What about Genomes? 

Larry H. Bernstein, MD, FCAP, Curator



Genome Digest

What researchers are learning as they sequence, map, and decode species’ genomes

By Karen Zusi | November 19, 2015



Unique fruit

Species: Cultivated pineapple (Ananas comosus) and wild pineapple (A. bracteatus)
Genome size: 526 million base pairs

Pineapple is one of the world’s most successful tropical crops, grown in more than 80 countries. Researchers sequenced the genomes of two cultivated varieties and the fruit’s wild relative to explore its lineage and photosynthetic pathway. The international team published its results earlier this month (November 2) in Nature Genetics.

Unlike most plants, pineapples use crassulacean acid metabolism (CAM) photosynthesis, which allows them to concentrate carbon dioxide at night and keep their stomata closed to conserve water during the day. The newly sequenced genomes revealed relationships between the genes for CAM photosynthesis and those for the pineapple’s circadian clock. “This is the first time scientists have found a link between regulatory elements of CAM photosynthesis genes and circadian clock regulation,” study coauthor Ray Ming ofthe University of Illinois at Urbana-Champaign said in a press release.

The researchers proposed that the pineapple’s CAM photosynthesis likely evolved by hijacking parts of molecular pathways involved in more common types of photosynthesis. “All plants contain the necessary genes for CAM photosynthesis, and the evolution of CAM simply requires rerouting of pre-existing pathways,” the authors concluded in their report.




Less-“beany” legume

Species: Adzuki bean (Vigna angularis)
Genome size: 542 billion base pairs

The adzuki bean contains more starch and less fat than other legumes do. To investigate the genetic basis for these differences, a team of researchers from China and India sequenced the genome of a Chinese adzuki bean cultivar. Their results, reported in PNAS last month (October 12), provide a basis for comparative genomic studies of other legumes.

The team found that, compared to soybeans, mature adzuki bean seeds show greater expression of genes involved in starch biosynthesis. The adzuki bean also has a low “beany flavor” compared to other legumes like soybeans, according to the authors, which is due to a lower expression level of enzymes called lipoxygenases.

“Generally, our results valuably reinforce the legume species genomes, provide insight into evolution and metabolic differences of legumes, and will accelerate studying of genetics and genomics for adzuki bean improvement,” the authors wrote in their paper.




Pernicious pest

Species: Asian tiger mosquito (Aedes albopictus)
Genome size: 1.97 million base pairs

Over time, the Asian tiger mosquito has invaded and thrived on every continent except Antarctica. It carries diseases such as dengue and Chikungunya fever. Seeking explanations for the insect’s adaptive nature, an international team led by researchers from the University of California, Irvine, published a draft genome for the Asian tiger mosquito last month (October 19) in PNAS.

The Asian tiger mosquito’s genome is the largest of any mosquito sequenced to date, mostly due to its high levels of repetitive DNA, according to the authors. “The genome size may account in part for why this mosquito is successful as an invasive species,” they wrote in their paper, explaining that a large bank of noncoding and coding DNA could allow an Asian tiger mosquito population to survive selection pressures and adapt in novel environments.

The team detected large numbers of genes involved in insecticide resistance and the sense of smell. The researchers also found viral genes integrated into the mosquito’s genome, which supports a shared evolutionary history between the insect and these mosquito-borne viruses, they noted.



Ancient cattle

Species: Aurochs (Bos primigenius)

Now extinct, the aurochs once roamed Europe, Asia, and North America, giving rise to today’s domestic cows. The last known aurochs died in Poland in 1627. But researchers have now extracted DNA from a 6,750-year-old skeleton and reconstructed an aurochs genome to investigate the history of European cattle. The group’s analysis was published last month (October 26) in Genome Biology.

The researchers compared the aurochs genome to those of modern cows, finding evidence of selection pressure on genes linked to neurobiology and muscle development, suggesting that early farmers domesticated the animals based on some behavioral and meat traits. The team also detected early interbreeding between the aurochs and domestic cows from Great Britain and Ireland.

“Our results show the ancestors of modern British and Irish breeds share more genetic similarities with this ancient specimen than other European cattle,” said coauthor David MacHugh from University College Dublin in a press release. “This suggests that early British farmers may have restocked their domesticated herds with wild aurochs.”




Save the date

Species: Date palm (Phoenix dactylifera)
Genome size: 690 million base pairs

The date palm serves as a significant crop in regions of the Middle East and North Africa. As part of a gene-sequencing project at New York University Abu Dhabi (NYUAD), an international team sequenced the genomes and catalogued molecular variations from 62 different date palm varieties to examine their histories and traits. The team published its results earlier this month (November 9) in Nature Communications.

The researchers suggested either that date palms were domesticated in the Middle East and spread westward, with a separate domestication event in North Africa, or that the plants reached North Africa from the original Middle Eastern population but were then backcrossed with a population of wild date palms.

The team discovered 56 genomic regions that were associated with selective pressures and pinpointed a genetic mutation governing fruit coloration (the date palm produces either red or yellow fruits). The tree shares this mutation with the oil palm (Elaeis guineensis), a distant cousin separated by roughly 60 million years.

The information “yields candidate mutations that present new opportunities for crop improvement,” the authors write in their report.

“We hope that some of these genomic technologies will allow people to think of creating ways to accelerate breeding processes in date palms that can be useful for the future,” study coauthor Michael Purugganan of NYUAD told Nature Middle East.

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

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






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

  1. Innervation of Heart and Heart Rate
  2. Action of hormones on the circulation
  3. Allogeneic Transfusion Reactions
  4. Graft-versus Host reaction
  5. Unique problems of perinatal period
  6. High altitude sickness
  7. Deep water adaptation
  8. Heart-Lung-and Kidney
  9. 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


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


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


Summary, Metabolic Pathways



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Altitude Adaptation

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



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.


Acute Mountain Sickness: Pathophysiology, Prevention, and Treatment

Chris Imraya, Alex Wright, Andrew Subudhie,, Robert Roache
Progress in Cardiovascular Diseases 52 (2010) 467–484

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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:

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

The response to oxygen lack comprises two phases

  1. defense, which includes metabolic arrest (a suppression of ATP-demand and ATP-supply) and channel arrest (decreases cell membrane permeability), and
  2. 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 131st residue of α1 chain and the 14th of the H. During (de-) oxygenation Hb switches between two major structural states:

  1. the high affinity oxygenated R (relaxed) state that prevails at the respiratory surfaces, and
  2. 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


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 (αD) 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

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

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

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

  1. Feng, B. Fan, K. Li, Q.D. Zhang, et al.
    Small Ruminant Research 75 (2008) 236–242

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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Evolution and Medicine

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



Excerpt of article

Cancer is an inescapable fact of life. All of us with either die from it or know someone who will. Cancer is so prevalent because it isn’t a disease in the way a flu or a cold is. No outside force or germ is needed to cause cancer (although it can). It arises from the very way we are put together.  Most of the genes that are needed for multicellular life have been found to be associated with cancer. Cancer is a result of our natural genetic machinery that has been built up over billions of years breaking down over time.


Cancer is not only a result of evolutionary processes, cancer itself follows evolutionary theory as it grows. The immune system places a selective pressure on cancer cells, keeping it in check until the cancer evolves a way to avoid it and surpass it in a process known as immunoediting. Cancers face selective pressures in the microenvironments in which they grow. Due to the fast growth of cancer cells, they suck up oxygen in the tissues, causing wildly fluctuating oxygen levels as the body tries to get oxygen to the tissues. This sort of situation is bad for normal tissues and so it is for cancer, at least until they evolve and adapt. At some point, some cancer cells will develop the ability to use what is called aerobic glycolysis to make the ATP we use for energy. Ordinarily, our cells only use glycolysis when they run out of oxygen because aerobic respiration (aka oxidative phosphorylation) is far more efficient. Cancer cells, on the other hand, learn to use glycolysis all the time, even in the presence of abundant oxygen. They may not grow as quickly when there is plenty of oxygen, but they are far better than normal cells at hypoxic, or low oxygen, conditions, which they create by virtue of their metabolism. Moreover, they are better at taking up nutrients because many of the metabolic pathways for aerobic respiration also influence nutrient uptake, so shifting those pathways to nutrient uptake rather than metabolism ensures cancer cells get first pick of any nutrients in the area. The Warburg Effect, as this is called, works by selective pressures hindering those cells that can’t do so and favoring those that can. Because cancer cells have loose genetic controls and they are constantly dividing, the cancer population can evolve, whereas the normal cells cannot.

Evolutionary theory can also be used to track cancer as it metastasizes. If a person has several tumors, it is possible to take biopsies of each one and use standard cladistic programs that are normally used to determine evolutionary relationships between organisms to find which tumor is the original tumor. If the original tumor is not one of those biopsied, it will tell you where the cancer originated within the body. You can thus track the progression of cancer throughout a person’s body. Expanding on this, one can even track the effect of cancer through its effects on how organisms interact within ecosystems, creating its own evolutionary stamp on the environment as its effects radiate throughout the ecosystem.

I’ve talked about cancer at decent length (although I could easily go one for many more pages) because it is less well publicly known than some of the other ways that evolutionary theory helps us out in medicine. The increasing resistance of bacteria and viruses to antibiotics is well known. Antibiotic resistance follows standard evolutionary processes, with the result that antibiotic resistant bacteria are expected to kill 10 million people a year by 2050.  People have to get a new flu shot every year because the flu viruses are legion and they evolve rapidly to bypass old vaccinations.  If we are to accurately predict how the viruses may adapt and properly prepare vaccines for the coming year, evolutionary theory must be taken into account. Without it, the vaccines are much less likely to be effective. Evolutionary studies have pointed out important changes in the Ebola virus and how those changes areaffecting its lethality, which will need to be taken into account for effective treatments. Tracking the origins of viruses, like the avian flu or swine flu, gives us information that will be useful in combating them or even stopping them at their source before they become a problem.




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Larry H Bernstein, MD, Reporter

Leaders in Pharmaceutical Intelligence

Special Achievement Award in Medical Science

Award Description

Mary-Claire King
For bold, imaginative, and diverse contributions to medical science and human rights — she discovered the BRCA1 gene locus that causes hereditary breast cancer and deployed DNA strategies that reunite missing persons or their remains with their families.

The 2014 Lasker~Koshland Award for Special Achievement in Medical Science honors a scientist who has made bold, imaginative, and diverse contributions to medical science and human rights. Mary-Claire King(University of Washington, Seattle) discovered the BRCA1 gene locus that causes hereditary breast cancer and deployed DNA strategies that reunite missing persons or their remains with their families. Her work has touched families around the world.

As a statistics graduate student in the late 1960s, King took the late Curt Stern’s genetics course just for fun. The puzzles she encountered there—problems posed by Stern—enchanted her. She was delighted to learn that people could be paid to solve such problems, and that mathematics holds their key. She decided to study genetics and never looked back.

During her Ph.D. work with the late Allan Wilson (University of California, Berkeley), King discovered that the sequences of human and chimpanzee proteins are, on average, more than 99 percent identical; DNA sequences that do not code for proteins differ only a little more. The two primates therefore are much closer cousins than suggested by fossil studies of the time. The genetic resemblance seemed to contradict obvious distinctions: Human brains outsize those of chimps; their limbs dwarf ours; and modes of communication, food gathering, and other lifestyle features diverge dramatically. King and Wilson proposed that these contrasts arise not from disparities in DNA sequences that encode proteins, but from a small number of differences in DNA sequences that turn the protein-coding genes on and off.

Just as genetic changes drive species in new directions, they also can propel cells toward malignancy. From an evolutionary perspective, the topic of breast cancer began to intrigue King. The illness runs in families and is clearly inherited, yet many affected women have no close relatives with the disease. It is especially deadly for women whose mothers succumbed to it—and risk increases for those who have a mother or sister with breast cancer, particularly if the cancer struck bilaterally or before menopause. Unlike the situation with lung cancer, no environmental exposure distinguishes sisters who get breast cancer from those who remain disease free.

By studying a rare familial cancer, Alfred Knudsen (Lasker Clinical Medical Research Award, 1998) had shown in the early 1970’s how an inherited genetic defect could increase vulnerability to cancer. In the model he advanced, some families harbor a damaged version of a gene that normally encourages proper cellular behavior. Genetic mishaps occur during a person’s lifetime, and a second “hit” in a cell with the first physiological liability nudges the injured cell toward malignancy. A similar story might play out in families with a high incidence of breast cancer, King reasoned. She began to hunt for the theoretical pernicious gene in 1974.

The hunt
Many geneticists doubted that susceptibility to breast cancer would map to a single gene; even if it did, finding the culprit seemed unlikely for numerous reasons. First, most cases are not familial and the disease is common—so common that inherited and non-inherited cases could occur in the same families. Furthermore, the malady might not strike all women who carry a high-risk gene, and different families might carry different high-risk genes. Prevailing views held that the ailment arises from the additive effects of multiple undefined genetic and environmental insults and from complicated interactions among them. No one had previously tacked such complexities, and an attempt to unearth a breast cancer gene seemed woefully naïve.

To test whether she could find evidence that particular genes increase the odds of getting breast cancer, King applied mathematical methods to data from more than 1500 families of women younger than 55 years old with newly diagnosed breast cancer. The analysis, published in 1988, suggested that four percent of the families carry a single gene that predisposes individuals to the illness.

The most convincing way to validate this idea was to track down the gene. Toward this end, King analyzed DNA from 329 participating relatives with 146 cases of invasive breast cancer. In many of the 23 families to which the participants belonged, the scourge struck young women, often in both breasts, and in some families, even men.

In late 1990, King (by then a professor at the University of California, Berkeley) hit her quarry. She had zeroed in on a suspicious section of chromosome 17 that carried particular genetic markers in women with breast cancer in the most severely affected families. Somewhere in that stretch of DNA lay the gene, which she named BRCA1.

This discovery spurred an international race to find the gene. Four years later, scientists at Myriad Genetics, Inc. isolated it. Alterations in either BRCA1 or a second breast-cancer susceptibility gene, BRCA2, found by Michael Stratton and colleagues (Institute of Cancer Research, UK) increase risk of ovarian as well as breast cancer. The proteins encoded by these genes help maintain cellular health by repairing marred DNA. When theBRCA1 or BRCA2 proteins fail to perform their jobs, genetic integrity is compromised, thus setting the stage for cancer.

About 12 percent of women in the general population get breast cancer at some point in their lives. In contrast, 65 percent of women who inherit an abnormal version of BRCA1 and about 45 percent of women who inherit an abnormal version of BRCA2 develop breast cancer by the time they are 70 years old. Individuals with troublesome forms of BRCA1 and BRCA2 can now be identified, monitored, counseled, and treated appropriately.

Harmful versions of other genes also predispose women to breast cancer, ovarian cancer, or both. Several years ago, King devised a scheme to screen for all of these genetic miscreants. This strategy allows genetic testing and risk determination for breast and ovarian cancer; it is already in clinical practice.

Genetic tools, human rights
King has applied her expertise to aid people who suffer from ills perpetrated by humans as well as genes. She helped find the “lost children” of Argentina—those who had been kidnapped as infants or born while their mothers were in prison during the military regime of the late 1970s and early 1980s. Some of these youngsters had been illegally adopted, many by military families. In 1983, King began identifying individuals, first with a technique that was originally designed to match potential organ transplant donors and recipients. She then developed an approach that relies on analysis of DNA from mitochondria—a cellular component that passes specifically from mother to child, and is powerful for connecting people to their female forebears. King helped prove genetic relationships and thus facilitated the reunion of more than 100 of the children with their families.

Later, the Argentinian government asked if she could help identify dead bodies of individuals thought to have been murdered. King harnessed the same method to figure out who had been buried in mass graves. She established that teeth, whose enamel coating protects DNA in the dental pulp from degradation, offer a valuable resource when attempting to trace remains in situations where long periods have elapsed since the time of death.

This and related approaches have been used to identify soldiers who went missing in action, including the remains of an American serviceman who was buried beneath the Tomb of the Unknowns in Arlington National Cemetery for 14 years, as well as victims of natural disasters and man-made tragedies such as 9/11.

Mary-Claire King has employed her intellect, dedication, and ethical sensibilities to generate knowledge that has catalyzed profound changes in health care, and she has applied her expertise to promote justice where nefarious governments have terrorized their citizens.

by Evelyn Strauss

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From Genomics of Microorganisms to Translational Medicine

Reporter and Curator: Demet Sag, PhD

Pharmacogenomics needs new materials that are inert against the host and specifically  active to modulate molecular metabolism towards wanted homeostasis of the physiological system.  These can come from natural resources or men-made.  That is why we must know the origin  to  improve.     Recently, Synthetic Biology, even though it is a developing upcoming field, it is generating mile stones for applications in the clinic, the biotechnology industry and in basic molecular research. As  a result, it created a multidisciplinary expertise from scientists to engineers.  Among other things extending the search to first life on Earth is one of the many alternatives.  Here I like to present how synthetic biology can be initiated onto Translational Medicine from adiscovery of molecules from the sea.

Microorganisms played a role in evolution to start a life.  99 % of our genome is related to microbial organisms. initially there was a classical  Microbiology, then evolved to Industrial Microbiology and Biotechnology then Microbial Genomics and now Microbiome and Health became the focus.  Finally,  the circle is getting tide into how microbiome involved with healthy and disease state of human? How they can be used that is what it really means to include microorganisms into human health for diagnostics and targeted therapies?

Or should we start from  scarcity?

Microbiology is my first formal education and  building block.  Simple but help to understand system biology and  the mechanism of life in a nut shell.   The closest field is embryonic stem cell biology for building “synthesizing” a whole new organism.  Then  system biology and developmental biology also gain interest.

The real  remember the month of October in 2001 when DOE reported that they sequenced 23 organisms in Walnut Creek.  Having seen presentation to identify microorganisms through complex crystal structure assays through chemical pathway  at the Microbial Genomics Meeting organized by ASM in Monterey, CA in 2001.

Discovery of microorganisms in marine life like in Mediterranean Sea, containing 38% salt,is very similar with finding circulating disease making cells.   Yet, they are similar since both search for a specific needle in the pile.  Furthermore, the unique behavior of enzymes from microbial organisms such as Taq polymerase or restriction enzymes made it possible for us to develop new technologies for copying and propagating significant sequences.  When these early molecular biology methods are combined with the power of genomics and knowledge of unique structures in molecular physiology, it is possible to design better and sensitive sensors or build an organism to rptect or fix the need of the body.  neither sensors nor synthesized organism model are complete since one is missing the basic element of life “transformation of information” the other is missing the integrity that once nature provided in a single simple cell.

Having sensory smart chip/band/nanomolecule to redesign the cells may also possible if only we know the combination.  Thus, we have options to deliver if we know what to be carried.

An external file that holds a picture, illustration, etc.<br /><br /><br /><br /><br /> Object name is marinedrugs-11-00700-g002.jpg

(Figure: The combined strategy of gene-based screening and bioactivity-based screening for marine microbial natural products (MMNPs) discovery, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3705366/figure/marinedrugs-11-00700-f002/)

As we come across, novel pathways or primary pathways of physiology gain significant interest to determine marine microbial compound for therapeutics since they are further away from the evolution three that gives an advantage for biomedical/translational scientist to avoid most part of th eimmune responses such as inflammation, toxicity. Yes, indeed these are not scientific tails but true since currently, 16 of 20 marine antitumor compounds under clinical trial are derived from microbial sources because marine microorganisms are a major source for MMNP discovery.  However, isolation of these organisms.  For example, pretreatment methods, enrichment, physical, and chemical techniques (e.g., dry heat, exposure to 1%–1.5% phenol, sucrose-gradient centrifugation, and filtration through cellulose membrane filters) can be applied to increase especially the less abundant specific groups of marine microorganisms, . A variety of pretreatment methods including recovery of these microorganisms.  This reminds me ecosystem of the soil, since in soil the trouble is identifying the specific culture among millions of others.

Regardless of the case,  nutrients are the key for selecting and isolating any organisms but specifically, as a result any marine microbes have specific nutrient requirements for growth (e.g., sponge extract ) or chemical (e.g., siderophores, signal molecules, non-traditional electron donors, and electron acceptors.  This also should remind us subject of Biology 101 Essential Vitamins and Minerals.  What we eat who we are.

For example, Bruns et al. employed technique where they employed different carbon substrates (agarose, starch, laminarin, xylan, chitin, and glucose) at low concentrations (200 μM each) so that they can  improve the cultivation efficiency of bacteria from the Gotland Deep in the central Baltic Sea. As a result of this growth medium they were able to elevate yield, which is created higher cultivation efficiencies (up to 11% in fluid media) compared to other studies.

Yet, another component must be addressed that is culture medium such as ionic strength for a microbila growth. For example, Tsueng et al. study on marine actinomycete genus Salinispora that can produce bioactive secondary metabolites such as desferrioxamine, saliniketals, arenamides, arenimycin and salinosporamide.  However, they observed that  three species of SalinisporaS. arenicolaS. tropica, and S. pacifica require a high ionic strength but  S. arenicolahas a lower growth requirement for ionic strength than S. tropica and S. pacificaUsing after assaying them against sodium chloride-based and lithium chloride-based media. As  aresult, there is a specificity for growth. 

In addition, energy must be supported imagine that in marine organisms the metabolism is very unique, may be slow and possibly.  However, the main criteria is  most of them grow under low oxygen conditions like tumors.  Warburg effect posed a  problem for human but helped microorganisms to survive and evolve.  One’s weakness the other’s strength make a great teamwork for solving diseases of human kind es especially for cancer. 

This reminds us to utilize minerals, electrons specifically after all the simplest form of carbon metabolism based on biochemical pathways like Crebs cycle, one carbon metabolism and amino acid metabolism etc. Even though 90% of human body made up off microbial origin there are microorganisms that are not cultured yet.

The irony is less than 1% of microorganisms can be cultured.  Furthermore, they are not included for representing the total phylogenetic diversity. Therefore, majority of work concentrated on finding and cultivating the uncultured majority of the microbial world for MMNPs’.  For example,  an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei  producing many antitumor compounds such as pederin, mycalamide A, and onnamide A.

In any conditions if any living needs to be recognized and remembered, their place would be either on top or the bottom of the stack. Microbiome searches for specificity among tone of other organisms to recognize the disease, changes in cell differentiation and pathways or marine microbiologist search for uncommon scarce organisms. Yet, both of them are beneficial with their unique way.

Then what is the catch or fuss?  The catch is screening to identify what makes this organism unique that can be use for human health. Translational medicine may start from the beginning of life from microorganisms created.  This can be called with its newly coined named”synthetic biology” but if we go further than the conventional screening methods which include bioactivity-guided screening and gene-guided screening  and increase the power with genomics we may call it “synthetic genomics”.

As  a result these signature sequences establishes the “unique” biomarkers  or therpaeutics to be used for drug discovery, making vaccines, and remodulating the targeted cells. How?

These microorganisms secrete these metabolites or proteins to their growth medium just like a soluble protein, if you will like a inflammation factor or any other secreted protein of our human body cells. Collecting substrate or extract the pellet could be the choice.   in a nut shell this require at least three steps: First, finding the bioactivity, apply bioactivity-guided screening for direct detection of  the activity such as antimicrobial, antitumor, antiviral, and antiparasitic activities.  Second, a bioinformatic assessment of the secondary metabolite biosynthetic potential in the absence of fully assembled pathways or genome sequences. Third, application on cell lines and possible onto model organisms can improve the process of MMNP discovery so that allow us to prioritize strains for fermentation studies and chemical analysis. 

In summary, establish the culture growth, analyze bioactivity and discover the new gene product to be used.  Here is an example, first they  isolated Marinispora sp from the saline culture.  Next step,  identify new sources of bioactive secondary metabolites, gene-guided screening has been deployed to search target genes associated with NPs biosynthetic pathways, e.g., the fragments between ketosynthase and methylmalonyl-CoA transferase of polyketides (PKS) type I, enediyne PKS ketosynthase gene, O-methyltransferase gene, P450 monooxygenase gene, polyether epoxidase gene, 3-hydroxyl-3-methylglutaryl coenzyme A reductase gene, dTDP-glucose-4,6-dehydratase (dTGD) gene, and halogenase gene. The, apply bioinformatics that includes synthesizing the knowledge with  homology-based searches and phylogenetic analyses, gene-based screening  to predict new secondary metabolites discovered by isolates or environments.  Finally, identify the sequnce for PCR and use against a cell line or model organisms. In this case,  CNQ-140 based on significant antibacterial activities  against drug-resistant pathogens (e.g., MRSA) and impressive and selective cancer cell cytotoxicities (0.2–2.7 μM of MIC50 values) against six melanoma cell lines provided significant outcome. They are recognized as antitumor antibiotics with a new structural class, marinomycins A–D

This is a great method but there are two botle necks: 1. 99% of microbial organisms are not cultured in the labs. 2. Finding the optimum microbial growth and screening takes time. Thus, assesments can me done through metagenomics.  However, metagenomics has its shortcomings since on face of living unless applications applied in vivo in vitro results may not be valid.  The disadvantage of  metagenomics can be listed as:  1. inability of efficient acquisition of intact gene fragment,  2. incompatibility of expression elements such as promoter in a heterologous host.  On the pther hand, there can be possible resolution to avoid these factors  so metagenomics-based MMNP discovery can be plausable such as development  in  synthetic biology by large DNA fragment assembly techniques for artificial genome synthesis and synthetic microbial chassis suitable for different classes of MMNP biosynthesis.

However, many gene clusters have been identified by combined power of genomics and biioinformatics for MNP discovery.  This is  mainly necessary since  secondary metabolites usually biosynthesized by large multifunctional synthases that acts in a sequential assembly lines like adding carboxylic acid and amino acid building blocks into their products.  


Simmons TL, Coates RC, Clark BR, Engene N, Gonzalez D, Esquenazi E, Dorrestein PC, Gerwick W

Proc Natl Acad Sci U S A. 2008 Mar 25; 105(12):4587-94.

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The immune response mechanism is the holy grail of the human defense system for health.   IDO, indolamine 2, 3-dioxygenase, is a key gene for homeostasis of immune responses and producing an enzyme catabolizing the first rate-limiting step in tryptophan degradation metabolism. The hemostasis of immune system is complicated.  In this review, the  properties of IDO such as basic molecular genetics, biochemistry and genesis will be discussed.

IDO belongs to globin gene family to carry oxygen and heme.  The main function and genesis of IDO comes from the immune responses during host-microbial invasion and choice between tolerance and immunegenity.  In human there are three kinds of IDOs, which are IDO1, IDO2, and TDO, with distinguished mechanisms and expression profiles. , IDO mechanism includes three distinguished pathways: enzymatic acts through IFNgamma, non-enzymatic acts through TGFbeta-IFNalpha/IFNbeta and moonlighting acts through AhR/Kyn.

The well understood functional genomics and mechanisms is important to translate basic science for clinical interventions of human health needs. In conclusion, overall purpose is to find a method to manipulate IDO to correct/fix/modulate immune responses for clinical applications.

The first part of the review concerns the basic science information gained overall several years that lay the foundation where translational research scientist should familiar to develop a new technology for clinic. The first connection of IDO and human health came from a very natural event that is protection of pregnancy in human. The focus of the translational medicine is treatment of cancer or prevention/delay cancer by stem cell based Dendritic Cell Vaccine (DCvax) development.

Table of Contents:

  • Abstract

1         Introduction: IDO gene encodes a heme enzyme

2        Location, location, location

3        Molecular genetics

4        Types of IDO:

4.1       IDO1,

4.2       IDO2,

4.3       IDO-like proteins

5        Working mechanisms of IDO

6        Infection Diseases and IDO

7. Conclusion

  1. 1.     Indoleamine 2, 3-dioxygenase (IDO) gene encodes a heme enzyme

IDO is a key homeostatic regulator and confined in immune system mechanism for the balance between tolerance and immunity.  This gene encodes indoleamine 2, 3-dioxygenase (IDO) – a heme enzyme (EC= that catalyzes the first rate-limiting step in tryptophan catabolism to N-formyl-kynurenine and acts on multiple tryptophan substrates including D-tryptophan, L-tryptophan, 5-hydroxy-tryptophan, tryptamine, and serotonin.

The basic genetic information describes indoleamine 2, 3-dioxygenase 1 (IDO1, IDO, INDO) as an enzyme located at Chromosome 8p12-p11 (5; 6) that active at the first step of the Tryptophan catabolism.    The cloned gene structure showed that IDO contains 10 exons ad 9 introns (7; 8) producing 9 transcripts.

After alternative splicing only five of the transcripts encode a protein but the other four does not make protein products, three of transcripts retain intron and one of them create a nonsense code (7).  Based on IDO related studies 15 phenotypes of IDO is identified, of which, twelve in cancer tumor models of lung, kidney, endometrium, intestine, two in nervous system, and one HGMD- deletion.

  1. 2.     Location, Location and Location

The specific cellular location of IDO is in cytosol, smooth muscle contractile fibers and stereocilium bundle. The expression specificity shows that IDO is present very widely in all cell types but there is an elevation of expression in placenta, pancreas, pancreas islets, including dendritic cells (DCs) according to gene atlas of transcriptome (9).  Expression of IDO is common in antigen presenting cells (APCs), monocytes (MO), macrophages (MQs), DCs, T-cells, and some B-cells. IDO present in APCs (10; 11), due to magnitude of role play hierarchy and level of expression DCs are the better choice but including MOs during establishment of three DC cell subset, CD14+CD25+, CD14++CD25+ and CD14+CD25++ may increase the longevity and efficacy of the interventions.

IDO is strictly regulated and confined to immune system with diverse functions based on either positive or negative stimulations. The positive stimulations are T cell tolerance induction, apoptotic process, and chronic inflammatory response, type 2 immune response, interleukin-12 production (12).  The negative stimulations are interleukin-10 production, activated T cell proliferation, T cell apoptotic process.  Furthermore, there are more functions allocating fetus during female pregnancy; changing behavior, responding to lipopolysaccharide or multicellular organismal response to stress possible due to degradation of tryptophan, kynurenic acid biosynthetic process, cellular nitrogen compound metabolic process, small molecule metabolic process, producing kynurenine process (13; 14; 15).

IDO plays a role in a variety of pathophysiological processes such as antimicrobial and antitumor defense, neuropathology, immunoregulation, and antioxidant activity (16; 17; 18; 19).


 3.     Molecular Genetics of IDO:

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3' untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database. (reference: http://atlasgeneticsoncology.org/Genes/IDO2ID44387ch8p11.html)

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3′ untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database.
(reference: http://atlasgeneticsoncology.org/Genes/IDO2ID44387ch8p11.html)

Molecular genetics data from earlier findings based on reporter assay results showed that IDO promoter is regulated by ISRE-like elements and GAS-sequence at -1126 and -1083 region (20).  Two cis-acting elements are ISRE1 (interferon sequence response element 1) and interferon sequence response element 2 (ISRE2).

Analyses of site directed and deletion mutation with transfected cells demonstrated that introduction of point mutations at these elements decreases the IDO expression. Removing ISRE1 decreases the effects of IFNgamma induction 50 fold and deleting ISRE1 at -1126 reduced by 25 fold (3). Introducing point mutations in conserved t residues at -1124 and -1122 (from T to C or G) in ISRE consensus sequence NAGtttCA/tntttNCC of IFNa/b inducible gene ISG4 eliminates the promoter activity by 24 fold (21).

ISRE2 have two boxes, X box (-114/1104) and Y Box 9-144/-135), which are essential part of the IFNgamma response region of major histocompatibility complex class II promoters (22; 23).  When these were removed from ISRE2 or introducing point mutations at two A residues of ISRE2 at -111 showed a sharp decrease after IFNgamma treatment by 4 fold (3).

The lack of responses related to truncated or deleted IRF-1 interactions whereas IRF-2, Jak2 and STAT91 levels were similar in the cells, HEPg2 and ME180 (3). Furthermore, 748 bp deleted between these elements did not affect the IDO expression, thus the distance between ISRE1 and ISRE2 elements have no function or influence on IDO (3; 24)

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

4.     There are three types of IDO in human genome:

IDO was originally discovered in 1967 in rabbit intestine (25). Later, in 1990 the human IDO gene is cloned and sequenced (7).  However, its importance and relevance in immunology was not created until prevention of allocation of fetal rejection and founding expression in wide range of human cancers (26; 27).

There are three types of IDO, pro-IDO like, IDO1, and IDO2.  In addition, another enzyme called TDO, tryptophan 2, 3, dehydrogenase solely degrade L-Trp at first-rate limiting mechanism in liver and brain.

4.1.  IDO1:

IDO1 mechanism is the target for immunotherapy applications. The initial discovery of IDO in human physiology is protection of pregnancy (1) since lack of IDO results in premature recurrent abortion (28; 26; 29).   The initial rate-limiting step of tryptophan metabolism is catalyzed by either IDO or tryptophan 2, 3-dioxygenase (TDO).

Structural studies of IDO versus TDO presenting active site environments, conserved Arg 117 and Tyr113, found both in TDO and IDO for the Tyr-Glu motif, but His55 in TDO replaced by Ser167b in IDO (30; 2). As a result, they are regulated with different mechanisms (1; 2) (30).  The short-lived TDO, about 2h, responds to level of tryptophan and its expression regulated by glucorticoids (31; 32).  Thus, it is a useful target for regulation and induced by tryptophan so that increasing tryptophan induces NAD biosynthesis. Whereas, IDO is not activated by the level of Trp presence but inflammatory agents with its interferon stimulated response elements (ISRE1 and ISRE2) in its (33; 34; 35; 36; 3; 10) promoter.

TDO promoter contains glucorticoid response elements (37; 38) and regulated by glucocorticoids and other available amino acids for gluconeogenesis. This is how IDO binds to only immune response cells and TDO relates to NAD biosynthesis mechanisms. Furthermore, TDO is express solely in liver and brain (36).  NAD synthesis (39) showed increased IDO ubiquitous and TDO in liver and causing NAD level increase in rat with neuronal degeneration (40; 41).  NAM has protective function in beta-cells could be used to cure Type1 diabetes (40; 42; 43). In addition, knowledge on NADH/NAD, Kyn/Trp or Trp/Kyn ratios as well as Th1/Th2, CD4/CD8 or Th17/Threg are equally important (44; 40).

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (http://www.pnas.org/content/103/8/2611/F3.expansion.html)

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (http://www.pnas.org/content/103/8/2611/F3.expansion.html)

4.2. IDO2:

The third type of IDO, called IDO2 exists in lower vertebrates like chicken, fish and frogs (45) and in human with differential expression properties. The expression of IDO2 is only in DCs, unlike IDO1 expresses on both tumors and DCs in human tissues.  Yet, in lower invertebrates IDO2 is not inhibited by general inhibitor of IDO, D-1-methyl-tryptophan (1MT) (46).   Recently, two structurally unusual natural inhibitors of IDO molecules, EXIGUAMINES A and B, are synthesized (47).  LIP mechanism cannot be switch back to activation after its induction in IDO2 (46).

Crucial cancer progression can continue with production of IL6, IL10 and TGF-beta1 to help invasion and metastasis.  Inclusion of two common SNPs affects the function of IDO2 in certain populations.  SNP1 reduces 90% of IDO2 catalytic activity in 50% of European and Asian descent and SNP2 produce premature protein through inclusion of stop-codon in 25% of African descent lack functional IDO2 (Uniport).

4.3. IDO-like proteins: The Origin of IDO:

Knowing the evolutionary steps will helps us to identify how we can manage the regulator function to protect human health in cancer, immune disorders, diabetes, and infectious diseases.

Bacterial IDO has two types of IDOs that are group I and group II IDO (48).  These are the earliest version of the IDO, pro-IDO like, proteins with a quite complicated function.  Each microorganism recognized by a specific set of receptors, called Toll-Like Receptors (TLR), to activate the IDO-like protein expression based on the origin of the bacteria or virus (49; 35).   Thus, the genesis of human IDO originates from gene duplication of these early bacterial versions of IDO-like proteins after their invasion interactions with human host.  IDO1 only exists in mammals and fungi.

Fungi also has three types of IDO; IDOa, IDO beta, and IDO gamma (50) with different properties than human IDOs, perhaps multiple IDO is necessary for the world’s decomposers.

All globins, haemoglobins and myoglobins are destined to evolve from a common ancestor, which  is only 14-16kDa (51) length. Binding of a heme and being oxygen carrier are central to the enzyme mechanism of this family.  Globins are classified under three distinct origins; a universal globin, a compact globin, and IDO-like globin (52) IDO like globin widely distributed among gastropodic mollusks (53; 51).  The indoleamine 2, 3-dioxygenase 1–like “myoglobin” (Myb) was discovered in 1989 in the buccal mass of the abalone Sulculus diversicolor (54).

The conserved region between Myb and IDO-like Myb existed for at least 600 million years (53) Even though the splice junction of seven introns was kept intact, the overall homolog region between Myb and IDO is only about 35%.

No significant evolutionary relationship is found between them after their amino acid sequence of each exon is compared to usual globin sequences. This led the hint that molluscan IDO-like protein must have other functions besides carrying oxygen, like myoglobin.   Alignment of S. cerevisiae cDNA, mollusk and vertebrate IDO–like globins show the key regions for controlling IDO or myoglobin function (55). These data suggest that there is an alternative pathways of myoglobin evolution.  In addition, understanding the diversity of globin may help to design better protocols for interventions of diseases.

Mechanisms of IDO:

The dichotomy of IDO mechanism lead the discovery that IDO is more than an enzyme as a versatile regulator of innate and adaptive immune responses in DCs (66; 67; 68). Meantime IDO also involve with Th2 response and B cell mediated autoimmunity showing that it has three paths, short term (acute) based on enzymatic actions, long term (chronic) based on non-enzymatic role, and moonlighting relies of downstream metabolites of tryptophan metabolism (69; 70).

IFNgamma produced by DC, MQ, NK, NKT, CD4+ T cells and CD8+ T cells, after stimulation with IL12 and IL8.  Inflammatory cytokine(s) expressed by DCs produce IFNgamma to stimulate IDO’s enzymatic reactions in acute response.  Then, TDO in liver and tryptophan catabolites act through Aryl hydrocarbon receptor induction for prevention of T cell proliferation. This mechanism is common among IDO, IDO2 (expresses in brain and liver) and TDO expresses in liver) provide an acute response for an innate immunity (30). When the pDCs are stimulated with IFNgamma, activation of IDO is go through Jak, STAT signaling pathway to degrade Trp to Kyn causing Trp depletion. The starvation of tryptophan in microenvironment inhibits generation of T cells by un-read t-RNAs and induce apoptosis through myc pathway.  In sum, lack of tryptophan halts T cell proliferation and put the T cells in apoptosis at S1 phase of cell division (71; 62).

The intermediary enzymes, functioning during Tryptophan degradation in Kynurenine (Kyn) pathway like kynurenine 3-hydroxylase and kynureninase, are also induced after stimulation with liposaccaride and proinflammatory cytokines (72). They exhibit their function in homeostasis through aryl-hydrocarbon receptor (AhR) induction by kynurenine as an endogenous signal (73; 74).  The endogenous tumor-promoting ligand of AhR are usually activated by environmental stress or xenobiotic toxic chemicals in several cellular processes like tumorigenesis, inflammation, transformation, and embryogenesis (Opitz ET. Al, 2011).

Human tumor cells constitutively produce TDO also contributes to production of Kyn as an endogenous ligand of the AhR (75; 27).  Degradation of tryptophan by IDO1/2 in tumors and tumor-draining lymph nodes occur. As a result, there are animal studies and Phase I/II clinical trials to inhibit the IDO1/2 to prevent cancer and poor prognosis (NewLink Genetics Corp. NCT00739609, 2007).

 IDO mechanism for immune response

Systemic inflammation (like in sepsis, cerebral malaria and brain tumor) creates hypotension and IDO expression has the central role on vascular tone control (63).  Moreover, inflammation activates the endothelial coagulation activation system causing coagulopathies on patients.  This reaction is namely endothelial cell activation of IDO by IFNgamma inducing Trp to Kyn conversion. After infection with malaria the blood vessel tone has decreases, inflammation induce IDO expression in endothelial cells producing Kyn causing decreased trp, lower arterial relaxation, and develop hypotension (Wang, Y. et. al 2010).  Furthermore, existing hypotension in knock out Ido mice point out a secondary mechanism driven by Kyn as an endogenous ligand to activate non-canonical NfKB pathway (63).

Another study also hints this “back –up” mechanism by a significant outcome with a differential response in pDCs against IMT treatment.  Unlike IFN gamma conditioned pDC blocks T cell proliferation and apoptosis, methyl tryptophan fails to inhibit IDO activity for activating naïve T cells to make Tregs at TGF-b1 conditioned pDCs (77; 78).

 Indoleamine-Pyrrole 2,3,-Dioxygenase; IDO dioxygenase; Indeolamine-2,3

The second role of the IDO relies on non-enzymatic action as being a signal molecule. Yet, IDO2 and TDO are devoid of this function. This role mainly for maintenance of microenvironment condition. DCs response to TGFbeta-1 exposure starts the kinase Fyn induce phosphorylation of IDO-associated immunoreceptor tyrosine–based inhibitory motifs (ITIMs) for propagation of the downstream signals involving non-canonical (anti-inflammatory) NF-kB pathway for a long term response. When the pDCs are conditioned with TGF-beta1 the signaling (68; 77; 78) Phospho Inositol Kinase3 (PIK-3)-dependent and Smad independent pathways (79; 80; 81; 82; 83) induce Fyn-dependent phosphorylation of IDO ITIMs.  A prototypic ITIM has the I/V/L/SxYxxL/V/F sequence (84), where x in place of an amino acid and Y is phosphorylation sites of tyrosines (85; 86).

Smad independent pathway stimulates SHP and PIK3 induce both SHP and IDO phosphorylation. Then, formed SHP-IDO complex can induce non-canonical (non-inflammatory) NF-kB pathway (64; 79; 80; 82) by phosphorylation of kinase IKKa to induce nuclear translocation of p52-Relb towards their targets.  Furthermore, the SHP-IDO complex also may inhibit IRAK1 (68). SHP-IDO complex activates genes through Nf-KB for production of Ido1 and Tgfb1 genes and secretion of IFNalpha/IFNbeta.  IFNa/IFNb establishes a second short positive feedback loop towards p52-RelB for continuous gene expression of IDO, TGFb1, IFNa and IFNb (87; 68).  However, SHP-IDO inhibited IRAK1 also activates p52-RelB.  Nf-KB induction at three path, one main and two positive feedback loops, is also critical.  Finally, based on TGF-beta1 induction (76) cellular differentiation occurs to stimulate naïve CD4+ T cell differentiation to regulatory T cells (Tregs).  In sum, TGF-b1 and IFNalpha/IFNbeta stimulate pDCs to keep inducing naïve T cells for generation of Treg cells at various stages, initiate, maintain, differentiate, infect, amplify, during long-term immune responses (67; 66).

Moonlighting function of Kyn/AhR is an adaptation mechanism after the catalytic (enzymatic) role of IDO depletes tryptophan and produce high concentration of Kyn induce Treg and Tr1 cell expansion leading Tregs to use TGFbeta for maintaining this environment (67; 76). In this role, Kyn pathway has positive-feedback-loop function to induce IDO expression.

In T cells, tryptophan starvation induces Gcn2-dependent stress signaling pathway, which initiates uncharged Trp-tRNA binding onto ribosomes. Elevated GCN2 expression stimulates elF2alfa phosphorylation to stop translation initiation (88). Therefore, most genes downregulated and LIP, an alternatively initiated isoform of the b/ZIP transcription factor NF-IL6/CEBP-beta (89).

This mechanism happens in tumor cells based on Prendergast group observations. As a result, not only IDO1 propagates itself while producing IFNalpha/IFNbeta, but also demonstrates homeostasis choosing between immunegenity by production of TH17or tolerance by Tregs. This mechanism acts like a see-saw. Yet, tolerance also can be broken by IL6 induction so reversal mechanism by SOC-3 dependent proteosomal degradation of the enzyme (90).  All proper responses require functional peripheral DCs to generate mature DCs for T cells to avoid autoimmunity (91).

Niacin (vitamin B3) is the final product of tryptophan catabolism and first molecule at Nicotinomic acid (NDA) Biosynthesis.  The function of IDO in tryptophan and NDA metabolism has a great importance to develop new clinical applications (40; 42; 41).  NAD+, biosynthesis and tryptophan metabolisms regulate several steps that can be utilize pharmacologically for reformation of healthy physiology (40).

IDO for protection in Microbial Infection with Toll-like Receptors

The mechanism of microbial response and infectious tolerance are complex and the origination of IDO based on duplication of microbial IDO (49).  During microbial responses, Toll-like receptors (TLRs) play a role to differentiate and determine the microbial structures as a ligand to initiate production of cytokines and pro-inflammatory agents to activate specific T helper cells (92; 93; 94; 95). Uniqueness of TLR comes from four major characteristics of each individual TLR by ligand specificity, signal transduction pathways, expression profiles and cellular localization (96). Thus, TLRs are important part of the immune response signaling mechanism to initiate and design adoptive responses from innate (naïve) immune system to defend the host.

TLRs are expressed cell type specific patterns and present themselves on APCs (DCs, MQs, monocytes) with a rich expression levels (96; 97; 98; 99; 93; 100; 101; 102; 87). Induction signals originate from microbial stimuli for the genesis of mature immune response cells.  Co-stimulation mechanisms stimulate immature DCs to travel from lymphoid organs to blood stream for proliferation of specific T cells (96).  After the induction of iDCs by microbial stimuli, they produce proinflammatory cytokines such as TNF and IL-12, which can activate differentiation of T cells into T helper cell, type one (Th1) cells. (103).

Utilizing specific TLR stimulation to link between innate and acquired responses can be possible through simple recognition of pathogen-associated molecular patterns (PAMPs) or co-stimulation of PAMPs with other TLR or non-TLR receptors, or even better with proinflammatory cytokines.   Some examples of ligand- TLR specificity shown in Table1, which are bacterial lipopeptides, Pam3Cys through TLR2 (92; 104; 105).  Double stranded (ds) RNAs through TLR3 (106; 107), Lipopolysaccharide (LPS) through TLR4, bacterial flagellin through TLR5 (108; 109), single stranded RNAs through TLR7/8 (97; 98), synthetic anti-viral compounds imiquinod through TLR 7 and resiquimod through TLR8, unmethylated CpG DNA motifs through TLR9 (Krieg, 2000).

IDO action

Then, the specificity is established by correct pairing of a TLR with its proinflammatory cytokines, so that these permutations influence creation and maintenance of cell differentiation. For example, leading the T cell response toward a preferred Th1 or Th2 response possible if the cytokines TLR-2 mediated signals induce a Th2 profile when combined with IL-2 but TLR4 mediated signals lean towards Th1 if it is combined with IL-10 or Il-12, (110; 111)  (112).

TLR ligand TLR Reference
Lipopolysaccharide, LPS TLR4 (96).  (112).
Lipopeptides, Pam3Cys TLR2 (92; 104; 105)
Double stranded (ds) RNAs TLR3 (106; 107)
Bacterial flagellin TLR5 (108; 109)
Single stranded RNAs TLR7/8 (97; 98)
Unmethylated CpG DNA motifs TLR9 (Krieg, 2000)
Synthetic anti-viral compounds imiquinod and resiquimod TLR7 and TLR8 (Lee J, 2003)

Furthermore, if the DCs are stimulated with IL-6, DCs relieve the suppression of effector T cells by regulatory T cells (113).

The modification of IDO+ monocytes manage towards specific subset of T cell activation with specific TLRs are significantly important (94).

The type of cell with correct TLR and stimuli improves or decreases the effectiveness of stimuli. Induction of IDO in monocytes by synthetic viral RNAs (isRNA) and CMV was possible, but not in monocyte derived DCs or TLR2 ligand lipopeptide Pam3Cys since single- stranded RNA ligands target TLR7/8 in monocytes derive DCs only (Lee J, 2003).  These data show that TLRs has ligand specificity, signal transduction pathways, expression profiles and cellular localization so design of experiments should follow these rules.


Overall our purpose of this information is to find a method to manipulate IDO to correct/fix/modulate immune responses for clinical applications.  This first part of the review concerns the basic science information gained overall several years that lay the foundation that translational research scientist should familiar to develop a new technology for clinic. The first connection of IDO and human health came from a very natural event that is protection of pregnancy in human. The focus of the translational medicine is treatment of cancer or prevention/delay cancer by stem cell based Dendritic Cell Vaccine (DCvax) development.


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