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Posts Tagged ‘liver’


Seven Cancers: oropharynx, larynx, oesophagus, liver, colon, rectum and breast are caused by Alcohol Consumption

Reporter: Aviva Lev-Ari, PhD, RN

Keywords:

  • Alcohol;
  • cancer;
  • cardiovascular disease;
  • causal inference;
  • cohort studies;
  • epidemiology;
  • evidence-based policy

Background and aims

There is increasing research evidence about the causal role of alcohol in cancer, accompanied by unclear and conflicting messages in the media. This paper aimed to clarify the strength of the evidence for alcohol as a cause of cancer, and the meaning of cause in this context.

Methods

Recent epidemiological and biological research on alcohol and cancer was reviewed and summarized, drawing upon published meta-analyses identified from the Medline database and the archives of the International Agency for Research on Cancer. More recent epidemiological studies not included in these publications were also reviewed. A brief description of the nature of causal inference in epidemiology was used to frame discussion of the strength of the evidence that alcohol causes cancer, and contrast this with the case for a protective association of alcohol with cardiovascular disease.

Results

The usual epidemiological understanding of a cause is a factor that increases the incidence of a condition in the population. In the context of a body of epidemiological evidence of an association of alcohol consumption with a disease, the inference that it is a causal association requires alternative explanations of the observed finding to be judged unlikely. Even without complete knowledge of biological mechanisms, the epidemiological evidence can support the judgement that alcohol causes cancer of the oropharynx, larynx, oesophagus, liver, colon, rectum and breast. The measured associations exhibit gradients of effect that are biologically plausible, and there is some evidence of reversibility of risk in laryngeal, pharyngeal and liver cancers when consumption ceases. The limitations of cohort studies mean that the true effects may be somewhat weaker or stronger than estimated currently, but are unlikely to be qualitatively different. The same, or similar, epidemiological studies also commonly report protection from cardiovascular disease associated with drinking but a high level of scepticism regarding these findings is now warranted.

Conclusions

There is strong evidence that alcohol causes cancer at seven sites in the body and probably others. Current estimates suggest that alcohol-attributable cancers at these sites make up 5.8% of all cancer deaths world-wide. Confirmation of specific biological mechanisms by which alcohol increases the incidence of each type of cancer is not required to infer that alcohol is a cause.

SOURCE

http://onlinelibrary.wiley.com/doi/10.1111/add.13477/full

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Marcela’s Story:  A Liver Transplant Gives the Gift of Life

Patient is HCV Positive, liver transplanted from a 22-year-old donor performed at age 70. Interview conducted 14 years post-liver transplant.

Author: Gail S. Thornton, M.A.

Co-Editor: The VOICES of Patients, HealthCare Providers, Caregivers and Families: Personal Experience with Critical Care and Invasive Medical Procedures

For Marcela Almada Calles of Valle de Bravo, Mexico, a picturesque town on the shores of Lake Avándaro about two hours outside of Mexico City where she has lived for 30 years, life is about seizing the moment and having “an open mind and positive attitude.”  An active woman in her 80’s, Marcela’s days are full of professional and personal achievements and a long list of activities still to accomplish. However, life wasn’t always so positive as she put her life on hold for two-and-a-half years to relocate to Los Angeles, California, so that she could have a liver transplant.

“My spirit and attitude have always been what has carried me through life and difficult situations. This time was no different.”

Image SOURCE: Photographs courtesy of Marcela Almada Calles.   

Marcela’s story started 20 years ago during a time when she operated a successful event planning and catering business for high-profile government and social dignitaries, pharmaceutical companies, and luxury department stores.

“I normally worked long hours from early morning until evening, until one day, I felt exceptionally tired and it became a huge effort to concentrate. My ankles were swollen and I was out of breath all the time and my skin was yellow. I felt sleepy and would sometimes become tired during the day. This was unusual for me. I knew something was not right.”

At that point, Marcela decided to make an appointment with her local physician and friend, Dr. Sergio Ulloa, a highly regarded rheumatologist and corporate and government affairs leader in Mexico, who examined her and took several blood tests. When the blood results came back, Dr. Ulloa immediately referred her to Dr. Sergio Kershenovich, a well-regarded hepatologist, at his private clinic, who checked her for symptoms of Hepatitis C. After that Marcela decided to get another opinion and went to see Dr. Fernando Quijano, a general surgeon, who immediately wanted her to have surgery because he had found a cancerous tumor in her liver.

“My doctors’ opinions were that I needed to have a liver transplant immediately because I was in liver failure. It appeared that I had a failing liver — and a tumor there as well and my liver was not working properly.”

Relocating Life to the United States

At that point, my six children – Marcela, Luis, Diego, Rodolfo, Gabriela, Mario — who live in parts of Mexico and Singapore became involved in my health care decisions and treatment plan.

“My son, Luis, believed the best treatment for me was to see a liver specialist in the United States so that I received the best care from a leading liver transplantation hospital. He made some connections with friends and that next day, Dr. Francisco Durazo, chief of Transplant Hepatology and medical director of the Dumont UCLA Liver Transplant Center in Los Angeles, told me to come immediately to see him. I remember my children were supportive and concerned, but were afraid for me as we all knew that I had a long road ahead of me.”

At that time, she was put on a national liver transplant list by the UCLA Transplant Center.

“What I didn’t know was that more than 9,000 potential recipients are currently awaiting liver transplants.”  http://transplants.ucla.edu/site.cfm?id=397

“Dr. Durazo was very concerned and told me that my liver was not working at all and I had to have a liver transplant as soon as possible, so he asked me to stay in Los Angeles, since I was now part of a transplant list.”

Evaluation By Transplant Team

Marcela’s case is no different than any other patient awaiting a liver transplant. According to their web site, the UCLA Transplant Center conducts evaluations over two or three days. During this time, the patients meets with a social worker, transplant hepatologist, surgeon, transplant coordinator, psychiatrist and dietitian, as well as other specialists as needed. The evaluation is customized to each patient’s medical condition. Once the evaluation is completed, each patient’s case is presented at a weekly meeting of the UCLA Liver Transplant Consultation Team. This group includes specialists from surgery, adult and pediatric hepatology, cardiology, pulmonary, nephrology, hematology, infectious disease, as well as transplant coordinators and social workers. At this time, the team determines if any other tests are required to ensure the patient’s candidacy for transplant, then the patient and the physician are notified of the recommendation made by the transplant team. http://transplants.ucla.edu/site.cfm?id=401

Waiting For Answers

Marcela arrived at UCLA in Los Angeles with her family on Mother’s Day — May 10, 1999 — for what she describes as “the best time in her life to be alive with the help of medicine and technology.” That meant that she needed to rent an apartment and live near the hospital in case the doctors received an anonymous donor who would give her the gift of life.

“I had to wear a beeper 24 hours a day and I was never alone. My children took turns over the next two-and-a-half years to give up their lives with their families to live with me and help me navigate the health care system and my upcoming surgery.”

Marcela filled her days at her new apartment in Los Angeles reading about her condition, meditating to quiet her mind, watching television, and talking with family, friends and neighbors.

“The doctors called me two times over the next few months, saying they had an anonymous liver donor and I needed to come now to the hospital for tests. Unfortunately, those blood tests and other diagnostic tests showed that I was not a good match, so the doctors sent me home. It was a frustrating time because I wanted to have the liver transplant surgery and move on with my life.”

Finally, after waiting eight months for a liver transplant, Marcela’s outlook on life was greatly improved when an anonymous donor gave her the gift of life – a new, healthy liver.

“The donor’s blood type was a match for me. The surgery took eight hours and it was successful. The doctors told me that my immune system might reject my new liver, so I was given a cocktail of medicines, such as anti-rejection drugs, corticosteroids, calcinurin inhibitors, mTOR inhibitors, and antibiotics and watched very closely in the hospital.”

Marcela was then permitted to leave the hospital only a week after her surgery.

“That was the happiest day of my life. My spirits were high and I had a life to live.”

Her children served as her strength.

“My children took turns flying back and forth to Los Angeles to stay with me. They had a long list of instructions from the doctor. I could take some walks and eat small meals for the next few weeks, but I couldn’t exert myself in any way. I developed a cold over the next few weeks, as my immune system was low, so I had to take special care to eat right, get enough sleep and, most of all, relax. My body, spirit and mind had much healing to do.”

For the next 1 ½ years, Los Angeles was my “second” home.

“I needed to remain there after the procedure so my doctors could monitor my progress. During that time, I felt stronger each day. The support of my family was a true blessing for me. They were my eyes and ears – and my greatest advocates. My doctor recommended that I come weekly for check-ups and go through a physical therapy program so that I could regain my liver function and physical strength. I followed all my doctor’s orders.”

Day by day, Marcela believed as if she could conquer the world.

“I decided, one day many months after the surgery, to become ‘irresponsible’ and spent time with a few good friends, Gabriela and Guadalupe, who traveled to see me. For a weekend, we went to Las Vegas to see shows and go to the casinos. I laughed, played and walked all I could. My children didn’t even know what I was up to, but I felt good and wanted to enjoy the world and my new freedom.”

Marcela was able to return home to Valle de Bravo with a fresh perspective, a long list of things to do, and many happy memories.

“Since that time, I have kept myself active and busy; I never let my mind and heart rest. I am also forever grateful to my anonymous liver donor because it is because of a 22-year-old young man who died in an unfortunate automobile accident that I am here today.”

Liver Transplant Facts

The liver is the body’s vital organ that you cannot live without. It serves many critical functions, including metabolism of drugs and toxins, removing degradation products of normal body metabolism and synthesis of many proteins and enzyme, which are necessary for blood to clot. Transplantation is the only cure for liver insufficiency or liver failure because no device or machine reliably performs all the functions of the liver. http://transplant.surgery.ucsf.edu/conditions–procedures/liver-transplantation.aspx

According to a hospital transplant web site, overall, outcomes for liver transplantation are very good, but vary significantly depending on the indication for liver transplant as well as factors associated with the donor. Currently, the overall patient survival one year after liver transplant is 88 percent. Patient survival five years after liver transplant is 73 percent. These results vary significantly based on the indication for liver transplantation. The encouraging trend is that over the past 20 years short- and long-term patient survival has continued to improve. With advances in surgical technique, organ preservation, peri-operative care, and immunosuppression, survival will hopefully continue to improve in the future. http://transplant.surgery.ucsf.edu/conditions–procedures/liver-transplantation.aspx

Life For Marcela Today

Science is helping rebalance medicine with the most innovative discoveries and new ways of treating illness.

“I am happy to be part of the solution with a happy ending, too.”

Today, Marcela leads a rich and full life.

“It’s been 14 years since my liver transplant. I continue to feel healthy and alive. Nothing will keep me from doing what I want to do.”

Marcela has an active social life. She takes frequent vacations around the world, including a three-month holiday to Asia, where she travels multiple times to Bali, Cambodia, China and Singapore, where her daughter lives. She is an avid golfer and organizes tournaments in many private golf courses. She is learning to speak French, which is an easy transition (she says) from speaking Spanish. She plays cards with a group of friends weekly, sings in a musical group, and takes dance lessons, too. Life is very, very good.

Editor’s note: We would like to thank Gabriela Contreras, a global communications consultant and patient advocate, for the tremendous help and support that she provided in locating and scheduling time to talk with Marcela Almada Calles.

Marcela Almada Calles provided her permission to publish this interview on July 21, 2016.

 

REFERENCE/SOURCE 

http://www.webmd.com/digestive-disorders/digestive-diseases-liver-transplantation

Other related articles:

Retrieved from http://transplants.ucla.edu/site.cfm?id=397

Retrieved from http://transplant.surgery.ucsf.edu/conditions–procedures/liver-transplantation.aspx

Retrieved from http://transplant.surgery.ucsf.edu/conditions–procedures/liver-transplantation.aspx

Other related articles were published in this Open Access Online Scientific Journal include the following: 

2016

AGENDA for Adoptive T Cell Therapy Delivering CAR, TCR, and TIL from Research to Reality, CHI’S 4TH ANNUAL IMMUNO-ONCOLOGY SUMMIT – SEPTEMBER 1-2, 2016 | Marriott Long Wharf Hotel – Boston, MA

https://pharmaceuticalintelligence.com/2016/07/15/adoptive-t-cell-therapy-delivering-car-tcr-and-til-from-research-to-reality-chis-4th-annual-immuno-oncology-summit-september-1-2-2016-marriott-long-wharf-hotel-boston-ma/

Technologies For Targeting And Delivering Chemotherapeutics Directly To The Tumour Site

https://pharmaceuticalintelligence.com/2016/04/25/technologies-for-targeting-and-delivering-chemotherapeutics-directly-to-the-tumour-site/

2015

3-D Printed Liver

https://pharmaceuticalintelligence.com/2015/11/16/3-d-printed-liver/

Newly discovered cells regenerate liver tissue without forming tumors

https://pharmaceuticalintelligence.com/2015/08/16/newly-discovered-cells-regenerate-liver-tissue-without-forming-tumors/

Novel Approaches to Cancer Therapy 

https://pharmaceuticalintelligence.com/2015/04/11/novel-approaches-to-cancer-therapy-7-12/

 

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Alzheimer Disease Developments – Spring 2015

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Cognitive Stimulation Modulates Platelet Total Phospholipases A2 Activity in Subjects with Mild Cognitive Impairment

 

JNK: A Putative Link Between Insulin Signaling and VGLUT1 in Alzheimer’s Disease

Omega-3 Fatty Acid Status Enhances the Prevention of Cognitive Decline by B Vitamins in Mild Cognitive ImpairmentOpenly Available
Oulhaj, Abderrahim | Jernerén, Fredrik | Refsum, Helga | Smith, A. David | de Jager, Celeste A.

Preliminary Study of Plasma Exosomal Tau as a Potential Biomarker for Chronic Traumatic EncephalopathyOpenly Available
Stern, Robert A. | Tripodis, Yorghos | Baugh, Christine M. | Fritts, Nathan G. | Martin, Brett M. | Chaisson, Christine | Cantu, Robert C. | Joyce, James A. | Shah, Sahil | Ikezu, Tsuneya | Zhang, Jing | Gercel-Taylor, Cicek | Taylor, Douglas D

AZD3293: A Novel, Orally Active BACE1 Inhibitor with High Potency and Permeability and Markedly Slow Off-Rate KineticsOpenly Available
Eketjäll, Susanna | Janson, Juliette | Kaspersson, Karin | Bogstedt, Anna | Jeppsson, Fredrik | Fälting, Johanna | Haeberlein, Samantha Budd | Kugler, Alan R. | Alexander, Robert C. | Cebers, Gvido

Predictive Value of Cerebrospinal Fluid Visinin-Like Protein-1 Levels for Alzheimer’s Disease Early Detection and Differential Diagnosis in Patients with Mild Cognitive Impairment
Babić Leko, Mirjana | Borovečki, Fran | Dejanović, Nenad | Hof, Patrick R. | Šimić, Goran

Plasma Phospholipid and Sphingolipid Alterations in Presenilin1 Mutation Carriers: A Pilot Study
Chatterjee, Pratishtha | Lim, Wei L.F. | Shui, Guanghou | Gupta, Veer B. | James, Ian | …… | Wenk, Marcus R. | Bateman, Randall J. | Morris, John C. | Martins, Ralph N.

Cognitive reserve in ageing and Alzheimer’s disease / Stern Y / Lancet Neurol. 2012 Nov; 11(11):1006-12. PMID: 23079557.

A mutation in APP protects against Alzheimer’s disease and age-related cognitive decline/ Jonsson T, Atwal JK, Steinberg S, Snaedal J, Jonsson PV, Bjornsson S, Stefansson H, Sulem P, Gudbjartsson D, Maloney J, et al. / Nature. 2012 Aug 2; 488(7409):96-9. PMID: 22801501.

 Propagation of tau pathology in a model of early Alzheimer’s disease / de Calignon A, Polydoro M, Suárez-Calvet M, William C, Adamowicz DH, Kopeikina KJ, Pitstick R, Sahara N, Ashe KH, Carlson GA, et al. / Neuron. 2012 Feb 23; 73(4):685-97. PMID: 22365544.

Stages of the pathologic process in Alzheimer disease: age categories from 1 to 100 years/ Braak H, Thal DR, Ghebremedhin E, Del Tredici K / J Neuropathol Exp Neurol. 2011 Nov; 70(11):960-9. PMID: 22002422.

Neuroinflammation in Alzheimer’s disease and mild cognitive impairment: a field in its infancy / McGeer EG, McGeer PL / J Alzheimers Dis. 2010; 19(1):355-61. PMID: 20061650.

Metallothioneins in Prion- and Amyloid-Related Diseases

MICROGLIA

Microglia are the immune cells of the CNS and account for approximately 10% of the CNS cellpopulation, with regional variation in density [27, 28]. During embryonic development, microglia originate from yolk sac progenitor cells that migrate into the developing CNS during early embryogenesis [29,30].Following construction of the blood-brain barrier (BBB), microglia are renewed by local turnover [31]. In the healthy brain, microglia actively support neurons through the release of insulin-like growth factor 1, nerve growth factor, ciliary neurotrophic factor, and brain-derived neurotrophic factor (BDNF) [32–34]. Microglia also provide indirect support to neurons by clearance of debris to maintain the extracellular environment, and phagocytosis of apoptotic cells to facilitate neurogenesis [35, 36]. In the adult brain, microglia coordinate much of their activity with astrocytes and activate in response to similar stimuli [37, 38]. Dysfunctional signaling between microglia and astrocytes often results in chronic inflammation, a characteristic of many neurodegenerative diseases [39, 40].

Historically, it has been thought that microglia ‘rest’ when not responding to inflammatory stimuli or damage [41, 42]. However, this notion is being increasingly recognized as inaccurate [43]. When not involved in active inflammatory signaling, microglia constantly patrol the neuropil by extension and retraction of their finely branched processes [44]. Microglial activation is often broadly classified into two states; pro-inflammatory (M1) or anti-inflammatory (M2) [36, 45], based on similar phenotypes in peripheral macrophages [46]. M1 activated microglia are characterized by increased expression of pro-inflammatory mediators and cytokines, including inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β, often under the control of the transcription factor nuclear factor-κB [45]. Pro-inflammatory microglia rapidly retract their processes and adopt an amoeboid morphology and often migrate closer to the site of injury [47]. Anti-inflammatory M2 activation of microglia, often referred to as alternative activation, represents the other side of microglial behavior. Anti-inflammatory activation is characterized by increased expression of cytokines including arginase 1 and interleukin-10, and is associated with increased ramification of processes [45]. The polarization of microglia into M1 or M2 throughout the brain is well characterized, especially in neurodegenerative diseases [48]. In the AD brain, microglia expressing markers of M1 activation are typically localized to brain regions such as the hippocampus that are most heavily affected in the disease [49]. However, it is important to note that M1 and M2 classifications of microglia may over-simplify microglial phenotypes and may only represent the extremes of microglial activation [50]. It has been more recently proposed that microglia likely occupy a continuum between these phenotypes [39, 51].

Do microglia have multiple roles in AD?

Classical pro-inflammatory activation of microglia has long been associated with AD [39, 49]. Samples taken from late-stage AD brains contain characteristic signs of inflammation, including amoeboid morphology of microglia, high levels of pro-inflammatory cytokines in the cerebrospinal fluid, and evidence of neuronal damage due to chronic exposure to pro-inflammatory cytokines and oxidative stress [52, 53]. The cause of this inflammation may be in response to direct toxicity of Aβ to neurons resulting in activation of nearby microglia and astrocytes [53, 54]. However, Aβ may also induce inflammatory activation of microglia and astrocytes. Activated immune cells are typically present surrounding amyloid plaques [55–57], with such peri-plaque cells exhibiting strong evidence of pro-inflammatory activation [56, 58–60]. The presence of undigested Aβ particles within these activated microglia may suggest that the Aβ peptide itself is a pro-inflammatory signal for microglia [61–64]. In vitro experiments provide supporting evidence for the in vivo studies, with Aβ promoting pro-inflammatory microglial activation [65, 66], and also acting as a potent chemotactic signal [67].

However, it is important to note that although widespread inflammation is characteristic of late-stage AD, it remains unclear what role inflammation could play in early stages of the disease. Some evidence suggests that reducing inflammation through the long-term use of some non-steroidal anti-inflammatory drugs (NSAIDs) can reduce the risk of AD [68]. However, these findings have not yet been verified in clinical trials [69, 70]. Little is understood about how NSAIDs and related compounds affect the delicate balance of pro- versus anti-inflammatory microglial activity within the brain. Although there is considerable evidence to suggest that chronic inflammation may contribute to pathology in the later stages of AD, it is important to note that inflammation normally only represents a small aspect of microglial function. The non-inflammatory functions of microglia may play a more important role in early disease; specifically, microglial functions relating to maintenance of the CNS.

Phagocytosis: A vital role of microglia that may be lost in AD    

SYNAPTIC PRUNING: MICROGLIA CAN REGULATE NETWORK ACTIVITY

Recently, a new function has been proposed for microglia. A number of studies have provided evidence that microglia prune synapses throughout life. Microglia are known to remove extraneous synapses during development to ensure that only meaningful connections remain [43]. It was, however, thought that differentiated astrocytes performed the majority of synaptic pruning in the adult brain [91]. The discovery that microglial processes are constantly active within the brain and are often positioned near synapses raised the question of whether microglial synaptic pruning continued throughout life [44, 47, 92–94]. This question was answered in 2014 in a study that demonstrated that microglia do prune synapses into adulthood, and that this activity is important for normal brain function [95]. These findings supported those found a year earlier in a study reporting that ablation of microglia from brain slices increases synapse density and results in abnormal firing of hippocampalneurons [96].

Altered microglial behavior may underlie altered neuronal firing in AD  

Altered neuronal activity is an early phenomenon in AD

The cause of DMN hypoactivity in AD is not yet clear; however studies performed in cohorts that are genetically predisposed to AD suggest that DMN hypoactivity is preceded by a period of hyperactivity and increased functional connectivity [123, 136], often manifesting as an absence of normal DMN deactivation during external tasks [137–140]. DMN hyperactivity may interfere with hippocampal memory encoding, leading to the memory deficits that are present in mild cognitive impairment [141, 142]. It has been proposed that hippocampal hyperexcitability in AD may develop as a protective mechanism against increased input from the DMN [142–144]. As AD progresses, the initial hyperexcitability of the DMN and hippocampus may result in hypoactivity due to exhaustion of compensatory mechanisms [123, 136]. Evidence from both transgenic AD mice and longitudinal human studies supports an exhaustion model of hyperactivation leading to later hypoactivation [143, 145–147]. Interestingly, a number of studies report a lower incidence of AD among those who regularly practice meditation which specifically ‘calms’ the DMN [148].

Our understanding of AD as a disease is changing. Historically considered to be primarily a disease of neuronal degeneration, this neurocentric view has widened to encompass non-neuronal cells such as astrocytes into our understanding of the disease process and pathogenesis. A proposed model for microglia in AD is shown in Fig. 2. Microglia perform a wide range of functions in the CNS and although this includes induction of an inflammatory reaction in response to damage, they also have critical roles for maintaining normal function in the brain. Recent evidence shows that microglia regulate neuronal activity through synaptic pruning throughout life as an extension on their normal phagocytosis behavior. The discovery of a large number of AD risk genes associated with reduced immune cell function suggests that perturbed microglial phagocytosis could lead to AD. In our model, altered microglial phagocytosis of synapses results in network dysfunction and onset of AD, occurring downstream of Aβ.

The immune system and microglia represent a novel target for intervention in AD. Importantly, a large number of anti-inflammatory drugs are already in use for other conditions. What is important to know at this stage is exactly how to best target immune cell function. The studies outlined here provide evidence that an indiscriminate dampening down of all microglial activity may result in a worse outcome for individuals by suppressing normal microglial regulatory functions. We currently do not know whether future microglial-based therapies should focus on reducing chronic inflammation or conversely, whether they should be aimed at boosting microglial phagocytosis. It is also likely that future treatment strategies may use a combination of approaches to target Aβ, immune cell phagocytosis and network activity. An increasing view in the AD field is that any drug or therapy needs to be provided very early in the disease process to maximize its beneficial effects. Although we are currently unable to effectively target those at risk of AD at such an early stage, advances in neuroimaging for subtle changes in network activity, or in assays for immune cell function, may provide new avenues for identification of early damage and risk of disease.

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Late-Onset Metachromatic Leukodystrophy with Early Onset Dementia Associated with a Novel Missense Mutation in the Arylsulfatase A Gene

Microbes and Alzheimer’s DiseaseOpenly Available
Itzhaki, Ruth F. | Lathe, Richard | Balin, Brian J. | Ball, Melvyn J. | Bearer, Elaine L. | Braak, Heiko | Bullido, Maria J. | Carter, Chris | Clerici, Mario | Cosby, S. Louise | Del Tredici, Kelly | Field, Hugh | Fulop, Tamas | Grassi, Claudio | Griffin, W. Sue T. | Haas, Jürgen | Hudson, Alan P. | Kamer, Angela R. | Kell, Douglas B. | Licastro, Federico | Letenneur, Luc | Lövheim, Hugo | Mancuso, Roberta | Miklossy, Judith | Otth, Carola | Palamara, Anna Teresa | Perry, George | Preston, Christopher | Pretorius, Etheresia | Strandberg, Timo | Tabet, Naji | Taylor-Robinson, Simon D. | Whittum-Hudson, Judith A.

Longitudinal Relationships between Caloric Expenditure and Gray Matter in the Cardiovascular Health StudyOpenly Available
Raji, Cyrus A. | Merrill, David A. | Eyre, Harris | Mallam, Sravya | Torosyan, Nare | Erickson, Kirk I. | Lopez, Oscar L. | Becker, James T. | Carmichael, Owen T. | Gach, H. Michael | Thompson, Paul M. | Longstreth Jr., W.T. | Kuller, Lewis H.

Preliminary Study of Plasma Exosomal Tau as a Potential Biomarker for Chronic Traumatic EncephalopathyOpenly Available
Stern, Robert A. | Tripodis, Yorghos | Baugh, Christine M. | Fritts, Nathan G. | Martin, Brett M. | Chaisson, Christine | Cantu, Robert C. | Joyce, James A. | Shah, Sahil | Ikezu, Tsuneya | Zhang, Jing | Gercel-Taylor, Cicek | Taylor, Douglas D.

Unraveling Alzheimer’s: Making Sense of the Relationship between Diabetes and Alzheimer’s Disease1Openly Available
Schilling, Melissa A.

Pain Assessment in Elderly with Behavioral and Psychological Symptoms of DementiaOpenly Available
Malara, Alba | De Biase, Giuseppe Andrea | Bettarini, Francesco | Ceravolo, Francesco | Di Cello, Serena | Garo, Michele | Praino, Francesco | Settembrini, Vincenzo | Sgrò, Giovanni | Spadea, Fausto | Rispoli, Vincenzo

Editor’s Choice from Volume 50, Number 4 / 2016

Post Hoc Analyses of ApoE Genotype-Defined Subgroups in Clinical Trials
Kennedy, Richard E. | Cutter, Gary R. | Wang, Guoqiao | Schneider, Lon S.

Protective Effect of Amyloid-β Peptides Against Herpes Simplex Virus-1 Infection in a Neuronal Cell Culture Model
Bourgade, Karine | Le Page, Aurélie | Bocti, Christian | Witkowski, Jacek M. | Dupuis, Gilles | Frost, Eric H. | Fülöp, Tamás

Association Between Serum Ceruloplasmin Specific Activity and Risk of Alzheimer’s Disease
Siotto, Mariacristina | Simonelli, Ilaria | Pasqualetti, Patrizio | Mariani, Stefania | Caprara, Deborah | Bucossi, Serena | Ventriglia, Mariacarla | Molinario, Rossana | Antenucci, Mirca | Rongioletti, Mauro | Rossini, Paolo Maria | Squitti, Rosanna

Effects of Hypertension and Anti-Hypertensive Treatment on Amyloid-β (Aβ) Plaque Load and Aβ-Synthesizing and Aβ-Degrading Enzymes in Frontal Cortex
Ashby, Emma L. | Miners, James S. | Kehoe , Patrick G. | Love, Seth

AZD3293: A Novel, Orally Active BACE1 Inhibitor with High Potency and Permeability and Markedly Slow Off-Rate KineticsOpenly Available
Eketjäll, Susanna | Janson, Juliette | Kaspersson, Karin | Bogstedt, Anna | Jeppsson, Fredrik | Fälting, Johannad | Haeberlein, Samantha Budd | Kugler, Alan R. | Alexander, Robert C. | Cebers, Gvido

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blocking copper transport in cancer cells

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

DC_AC50, selective way of blocking copper transport in cancer cells

http://newdrugapprovals.org/2015/11/11/dc_ac50-selective-way-of-blocking-copper-transport-in-cancer-cells/

Dr. Melvin Crasto, World Drug Tracker

Jing Chen of Emory University School of Medicine, Hualiang Jiang of the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences, Chuan He of the University of Chicago, and coworkers have now developed a selective way of blocking copper transport in cancer cells (Nat. Chem. 2015, DOI: 10.1038/nchem.2381). By screening a database of 200,000 druglike small molecules, the researchers discovered a promising compound, DC_AC50, for cancer treatment. They zeroed in on the compound by testing how well database hits inhibited a protein-protein interaction leading to copper transport and reduced proliferation of cancer cells.

 

Figure imgf000094_0001

DC_AC50

3-amino-N-(2-bromo-4,6-difluorophenyl)-6,7-dihydro-5H- cyclopenta [b] thieno [3,2-e] pyridine-2-carboxamide

licensed DC_AC50 to Suring Therapeutics, in Suzhou, China

INNOVATORS  Jing Chen of Emory University School of Medicine, Hualiang Jiang of the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences, Chuan He of the University of Chicago, and coworkers

 

Developing small molecules that specifically inhibit human copper-trafficking proteins and an overview of the screening process.

http://www.nature.com/nchem/journal/vaop/ncurrent/images/nchem.2381-f1.jpg

 

COPPER TRANSPORT
Chaperone proteins (green) transfer copper ions to copper-dependent proteins (lilac) via ligand exchange between two cysteines (-SH groups) on each protein. DC_AC50 binds the chaperone and inhibits this interaction.
Credit: Nat. Chem.

Jing Chen of Emory University School of Medicine, Hualiang Jiang of the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences,Chuan He of the University of Chicago, and coworkers have now developed a selective way of blocking copper transport in cancer cells (Nat. Chem. 2015, DOI: 10.1038/nchem.2381). By screening a database of 200,000 druglike small molecules, the researchers discovered a promising compound, DC_AC50, for cancer treatment. They zeroed in on the compound by testing how well database hits inhibited a protein-protein interaction leading to copper transport and reduced proliferation of cancer cells.

20151109lnp1-dca

http://cen.acs.org/content/cen/articles/93/web/2015/11/Agent-Fight-Cancer-Inhibiting-Copper/_jcr_content/articlebody/subpar/articlemedia_0.img.jpg/1447092911801.jpg

 

Scientists had already found a molecule, tetrathiomolybdate, that interferes with copper trafficking and have tested it in clinical trials against cancer. But tetrathiomolybdate is a copper chelator: It inhibits copper transport in cells by nonselectively sequestering copper ions. Sometimes, the chelator snags too much copper, inhibiting essential copper-based processes in normal cells and causing side effects.

In contrast, DC_AC50 works by inhibiting interactions between proteins in the copper-trafficking pathway: It prevents chaperone proteins, called Atox1 and CCS, from passing copper ions to enzymes that use them to run vital cellular processes. Cancer cells are heavy users of Atox1 and CCS, so DC_AC50 affects cancer cells selectively.

The team has licensed DC_AC50 to Suring Therapeutics, in Suzhou, China, for developing anticancer therapies. The group also plans to further tweak DC_AC50 to develop more-potent versions.

Thomas O’Halloran of Northwestern University, who has studied tetrathiomolybdate, comments that “the challenge in drug design is hitting one of these copper-dependent processes without messing with housekeeping functions that normal cells depend upon. DC_AC50 appears to block the function of copper metallochaperone proteins without interacting directly with their cargo, copper ions. As the first member of a new class of inhibitors, it provides a new way to interrogate the physiology of copper trafficking disorders and possibly intervene.”

PATENT

http://www.google.com/patents/WO2014116859A1?cl=en

 

Figure imgf000053_0003

COMPD IS LC-1 COMPD 50

 

NMR and mass spectral data: LC-1 (Compound 50)- 3-amino-N-(2-bromo-4,6-difluorophenyl)-6,7-dihydro-5H- cyclopenta [b] thieno [3,2-e] pyridine-2-carboxamide

Figure imgf000075_0001

1H NMR (CDCI3, 400 MHz) δ 9.15 (s, 1H), 7.61 (s, 1H), 7.13(m, 1H), 6.60 (m, 1H), 6.27 (s, 2H), 3.20 (t, 2H), 2.98 (t, 2H), 2.39 (m, 2H); ESI-MS (EI) m/z 422 (M+)

 

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Liver tissues free of tumors found in newly discovered cells

Reported by: Irina Robu, PhD

Researchers at University of San Diego, School of Medicine discovered a new population of liver cells that are better at regenerating liver tissues than original liver cells, hepatocytes. The article published in August 13 in cells were identified as hybrid hepatocytes and are able to regenerate liver tissue without giving rise to cancer. Of all major organs, the liver has the highest capacity to regenerate—that’s why many liver diseases, including cirrhosis and hepatitis, can often be cured by transplanting a piece of liver from a healthy donor.

A study done recently by Michael Karin, PhD, Distinguished Professor of Pharmacology and Pathology, researchers traced the cells responsible for replenishing hepatocytes following chronic liver injury induced by exposure to carbon tetrachloride.They found a unique population of hepatocytes located in the portal triad which undergo extensive proliferation and replenish liver mass after chronic liver injuries. Since the cells are similar to normal hepatocytes, but express low levels of bile duct cell-specific genes, the researchers called them “hybrid hepatocytes.”

“Hybrid hepatocytes represent not only the most effective way to repair a diseased liver, but also the safest way to prevent fatal liver failure by cell transplantation,” Karin said.

Source

http://medicalxpress.com/news/2015-08-newly-cells-regenerate-liver-tissue.html#nRlvliver

Image Source

http://medicalxpress.com/news/2015-08-newly-cells-regenerate-liver-tissue.html#nRlv

 

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Larry H. Bernstein, MD, FCAP, Author and Curator

Isozymes

An example of an isozyme is glucokinase, a variant of hexokinase which is not
inhibited by glucose 6-phosphate.  Its different regulatory features and lower
affinity for glucose (compared to other hexokinases), allows it to serve different
functions in cells of specific organs, such as

  • control of insulinrelease by the beta cells of the pancreas, or
  • initiation ofglycogen synthesis by liver
  • Both of these processes must only occur when glucose is abundant,or
    problems occur.

Isozymes or Isoenzymes are proteins with different structure which catalyze
the same reaction. Frequently they are oligomers made with different
polypeptide chains, so they usually differ in regulatory mechanisms and in
kinetic characteristics.

From the physiological point of view, isozymes allow the existence of similar
enzymes with different characteristics, “customized” to specific tissue
requirements or metabolic conditions.

One example of the advantages of having isoenzymes for adjusting the
metabolism to different conditions and/ or in different organs is the following:

Glucokinase and Hexokinase are typical examples of isoenzymes. In fact,
there are four Hexokinases: I, II, III and IV. Hexokinase I is present in all
mammalian tissues, and Hexokinase IV, aka Glucokinase, is found mainly
in liver, pancreas  and brain.

Both enzymes catalyze the phosphorylation of Glucose:

Glucose + ATP —–à Glucose 6 (P) + ADP

Hexokinase I has a low Km and is inhibited by glucose 6 (P).  Glucokinase
is not inhibited by Glucose 6 (P) and his Km is high. These two facts
indicate that the activity of glucokinase depends on the availability
of substrate and not on the demand of the product.

Since Glucokinase is not inhibited by glucose 6 phosphate, in
conditions of high concentrations of glucose this enzyme
continues phosphorylating glucose, which can be used for
glycogen synthesis in liver. Additionally, since Glucokinase
has a high Km, its activity does not compromise the supply
of glucose to other organs; in other words, if Glucokinase
had a low Km, and since it is not inhibited by its product, it
would continue converting glucose to glucose 6 phosphate
in the liver,  making glucose unavailable for other organs
(remember that after meals, glucose arrives first to the liver
through the portal system).

The enzyme Lactate Dehydrogenase is made of two (H-
and M-)  sub units, combined in different Permutations
and 
Combinations  depending on the tissue in which it
is present as shown in table,

Type Composition Location
LDH1 HHHH Heart and Erythrocyte
LDH2 HHHM Heart and Erythrocyte
LDH3 HHMM Brain and Kidney
LDH4 HMMM Skeletal Muscle and Liver
LDH5 MMMM Skeletal Muscle and Liver
  • While isozymes may be almost identical in function
    (defined by Michaelis constant, KM)
  • they differ in amino acidsubstitutions that change the
    electric charge of the enzyme (such as replacing
    aspartic acid with glutamic acid)
  • The sum of zwitterion charges result in identifyjng
    difference inmigratiion toward the anode by gel
    electrophoresis
    , and this forms the basis for the use
    of isozymes as molecular markers.
  • To identify isozymes, a crude protein extract is made by
    grinding animal or plant tissue with an extraction buffer,
    and the components of extract are separated according
    to their charge by gel electrophoresis.
  • They were classically purified by ion-exchange column
    chromatography after first precipitation with ammonium
    sulfate, followed by dialysis.

The cytochrome P450 isozymes play important roles in
metabolism and steroidogenesis. The multiple forms of
phosphodiesterase also play major roles in various
biological processes.

These isoforms of the enzyme are unequally distributed
in the various cells of an organism.

Further the main isoenzymes may have closely grouped
“isoforms” having unclear significance.

There are many examples of isoenzymes in cell
metabolism that distinguish cells:

  • Adenylate kinase (AL in liver, and myokinase) – that
    are distinguished by reactivity with sulfhydryl reagents
  • Pyruvate kinase
  • AMPK, and Calmodulin kinase
  • Malate, isocitrate, alcohol, and aldehyde dehydrogenase
  • Nitric oxide synthase (i, e, and n)…

References[edit]

Hunter, R. L. and C.L. Markert. (1957) Histochemical
demonstration of enzymes separated by zone electrophoresis
in starch gels. Science 125: 1294-1295

Uzunov, P. and Weiss, B.(1972) “Separation of multiple
molecular forms of cyclic adenosine 3′,5′-monophosphate
phosphodiesterase in rat cerebellum by polyacrylamide
gel electrophoresis.”  Biochim. Biophys. Acta 284:220-226.

Uzunov, P., Shein, H.M. and Weiss, B.(1974) “Multiple
forms of cyclic 3′,5′-AMP phosphodiesterase
of rat cerebrum and cloned astrocytoma and
neuroblastoma cells.” Neuropharmacology 13:377-391.

Weiss, B., Fertel, R., Figlin, R. and Uzunov, P. (1974)
“Selective alteration of the activity of the multiple forms
of adenosine 3′,5′-monophosphate phosphodiesterase
of rat cerebrum.” Mol. Pharmacol.10:615-625.

Lactate dehydrogenase

In cells, the immediate energy sources involve glucose oxidation. In anaerobic metabolism, the donor of the phosphate group is adenosine triphosphate (ATP), and the reaction is catalyzed via the hexokinase or glucokinase: Glucose +ATP-Mg²+ = Glucose-6-phosphate (ΔGo = – 3.4 kcal/mol with hexokinase as the co-enzyme for the reaction.).
In the following step, the conversion of G-6-phosphate into F-1-6-bisphosphate is mediated by the enzyme phosphofructokinase with the co-factor ATP-Mg²+. This reaction has a large negative free energy difference and is irreversible under normal cellular conditions. In the second step of glycolysis, phosphoenolpyruvic acid in the presence of Mg²+ and K+ is transformed into pyruvic acid. In cancer cells or in the absence of oxygen, the transformation of pyruvic acid into lactic acid alters the process of glycolysis.
The energetic sum of anaerobic glycolysis is ΔGo = -34.64 kcal/mol. However a glucose molecule contains 686kcal/mol and, the energy difference (654.51 kcal) allows the potential for un-controlled reactions during carcinogenesis. The transfer of electrons from NADPH in each place of the conserved unit of energy transmits conformational exchanges in the mitochondrial ATPase. The reaction ADP³+ P²¯ + H²–à ATP + H2O is reversible. The terminal oxygen from ADP binds the P2¯ by forming an intermediate pentacovalent complex, resulting in the formation of ATP and H2O. This reaction requires Mg²+ and an ATP-synthetase, which is known as the H+-ATPase or the Fo-F1-ATPase complex. Intracellular calcium induces mitochondrial swelling and aging. [12].
The known marker of monitoring of treatment in cancer diseases, lactate dehydrogenase (LDH) is an enzyme that is localized to the cytosol of human cells and catalyzes the reversible reduction of pyruvate to lactate via using hydrogenated nicotinamide deaminase (NADH) as co-enzyme.
The causes of high LDH and high Mg levels in the serum include neoplastic states that promote the high production of intracellular LDH and the increased use of Mg²+ during molecular synthesis in processes pf carcinogenesis (Pyruvate acid>> LDH/NADH >>Lactate acid + NAD), [13].
LDH is released from tissues in patients with physiological or pathological conditions and is present in the serum as a tetramer that is composed of the two monomers LDH-A and LDH-B, which can be combined into 5 isoenzymes: LDH-1 (B4), LDH-2 (B3-A1), LDH-3 (B2-A2), LDH-4 (B1-A3) and LDH-5 (A4). The LDH-A gene is located on chromosome 11, whereas the LDH-B gene is located on chromosome 12. The monomers differ based on their sensitivity to allosteric modulators. They facilitate adaptive metabolism in various tissues. The LDH-4 isoform predominates in the myocardium, is inhibited by pyruvate and is guided by the anaerobic conversion to lactate.
Total LDH, which is derived from hemolytic processes, is used as a marker for monitoring the response to chemotherapy in patients with advanced neoplasm with or without metastasis. LDH levels in patients with malignant disease are increased as the result of high levels of the isoenzyme LDH-3 in patients with hematological malignant diseases and of the high level of the isoenzymes LDH-4 and LDH-5, which are increased in patients with other malignant diseases of tissues such as the liver, muscle, lungs, and conjunctive tissues. High concentrations of serum LDH damage the cell membrane [11, 31].

Relation between LDH and Mg as Factors of Interest in the Monitoring and Prognoses of Cancer

Aurelian Udristioiu, Emergency County Hospital Targu Jiu Romania, Clinical Laboratory Medical Analyses, E-mail: aurelianu2007@yahoo.com

Lactate Dehydrogenase (LDH) is ubiquitous in animals and
man, and  it occurs in different organs of the body, each
region having a unique conformation of the subunits, but
the significance was once disputed. Perhaps the experiments
of Jakob and Monod on the lac 1 operon put to rest any
notions that isoenzymes and their conformational forms are
something of no real significance.  This concept does not
necessarily apply in all cases of isoenzyme differences, by
which I mean that there may be a difference in reactivity at
the active site.

For that matter, Jakob and Monod discovered and elucidated
allosterism.

300px-Enzyme_Model  allosterism
In biochemistryallosteric regulation is the regulation of a
protein by binding an effector molecule at a site other than
the protein’s active site.

The site the effector binds to is termed the allosteric site.
Allosteric sites allow effectors to bind to the protein, often
resulting in a conformational change. Effectors that enhance
the protein’s activity are referred to as allosteric activators,
whereas  those that decrease the protein’s activity are called
allosteric inhibitors.

Allosteric regulations are a natural example of control loops,
such as feedback from downstream products or feedforward
 from upstream substrates. Long-range allostery is especially
important in cell signaling. Allosteric regulation
is also particularly important in the cell’s ability to adjust
enzyme activity.

The term allostery comes from the Greek allos (ἄλλος), “other,”
and stereos (στερεὀς), “solid (object).” This is in reference
to the fact that the regulatory site of an allosteric protein is
physically distinct from its active site.

Jacob and Monod model of transcriptional regulation of the lac operon by lac repressor

Jacob and Monod model of  lac repressor

Most allosteric effects can be explained by the concerted
MWC model put forth by Monod, Wyman, and Changeux[2]
or by the sequential model described by Koshland, Nemethy,
and Filmer.[3] Both postulate that enzyme subunits exist in
one of two conformations, tensed (T) or relaxed (R), and
that relaxed subunits bind substrate more readily than
those in the tense state. The two models differ most in
their assumptions about subunit interaction and the pre-
existence of both states.

Allosteric_Regulation Model

Allosteric_Regulation Model

  1.  Monod, J. Wyman, J.P. Changeux. (1965). On the nature of
    allosteric transitions:A plausible model. J. Mol. Biol.;12:88-118.
  2. E. Jr Koshland, G. Némethy, D. Filmer (1966). Comparison of
    experimental binding data and theoretical models in proteins
    containing subunits. Biochemistry. Jan;5(1):365-8

The sequential model (2) of allosteric regulation holds that subunits
are not connected in such a way  that a  conformational change in
one induces a similar change in the others. Thus, all enzyme
subunits do not necessitate the  same conformation. Moreover,
the sequential model dictates that molecules of substrate
bind via an
 induced fit  protocol. In general, when a subunit
randomly collides with a molecule of substrate, the active site,
in essence, forms a  glove around its substrate.

While such an induced fit converts a subunit from the tensed
state to relaxed state, it does not propagate the conformational
change to adjacent subunits. Instead, substrate-binding at
one subunit  only slightly  alters the structure of other
subunits so that their binding sites are more receptive to
substrate.
To summarize:

  • subunits need not exist in the same conformation
  • molecules of substrate bind via induced-fit protocol
  • conformational changes are not propagated to all
    subunits

The discovery of morpheeins has revealed a previously
unforeseen mechanism to target universally essential
enzymes for species-specific drug design and discovery.
A morpheein-based inhibitor would function by  binding
to and stabilizing  the inactive morpheein form of the
enzyme, thereby shifting the equilibrium to favor that form (3).

  1. K. Jaffe, S.H. Lawrence (2008). “Expanding the
    concepts in protein structure-function relationships
    and  enzyme kinetics: Teaching using morpheeins”
    .
    Biochemistry and Molecular Biology  Education36 (4)
    : 274–283. http://dx.doi.org:/10.1002/bmb.20211.
    PMC 2575429PMID 19578473

Important related points are:

Non-regulatory allostery

A non-regulatory allosteric site refers to any non-regulatory
component of an enzyme (or any protein), that is not  itself
an amino acid. For instance, many enzymes require sodium
binding to ensure proper function. However, the sodium
does not necessarily act as a regulatory subunit; the sodium
is always present and there are no known biological processes
to add/remove sodium to regulate enzyme activity. Non-
regulatory allostery could comprise any other  ions besides
sodium (calcium, magnesium, zinc), as well as other chemicals
and possibly vitamins.

Lactate and malate dehydrogenases

LDH is a key enzyme in glycolysis. Anaerobic glycolysis is the
conversion of pyruvate into lactate acid in the absence
of oxygen. This pathway is important to glycolysis in two main
ways. The first is that

  • if pyruvate were to build up glycoysis
  • the generation of ATP would slow.

The second is anaerobic respiration

  • allows for the regeneration of NAD+ from NADH.

NAD+ is required when glyceraldehyde-3-phosphate
dehydrogenase oxidizes glyceraldehyde-3-phosphate in
glycolysis, which generates NADH. Lactate dehydrogenase
is responsible for the anaerobic conversion of NADH to
NAD+. Click here to see the residues which form
inter
actions with pyruvate in the Lactate Dehydrogenase
from Cryptosporidium  parvum (2fm3). (Wikipedia)

Glycolysis ends with the synthesis of pyruvate.  But, to be
self-functioning, it must end with lactate.  Why?  Anaerobic
means “without oxygen”.  This is tantamount to saying
“without mitochondria”.

  1. The mitochondria are especially adept at oxidizing
    NADH to NAD+. NAD+ is needed to keep the glyceraldehyde-
    3-PO4 dehydrogenase reaction functioning.
  2. If glycolysis is to continue when no oxygen is present or in
    short supply (as in a working muscle), an alternative means
    of oxidizing NADH must occur.

Pyruvate has 2 metabolic fates:

  • it can either be converted into lactate or to acetyl-CoA .
    Note that in animals and plants the electrons in  NADH
    are transferred  to pyruvate which reduces the carbonyl
    carbon in the pyruvate molecule to an alcohol. The
    reaction is catalyzed by the enzyme lactate dehydrogenase.
    Lactate (or L-lactate to be more precise)  is thus  a
    “waste product”, since it has no metabolic fate other
    than to be converted back into pyruvate in a reverse of
    the  forward reaction.
  • More importantly, the NAD+ feeds back to the glyceraldehyde-
    3-PO4 dehydrogenase reaction, which  allows glycolysis
    to continue.  Were it not for lactate formation, glycolysis
    as a self-functioning pathway could not exist.

In yeast a slightly different end of glycolysis becomes apparent.
Yeast do not synthesize lactate.  They do, however, oxidize
NADH back to NAD+ anaerobically.  How do they do this?  The
answer is they make ethanol.  In the reaction the pyruvate is
converted into acetaldehyde.  The reaction is catalyzed by a
lyase enzyme, pyruvate decarboxylase, which removes the
carboxyl group as a CO2.  Acetaldehyde is formed because
the electron pair that bonds the –COO group is not removed
by the decarboxylation.  A proton is plucked from the
environment giving the final product, acetaldehyde.
Acetaldehyde is now the substrate that will oxidize NADH to
NAD+ and in the process ethanol is formed.

There is another advantage to the pyruvate-lactate interchange.
The lactate formed by lactate  dehydrogenase  can  be
reconverted. This allows a cell to synthesize glucose from lactate.
Converting lactate to glucose is a major feature of gluconeogenesis,
an anabolic pathway that synthesizes glucose from smaller
precursors such as lactate. This is important because acetyl-CoA
cannot be converted back to pyruvate and hence cannot be a
source of carbons  for glucose biosynthesis.

ADP.  ADP is required in the 3-phosphoglycerate kinase reaction
and in the pyruvate kinase reaction.  It is formed from ATP in the
hexokinase reaction and the phosphofructokinase-I reaction.

NADH, ADP and PO4.   NADH oxidation is important in glycolysis.
NADH is converted into NAD+ in the mitochondria.  That
reaction is promoted by O2 ; NAD+ stays in the mitochondria.
Also in the mitochondria, ATP is formed by condensing ADP
with PO4.  Thus, O2 allows mitochondria to out-compete the
cytosol for ADP,  NADH and PO4, all limiting  substrates or
coenzymes.

In vertebrates, gluconeogenesis takes place mainly in the liver
and, to a lesser extent, in the cortex of kidneys. In many
animals, the process occurs during periods of fasting,
starvationlow-carbohydrate diets, or intense exercise.
The process is highly endergonic until it is coupled to the
hydrolysis of ATP or GTP, effectively making the process
exergonic. For example, the pathway leading from pyruvate
to glucose-6-phosphate requires 4 molecules of  ATP and
2 molecules of GTP to proceed spontaneously. Gluco-
neogenesis is a target of therapy for type II diabetes,
such as metformin, which inhibits glucose formation
and stimulates glucose uptake by cells.

Lactate is formed at the endstage of glycolysis with insufficient
oxygen is transported to the liver where it is converted into
pyruvate by the Cori cycle using the enzyme lactate
dehydrogenase
. In this reaction lactate loses two electrons
(becomes oxidized) and is converted to pyruvate. NAD+
gains two electrons (is reduced) and is converted to NADH.

Both lactate and NAD+ bind to the active site of the enzyme
lactate dehydrogenase and both lactate and NAD+ participate
in the catalysis reaction. In fact, catalysis could not occur
unless the coenzyme NAD+ bound to the active site.

lactat-pyr.LDH

lactat-pyr.LDH

http://academic.brooklyn.cuny.edu/biology/bio4fv/page/couple.gif

What is not shown:

  1. The liver LDH is composed of predominantly M-type subunits.
  2. The forward reaction is regulated in the H-type LDH, but not
    the M-type   enzyme by the formation of a ternary complex
    of LDH-ox. NAD-lactate
  3. The formation and breakup of the ternary complex is
    dependent on the pyruvate in the forward reaction in a
    concentration dependent manner.
  4. The M-type LDH doesn’t have this tight binding of the LDH –
    NAD+ – lactate  (see catalysis below)
  5. As lactate concentration builds in the circulation from heavy
    muscle production (M-type), or from circulatory insufficiency,
    the circulating lactic acid reaches the liver.
  6. The lactic acid is taken up by the liver, and the high
    concentration of lactic acid drives the backward reaction,
    unrestricted.

Pyruvate, the first designated substrate of the gluconeogenic
pathway, can then be used to generate glucose. Transamination
or deamination of amino acids facilitates entering of their
carbon skeleton into the cycle directly  (as pyruvate or
oxaloacetate), or indirectly via the citric acid cycle.  It is
known that odd-chain fatty acids can be  oxidized to yield
propionyl-CoA, a precursor for succinyl-CoA, which can
be converted to  pyruvate and  enter  into gluconeogenesis.

gluconeogenesis

gluconeogenesis

http://upload.wikimedia.org/wikipedia/commons/thumb/6/63/Amino_acid_catabolism.svg/300px-Amino_acid_catabolism.svg.png

Catalysis

Studies have shown that the reaction mechanism of LDH follows an ordered sequence.

mechanism of LDH reaction

mechanism of LDH reaction

In the forward reaction

  1. NADH must bind to the enzyme  Several residues are
    involved in the binding of NADH
    . Once the NADH is
    bound to the enzyme,
  2. pyruvatebinds (substrate oxamate is shown; the CH3
    group is replaced by NH2 to form oxamate). (see the
    direction of the arrow)
  3. binds to the enzyme between the nicotinamide ring
    and several LDH residues.-
  4. transfer of a hydride ion then happens quickly
  5. in either direction giving a mixture of the two ternary
    complexes,
  6. enzyme-NAD+-lactate and enzyme-NADH-pyruvate .
  7. finally L-lactate dissociates from the enzyme followed
    by NAD+[2].

What is not shown is:

  1. The dissocation of NAD+ and lactate from the H-type LDHs
    is  dependent on the pyruvate  in the forward reaction in a
    concentration dependent manner
  2. This results in inhibition of the reaction as it proceeds as
    a result of the abortive ternary complex that forms in about
    500 msec carried out in the Aminco-Morrow stop flow analyzer.
  3. The regulatory effect of the tighter binding of the LDH (H)-
    NAD+-lactate is not seen with the M-type LDH.
  4. The result of this is that the H-type LDH is regulated by the
    formation of oxidized coenzyme  bound with reduced substrate.

Genetics and Mutagenesis of Fish 1973, pp 243-276.
Developmental and Biochemical Genetics of Lactate
Dehydrogenase Isozymes in Fishes
.
G. S. WhittE. T. MillerJ. B. Shaklee
 http://link.springer.com/article/10.1007%2F978-3-642-
65700-9_23/lookinside/000.png

In the teleost there are only three of the isoenzymes.  LDH-1,
3, and 5 (H4, H2M2, M4).

 teleost

Lactic dehydrogenase isozymes in lens and cornea 
Larry BernsteinMichael KerriganHarry Maisel
Experimental Eye Research Oct 1966; 5, (4): Pp 309–314, IN23–IN28
http://dx.doi.org:/10.1016/S0014-4835(66)80041-6

Lactic dehydrogenase isozymes of bovine and rabbit lens and
cornea were analyzed by starch gel electrophoresis.
Although there was a progressive loss of enzyme activity in
the lenses of both species with increasing age, the loss of
isozymes was more clearly evident in the bovine lens. In
the adult bovine lens, 

  • lactic dehydrogenase isozyme Iwas predominant,
  • while in the adult rabbit lens, isozymes 3–5were mainly present.

The mobility of lens isozymes was identical to that of isozymes
in other tissues. Furthermore, the isozymes were not  localized
to any major specific lens crystallin.

Lactate Dehydrogenase Isozyme Patterns of Human
Platelets and Bovine Lens Fibers

Elliot S. Vesell
Science 24 Dec 1965; 150(3704): pp.1735-1737   
http://dx.doi.org:/10.1126/science

Since the platelets and lens fibers, like mature human erythrocytes,
lack a nucleus, the results strengthen the case for a

  • previously developed association between LDH-5 and the
    cell nucleus.

These three cell types are mainly anaerobic, and therefore

  • their isozyme patterns are incompatible with the theory
    that anaerobic `  tissues exhibit predominantly LDH-5
    and aerobic tissues mainly LDH-1.

Lactate dehydrogenase isozymes and their relationship
to lens cell differentiation 

James A. StewartJohn Papaconstantinou
Biochimica et Biophysica Acta (BBA) – General Subjects
26 May 1966; 121,(1): Pp 69–78
http://dx.doi.org:/10.1016/0304-4165(66)90349-7

Changes in the activity of lactate dehydrogenase (LDH) (l-lactate:
NAD+ oxidoreductase EC 1.1.1.27) isozymes are associated with
the growth and differentiation of bovine lens cells. Calf and adult
lens epithelial cells contain all 5 isozymes. The cathodal forms are
most active in the calf-epithelial cells; the anodal forms are most
active in the fiber cells
. This transition from cathodal to anodal
forms of lactate dehydrogenase in the epithelial cells is associated
with cellular aging.

During the differentiation of an epithelial cell to a fiber cell, in calf
and adult lenses there is an enhancement of 

  • the transition from cathodal forms to anodal forms. 

The regulation of lactate dehydrogenase subunit synthesis may
be associated, therefore, with

  • the replicative activity of these cells.

In cells having the greatest replicative activity (calf epithelial
cells) the cathodal isozymes are most active; in cells having a
decreased mitotic activity (adult epithelial cells) the anodal
isozymes are most active. The non-replicative

  • fiber cell of calf and adult shows a transition toward the
    anodal forms.

Although lens fiber cells have a low rate of oxidative metabolism
lactate dehydrogenase-I is the most active isozyme in these
cells. Kinetically,

  • lactate dehydrogenase-I factors other than, or in addition
    to, the regulation of carbohydrate metabolism
  • are involved in regulating the synthesis of lactate dehydrogenase subunits.

Abbreviations   LDH; lactate dehydrogenase

What is not examined to resolve the discrepancy (see the next item):

The Vessell paper was a challenge to the work in Nathan
Kaplan’s lab.  However, there is sufficient complexity revealed
in these works that there is no conceptual foundation.

  1. The analogy is to the loss of cell nuclei in crystallin lens
    fiber formation with the LDH-H type subunits (aerobic?)
  2. The findings are reproduced in several laboratories.
  3. In the lens, glucose is catabolized primarily to lactic
    acid, and is not appreciably combusted to CO2
    (J Kinoshita. Glucose metabolism of Lens)
  4. However, synthetic processes, including nuclear DNA and
    cell replication requires TPNH. This is produced by means
    of the Pentose Shunt.
  5. The most favorable conditions for the lens are achieved
    by incubating in a medium containing glucose in the
    presence of oxygen. Under these conditions of
    incubation (Kinoshita)
  • the lens remains completely transparent,
  • it maintains normal levels of high energy phosphate
    bonds and cations, and
  • it shows a high rate of arginine incorporationinto protein.

incubation in the absence of glucose, but in the presence of oxygen

  • a haze is found in the lens,
  • a drop in high energy phosphate level is observed, and
  • Changes in cation levels are apparent.
  • A 50 percent decrease in the incorporation of arginine
    into lens protein is also observed.

the most unfavorable condition for the lens is an anaerobic
incubation in a medium without glucose

Pirie2 observed that a-glycerophosphate is one of the end products
of lens metabolism. Its oxidation with DPN as the cofactor could
channel its electrons directly into the ETC to produce energy without
involving the Krebs cycle. a-Glycerophosphate is formed from intermediates of the glycolytic scheme by reduction of dihydroxy-
acetone phosphate, one of the triose phosphates produced in
glycolysis.

the dehydrogenase of the mitochondria catalyzes the transfer
of elections to form DPNH by the following reactions:

a-glycerophosphate + DPN+ ± dihydroxyacetone ……..

phosphate + DPNH.

The DPNH is channeled into the oxidative phosphorylation
mechanism to form ATP. The dihydroxyacetone phosphate
then diffuses out into the soluble cytoplasm, interacts with
the glycolytic intermediates by the reversal of the above reaction,

  • and the cyclic mechanism is begunover again.

That this type of electron transport system functions in the
lens was proposed by Pirie.
http://www.iovs.org/content/4/4/619.full.pdf

Lactate dehydrogenase activity and its isoenzymes in
concentric layers of adult bovine and calf lenses.
  
Sempol DOsinaga EZigman SKorc IKorc BSans ARadi R, et al.
Curr Eye Res. 1987 Apr;6(4):555-60.

The activity of lactate dehydrogenase (LDH) and its isoenzyme
pattern were studied in four concentric layers of adult
bovine and calf lenses. In both groups the specific activity of
the total LDH diminished progressively toward the internal
nuclear layer; the decrease was greater in the adult lenses.
The enzyme activities in the cortical layers of the calf lens
were lower than in the adult lens, but in the inner nuclear layers,
the opposite was found. All of the 5 LDH isoenzymes were found
in each layer. In both groups of animals the LDH1 isoenzyme
prevailed, followed by LDH2. No differences were found in the
percentage of each isoenzyme in the different lens layers.
The differences in the activitie(s) of LDH found may be due

  • to post-translational or post-synthetic modifications which
    may occur during the aging process.

Structural basis for altered activity of M- and H-isozyme
forms of human lactate dehydrogenase.

Read JA1, Winter VJEszes CMSessions RBBrady RL.
Author information  Proteins. 2001 May 1;43(2):175-85

Lactate dehydrogenase (LDH) interconverts pyruvate and
lactate with concomitant interconversion of NADH and NAD(+).
Although crystal structures of a variety of LDH have previously
been described, a notable absence has been any of the
three known human forms of this glycolytic enzyme. We have
now determined the crystal structures of two isoforms of
human LDH-the M form, predominantly found in muscle; and
the H form, found mainly in cardiac muscle. Both structures
have been crystallized as ternary complexes in the presence
of the NADH cofactor and oxamate, a substrate-like inhibitor.

Although each of these isoforms has different kinetic properties,
the domain structure, subunit association, and active-site regions
are indistinguishable between the two structures.

The pK(a) that governs the K(M) for pyruvate for the two isozymes
is found to differ by about 0.94 pH units, consistent with variation in
pK(a) of the active-site histidine.

The close similarity of these crystal structures suggests the distinctive
activity of these enzyme isoforms is likely to result

  • directly from variation of charged surface residues peripheral to the active site,
  • a hypothesis supported by electrostatic calculations based on each structure.

Proteins 2001;43:175-185.

Mechanistic aspects of biological redox reactions involving NADH.
Part 4. Possible mechanisms and corresponding intermediates for
the catalytic reaction in L-lactate dehydrogenase

J Molec Structure: THEOCHEM,25 Feb 1993; 279, Pp 99-125
Kathryn E. Norris, Jill E. Gready

The catalytic step in the conversion of pyruvate to L-lactate in the
enzyme L-lactate dehydrogenase involves the transfer of both a
proton and a hydride ion (A.R. Clarke, T. Atkinson and J.J. Holbrook,
TIBS, 14 (1989) 101.) However, it is not known whether the
reaction is concerted or, if a multistep process, the order in
which the transfers of the proton and the hydride ions take
place. Four possible non-concerted mechanisms can be
proposed, which differ in the order of the transfers of the
proton and hydride ion and the protonation state of the substrate
carboxylate group during the transfers. The energies and
optimized geometries of the corresponding intermediates,
protonated pyruvate, protonated pyruvic acid, deprotonated
L-lactate and deprotonated L-lactic acid, are computed using
the semiempirical AM 1 and ab initio SCF/3–21 G – methods.
These calculations are complementary to the study of
the substrates for the enzyme discussed in a previous paper
(K.E. Norris and J.E. Gready, J. Mol. Struct. (Theochem),
258 (1992) 109.) The structures and energetics of protonated
pyruvate and deprotonated L-lactate provide some
important insights into the requirements for enzymic reaction
and the characteristics of the transition state.

Pyruvate production by Enterococcus casseliflavus A-12
from gluconate in an alkaline medium

J Fermentation and Bioengineering, 1992; 73(4):287-291
H Yanase, N Mori, M Masuda, K Kita, M Shimao, N Kato

A newly isolated lactic acid bacterium, Enterococcus casseliflavus
A-12, produced pyruvic acid (16 g/l) during aerobic culture in
an alkaline medium containing sodium gluconate (50 g/l) as
the carbon source. The production was dependent on the pH
of the culture, the optimum initial pH being 10.0. With static
culture, the organism produced lactic acid (2.7 g/l) from both
gluconate and glucose. Pyruvate did not accumulate in growing
cultures on glucose, but resting cells obtained from a culture
on gluconate produced pyruvate from glucose as well as
gluconate. The enzyme profiles of the organism, which
grew on gluconate and glucose, suggested that gluconate
was metabolized via the Entner-Doudoroff and Embdem-
Meyerhof-Parnas pathways in aerobic culture, and that glucose
was oxidized mainly via the latter pathway under both aerobic
and anaerobic conditions. Gluconokinase, a key enzyme in
the aerobic metabolism of gluconate, was partially purified
from this strain and characterized.

A specific, highly active malate dehydrogenase by redesign
of a lactate dehydrogenase framework

HM WilksKW HartR FeeneyCR DunnH MuirheadWN Chiaet al.

Department of Biochemistry, University of Bristol, United Kingdom.
Science 16 Dec1988: 242(4885),  pp. 1541-1544
http://dx.doi.org:/10.1126/science.3201242

 Three variations to the structure of the nicotinamide adenine
dinucleotide (NAD)-dependent L-lactate dehydrogenase
from Bacillus stearothermophilus were made to try to
change the substrate specificity from lactate to malate:
Asp197—-Asn, Thr246—-Gly, and Gln102—-Arg).

Each modification shifts the specificity from lactate to malate, although

  • only the last (Gln102—-Arg) provides an effective and
    highly specific catalyst for the new substrate.

This synthetic enzyme has a ratio of catalytic rate (kcat) to
Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1,

  • equal to that of native lactate dehydrogenase for its natural
    substrate, pyruvate, and a maximum velocity (250 s-1),
    which is double that reported for a natural malate from B.
    stearothermophilus.

Malate dehydrogenase: distribution, function and properties.

Musrati RA1, Kollárová MMernik NMikulásová D.
Author information
Gen Physiol Biophys. 1998 Sep;17; (3):193-210.

Malate dehydrogenase (MDH) (EC 1.1.1.37) catalyzes the
conversion of oxaloacetate and malate. This reaction is
important in cellular metabolism, and it is coupled with
easily detectable cofactor oxidation/reduction. It is a
rather ubiquitous enzyme, for which several isoforms
have been identified, differing in their subcellular
localization and their specificity for the cofactor NAD
or NADP. The nucleotide binding characteristics can
be altered by a single amino acid change. Multiple
amino acid sequence alignments of MDH show there is a

  • low degree of primary structural similarity, apart from
    several positions crucial for catalysis, cofactor binding
    and the subunit interface.
  • Despite the low amino acids sequence identity their
    3-dimensional structures are very similar.
  • MDH is a group of multimeric enzymes consisting of
    identical subunits usually organized as either dimer
    or tetramers with subunit molecular weights of 30-35 kDa.

Malate dehydrogenase, mitochondrial (MDH2)

UniProt Number: P40926
Alternate Names: Malate DH

Structure and Function:
Malate dehydrogenase (MDH2) is an enzyme in the citric
acid cycle that catalyzes the conversion of malate into
oxaloacetate (using NAD+) and vice versa (this is a
reversible reaction). Malate dehydrogenase is also
involved in gluconeogenesis, the synthesis of glucose
from smaller molecules.Pyruvate in the mitochondria is acted upon by pyruvate
carboxylase  to form oxaloacetate, a citric acid cycle
intermediate.In order to get the oxaloacetate out of the mitochondria,
malate dehydrogenase reduces it to malate, and it then
traverses the inner mitochondrial membrane.Once in the cytosol, the malate is oxidized back to
oxaloacetate by cytosolic malate dehydrogenase.

Finally, phosphoenol-pyruvate carboxy kinase (PEPCK)
converts oxaloacetate to phosphoenol pyruvate.

Malate Dehydrogenase (MDH)(PDB entry 2x0i) is most known
for its role in the metabolic pathway of the tricarboxylic acid cycle,
critical to cellular respiration; The enzyme has other metabolic roles in –

  •  glyoxylate bypass,
  • amino acid synthesis,
  • glucogenesis, and
  • oxidation/reduction balance .

An oxidoreductase, MDH has been extensively studied due to its
isozymes The enzyme exists in two places inside a cell:

  • the mitochondria and cytoplasm.
  • In the mitochondria, the enzyme catalyzes the reaction of
    malate to oxaloacetate;
  • in the cytoplasm, the enzyme catalyzes oxaloacetate to
    malate to allow transport.

The enzyme malate dehydrogenase is composed of either
a dimer or tetramer depending on the location of the enzyme
and the organism it is located in. During catalysis, the enzyme
subunits are

  • non-cooperative between active sites.

The mitochondrial MDH is complexly,

  • allosterically controlled by citrate, but no other known
    metabolic regulation mechanisms have been discovered.
  • the exact mechanism of regulation has yet to be discovered.

Kinetically, the pH of optimization is 7.6 for oxaloacetate
conversion and 9.6 for malate conversion. The reported
K(m) value for malate conversion is 215 uM and the V(max)
value is 87.8 uM/min.

Comment:

The mMDH and the cMDH both form ternary complex
of MDH-NAD+-OAA formed during the forward reaction,
like the LDH H-type isozyme LDH-NAD+-PYR (mot the M-type).
However, the binding of the Enz-coenzyme-substrate is not
as strong as for the H-type LDH.  .The regulatory role has
not been established.

References

  1. Minarik P, Tomaskova N, Kollarova M, Antalik M. Malate
    dehydrogenases–structure and function. Gen Physiol Biophys.
    2002 Sep;21(3):257-65. PMID:12537350
  2. Musrati RA, Kollarova M, Mernik N, Mikulasova D.
    Malate dehydrogenase: distribution, function and properties.
    Gen Physiol Biophys. 1998 Sep;17(3):193-210. PMID:9834842
  3. Boernke WE, Millard CS, Stevens PW, Kakar SN, Stevens FJ,
    Donnelly MI. Stringency of substrate specificity of
    Escherichia coli malate dehydrogenase. Arch Biochem
    Biophys. 1995 Sep 10;322(1):43-52. PMID:7574693
    doi:http://dx.doi.org/10.1006/abbi.1995.1434
  4. Goward CR, Nicholls DJ. Malate dehydrogenase: a model
    for structure, evolution, and catalysis. Protein Sci. 1994
    Oct;3(10):1883-8. PMID:7849603
    doi:http://dx.doi.org/10.1002/pro.5560031027

Kinetic determination of malate dehydrogenase isozymes.

L H Bernstein, M B Grisham

Journal of Molecular and Cellular Cardiology (Impact Factor: 5.15).
11/1978; 10(10):931-44. http://dx.doi.org/10.1016/0022-2828(78)90339-5

Source: PubMed

ABSTRACT These studies determine the levels of malate
dehydrogenase isoenzymes in cardiac muscle by a steady
state kinetic method which depends on the differential inhibition
of these isoenzyme forms by high concentrations of oxaloacetate.
This inhibition is similar to that exhibited by lactate dehydrogenase
in the presence of high concentrations of pyruvate. The results
obtained by this method are comparable in resolution to those
obtained by CM-Sephadex fractionation and by differential
centrifugation for the analyses of mitochondrial malate
dehydrogenase and cytoplasmic malate dehydrogenase in
tissues. The use of standard curves of percent inhibition of
malate dehydrogenase activity plotted against the ratio of
mitochondrial MDH activity to the total of mMDH and cMDH
activities [ malate dehydrogenase ratio] (percent m-type) is
introduced for studies of comparative mitochondrial
function in heart muscle of different species or in different
tissues of the same species.

Calmodulin and Protein Kinase C Increase Ca21-stimulated
Secretion by Modulating Membrane-attached Exocytic Machinery

YA Chen, V Duvvuri, H Schulmani, and RH Scheller
Hughes Medical Institute, Department of Molecular and Cellular
Physiology, and the iDepartment of Neurobiology, Stanford
University School of Medicine, Stanford, California 94305-5135
JBC Sep 10, 1999; 274( 37): 26469–26476

Using a reconstituted [3H]norepinephrine
release assay in permeabilized PC12 cells, we
found that essential proteins that support the triggering
stage of Ca21-stimulated exocytosis are enriched in an
EGTA extract of brain membranes. Fractionation of this
extract allowed purification of two factors that stimulate
secretion in the absence of any other cytosolic proteins.
These are calmodulin and protein kinase Ca
(PKCa). Their effects on secretion were confirmed using
commercial and recombinant proteins. Calmodulin enhances
secretion in the absence of ATP, whereas PKC
requires ATP to increase secretion, suggesting that
phosphorylation is involved in PKC- but not calmodulin
mediated stimulation. Both proteins modulate release
events that occur in the triggering stage of exocytosis.

Endothelial nitric oxide synthase (eNOS) variants in
cardiovascular disease: pharmacogenomic implications  

Indian J Med Res  May 2011;  133:  464-466

Commentary

Manjula Bhanoori

Department of Biochemistry, University College of Science,
Osmania University, Hyderabad 500 007, India

 

The maintenance of regular vascular tone substantially
depends on the bioavailability of endothelium-derived
nitric oxide (NO) synthesized by eNOS. The essential
role of NO, as the elusive endothelium-derived relaxing
factor (EDRF), was the topic of research that won the
1998 Nobel Prize in Physiology or Medicine. The eNOS
gene, as a candidate gene in the investigations on
hypertension genetics, has attracted the attention of
several researchers because of the established role
of NO in vascular homeostasis. The eNOS variants
located in the 7q35-q36 region have been investigated
for their association with CVD, particularly hypertension.
Three variants, viz., (i) G894T substitution in exon 7
resulting in a Glu to Asp substitution at codon 298 (rs1799983),
(ii) an insertion-deletion in intron 4 (4a/b) consisting of two
alleles (the a*-deletion which has four tandem 27-bp repeats
and the b*-insertion having five repeats), and (iii) a T786C
substitution in the promoter region (rs2070744), have been
extensively studied20-22. Individual SNPs often cause only
a modest change in the resulting gene expression or function.
It is, therefore, the concurrent presence of a number of SNPs
or haplotypes within a defined region of the chromosome that
determines susceptibility to disease development and progression,
particularly in case of polygenic diseases.

Shankarishan et al24 analysed for the first time the prevalence
of eNOS exon 7 Glu298Asp polymorphism in tea garden community
of North Eastern India, who are a high risk group for CVD. This study
also included indigenous Assamese population and found no
significant difference between the distribution patterns of eNOS
exon 7 Glu298Asp variants between the communities. They have
rightly mentioned that for developing public health policies and
programmes it is necessary to know the prevalence and distribution
of the candidate genes in the population, as well as trends in
different population groups. They have also observed that the
eNOS exon 7 homozygous GG wild genotype (75.8%) was
predominant in the study population followed by heterozygous
GT genotype (21.5%) and homozygous TT genotype (2.7%).
The frequency distribution of the homozygous GG, heterozygous
GT and homozygous mutant TT genotypes were comparable to
that of the north Indian and south Indian population.

Polymorphisms in the endothelial nitric oxide synthase gene have
been associated inconsistently with cardiovascular diseases.
Varying distribution of eNOS variants among ethnic groups may
explain inter-ethnic differences in nitric oxide mediated vasodilation
and response to drugs28. Different population studies showed
association of eNOS polymorphisms with variations in NO
formation and response to drugs. Cardiovascular drugs including
statins increase eNOS expression and upregulate NO formation
and this effect may be responsible for protective, pleiotropic
effects produced by statins31. With respect to hypertension,
studies have reported interactions between diuretics and
polymorphisms in eNOS gene. Particularly, the Glu298Asp
polymorphism made a statistically significant contribution to
predicting blood pressure response to diuretics.

Neuronal Nitric Oxide Synthase and Its Interaction
With Soluble Guanylate Cyclase Is a Key Factor for
the Vascular Dysfunction of Experimental Sepsis

GM. Nardi, K Scheschowitsch, D Ammar, SK de
Oliveira, TB. Arruda; J Assreuy

Vascular dysfunction plays a central role in sepsis, and it is
characterized by hypotension and hyporesponsiveness to
vasoconstrictors. Nitric oxide is regarded as a central element
of sepsis vascular dysfunction. The high amounts of nitric
oxide produced during sepsis are mainly derived from the
inducible isoform of nitric oxide synthase 2.
We have previously shown that nitric oxide synthase 2 levels
decrease in later stages of sepsis, whereas levels and activity
of soluble guanylate cyclase increase. Therefore, we studied
the putative role of other relevant nitric oxide sources, namely,

  • the neuronal (nitric oxide synthase 1) isoform, in sepsis
  • and its relationship with soluble guanylate cyclase.

We also studied the consequences of

  • nitric oxide synthase 1 blockade in the hyporesponsiveness
    to vasoconstrictors.

1) Both nitric oxide synthase 1 and soluble guanylate cyclase
are expressed in higher levels in vascular tissues during sepsis;

2) both proteins physically interact and nitric oxide synthase 1
blockade inhibits cyclic guanosine monophosphate production;

3) pharmacological blockade of nitric oxide synthase 1 using
7-nitroindazole or S-methyl-l-thiocitrulline reverts the hypo
responsiveness to phenylephrine and increases the vaso
constrictor effect of norepinephrine and phenylephrine.

Sepsis induces increased expression and physical association
of nitric oxide synthase 1/soluble guanylate cyclase and a higher
production of cyclic guanosine monophosphate that together
may help explain sepsis-induced vascular dysfunction.

In addition, selective inhibition of nitric oxide synthase 1
restores the responsiveness to vasoconstrictors.

Therefore, inhibition of nitric oxide synthase 1 (and possibly
soluble guanylate cyclase) may represent a valuable
alternative to restore the effectiveness of vasopressor
agents during late sepsis.  (Crit Care Med 2014; XX:00–00)

Nitric Oxide Synthase Inhibitors That Interact with Both Heme
Propionate and Tetrahydrobiopterin Show High Isoform Selectivity

S Kang, W Tang, H Li, G Chreifi, P Martásek, LJ. Roman,
TL. Poulos, and RB. Silverman

†Department of Chemistry, Department of Molecular Biosciences,
Chemistry of Life Processes Institute, Center for Molecular Innovation
and Drug Discovery, Northwestern University, Evanston, Illinois
‡Departments of Molecular Biology and Biochemistry, Pharmaceutical
Sciences, and Chemistry, University of California, Irvine, California,
Department of Biochemistry, University of Texas Health Science Center,
San Antonio, Texas

Overproduction of NO by nNOS is implicated in the pathogenesis of
diverse neuronal disorders. Since NO signaling is involved in
diverse physiological functions, selective inhibition of nNOS
over other isoforms is essential to minimize side effects. A series of
α-amino functionalized aminopyridine derivatives (3−8) were
designed to probe the structure−activity relationship between ligand,
heme propionate, and H4B. Compound 8R was identified as the
most potent and selective molecule of this study, exhibiting a Ki of
24 nM for nNOS, with 273-fold and 2822-fold selectivity against
iNOS and eNOS, respectively.Although crystal structures of 8R
complexed with nNOS and eNOS revealed a similar binding mode,
the selectivity stems from the distinct electrostatic environments in
two isoforms that result in much lower inhibitor binding free energy
in nNOS than in eNOS. These findings provide a basis for further
development of simple, but even more selective and potent, nNOS
inhibitors

  • Aurelian Udristioiu

    Aurelian

    Aurelian Udristioiu

    Lab Director at Emergency County Hospital Targu Jiu

    In cells, the immediate energy sources involve glucose oxidation. In anaerobic metabolism, the donor of the phosphate group is adenosine triphosphate (ATP), and the reaction is catalyzed via the hexokinase or glucokinase: Glucose +ATP-Mg²+ = Glucose-6-phosphate (ΔGo = – 3.4 kcal/mol with hexokinase as the co-enzyme for the reaction.).
    In the following step, the conversion of G-6-phosphate into F-1-6-bisphosphate is mediated by the enzyme phosphofructokinase with the co-factor ATP-Mg²+. This reaction has a large negative free energy difference and is irreversible under normal cellular conditions. In the second step of glycolysis, phosphoenolpyruvic acid in the presence of Mg²+ and K+ is transformed into pyruvic acid. In cancer cells or in the absence of oxygen, the transformation of pyruvic acid into lactic acid alters the process of glycolysis.
    The energetic sum of anaerobic glycolysis is ΔGo = -34.64 kcal/mol. However a glucose molecule contains 686kcal/mol and, the energy difference (654.51 kcal) allows the potential for un-controlled reactions during carcinogenesis. The transfer of electrons from NADPH in each place of the conserved unit of energy transmits conformational exchanges in the mitochondrial ATPase. The reaction ADP³+ P²¯ + H²–à ATP + H2O is reversible. The terminal oxygen from ADP binds the P2¯ by forming an intermediate pentacovalent complex, resulting in the formation of ATP and H2O. This reaction requires Mg²+ and an ATP-synthetase, which is known as the H+-ATPase or the Fo-F1-ATPase complex. Intracellular calcium induces mitochondrial swelling and aging. [12].
    The known marker of monitoring of treatment in cancer diseases, lactate dehydrogenase (LDH) is an enzyme that is localized to the cytosol of human cells and catalyzes the reversible reduction of pyruvate to lactate via using hydrogenated nicotinamide deaminase (NADH) as co-enzyme.
    The causes of high LDH and high Mg levels in the serum include neoplastic states that promote the high production of intracellular LDH and the increased use of Mg²+ during molecular synthesis in processes pf carcinogenesis (Pyruvate acid>> LDH/NADH >>Lactate acid + NAD), [13].
    LDH is released from tissues in patients with physiological or pathological conditions and is present in the serum as a tetramer that is composed of the two monomers LDH-A and LDH-B, which can be combined into 5 isoenzymes: LDH-1 (B4), LDH-2 (B3-A1), LDH-3 (B2-A2), LDH-4 (B1-A3) and LDH-5 (A4). The LDH-A gene is located on chromosome 11, whereas the LDH-B gene is located on chromosome 12. The monomers differ based on their sensitivity to allosteric modulators. They facilitate adaptive metabolism in various tissues. The LDH-4 isoform predominates in the myocardium, is inhibited by pyruvate and is guided by the anaerobic conversion to lactate.
    Total LDH, which is derived from hemolytic processes, is used as a marker for monitoring the response to chemotherapy in patients with advanced neoplasm with or without metastasis. LDH levels in patients with malignant disease are increased as the result of high levels of the isoenzyme LDH-3 in patients with hematological malignant diseases and of the high level of the isoenzymes LDH-4 and LDH-5, which are increased in patients with other malignant diseases of tissues such as the liver, muscle, lungs, and conjunctive tissues. High concentrations of serum LDH damage the cell membrane [11, 31].

    Relation between LDH and Mg as Factors of Interest in the Monitoring and Prognoses of Cancer

    Aurelian Udristioiu, Emergency County Hospital Targu Jiu Romania, Clinical Laboratory Medical Analyses, E-mail: aurelianu2007@yahoo.com

    Larry Bernstein likes this

  • Larry Bernstein

    Larry Bernstein

    CEO/CSO at Triplex Consulting

    The inhibition be pyruvate is related by a ternary complex formed by NAD+ formed in the catalytic forward reaction Pyruvate + NADH –> Lactate + NAD(+). The reaction can be followed in an Aminco-Morrow stop-flow analyzer and occurs in ~ 500 msec. The reaction does not occur with the muscle type LDH, and it is regulatory in function. I did not know about the role of intracellular Mg(2+) in the catalysis, as my own work was in Nate Kaplan’s lab in 1970-73.

    This difference in the behavior of the isoenzyme types was considered to be important then in elucidating functional roles, but it was challenged by Vessell earlier. The isoenzymes were first described by Clement Markert at Yale. I think, but don’t know, that the Mg++ would have a role in driving the forward reaction, but I can’t conceptualize how it might have any role in the difference between muscle and heart.

    I didn’t quite know why oncologists used it specifically. Cancer cells exhibit the reliance on the anaerobic (muscle) type enzyme, which is also typical of liver, but with respect to the adenylate kinases – the liver AK and muscle AK (myokinase) are different. That difference was discovered by Masahiro Chiga, and differences in the reaction with sulfhydryl reagents were identified by Percy Russell.

    Oddly enough, Vessell had a point. The RBC has the heart type predominance, not the M-type. He thought that it was related to the loss of nuclei from the reticulocyte. I did not buy that, and I had worked on the lens of the eye at the time.

  • Aurelian Udristioiu

    Aurelian

    Aurelian Udristioiu

    Lab Director at Emergency County Hospital Targu Jiu

    Very interesting scientific comments. Thanks. !

  • Aurelian Udristioiu

    Aurelian

    Aurelian Udristioiu

    Lab Director at Emergency County Hospital Targu Jiu

    The IDH1 and IDH2 genes are mutated in > 75% of different malignant diseases. Two distinct alterations are caused by tumor-derived mutations in IDH1 or IDH2,
    IDH1 and IDH2 mutations have been observed in myeloid malignancies, including de novo and secondary AML (15%–30%), and in pre-leukemic clone malignancies, including myelodysplastic syndrome and myeloproliferative neoplasm (85% of the chronic phase and 20% of transformed cases in acute leukemia.
    Aurelian Udristioiu, M.D
    City Targu Jiu, Romania
    AACC, NACB, Member, USA.

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 What is the key method to harness Inflammation to close the doors for many complex diseases?

 

Author and Curator: Larry H Bernstein, MD, FCAP

 

The main goal is to  have a quality of a healthy life.

When we look at the picture 90% of main fluid of life, blood, carried by cardiovascular system with two main pumping mechanisms, lung with gas exchange and systemic with complex scavenger actions, collection of waste, distribution of nutrition and clean gases etc.  Yet without lymphatic system body can’t make up the 100% fluid.  Therefore, 10% balance is completed by lymphatic system as a counter clockwise direction so that not only the fluid balance but also mass balance is  maintained. Finally, the immune system patches the  remaining mechanism by providing cellular support to protect the body because it contains 99% of white cells to fight against any kinds of invasion, attack, trauma.

These three musketeers, ccardiovascular, lyphatic and immune systems, create the core mechanism of survival during human life.

However, there is a cellular balance between immune and cardiovascular system since blood that made up off 99% red cells and 1% white blood cells that are used to scavenger hunt circulating foreign materials.   These three systems are acting with a harmony not only defend the body but provide basic needs of life.  Thus, controlling angiogenesis and working mechanisms in blood not only helps to develop new diagnostic tools but more importantly establishes long lasting treatments that can harness Immunomodulation.

The word inflammation comes from the Latin “inflammo”, meaning “I set alight, I ignite”.

Medical Dictionary description is:

“A fundamental pathologic process consisting of a dynamic complex of histologically apparent cytologic changes, cellular infiltration, and mediator release that occurs in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical, or biologic agent, including the local reactions and resulting morphologic changes; the destruction or removal of the injurious material; and the responses that lead to repair and healing.”

The five elements makes up the signature of  inflammation:  rubor, redness; calor, heat (or warmth); tumor swelling; and dolor, pain; a fifth sign, functio laesa, inhibited or lost function.   However, these indications may not be present at once.

Please click on to the following link for genetic association of autoimmune diseases (Cho Et al selected major association signals in autoimmune diseases) from Cho JH, Gregersen PK. N Engl J Med 2011;365:1612-1623.

Inflammatory diseases grouped under two classification: the immune system related due to  inflammatory disorders, such as both allergic reactions  and some myopathies, with many immune system disorders.  The examples of inflammatory disorders  include Acne vulgaris, asthma, autoimmune disorders, celiac disease, chronic prostatitis, glomerulonepritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory diseases, reperfusion diseases, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis, interstitial cyctitis, The second kind of inflammation are related to  non-immune diseases such as cancer, atherosclerosis, and ischaemic heart disease.

This seems simple yet at molecular physiology and gene activation levels this is a complex response as an innate immune response from body.  There can be acute lasting few days after exposure to bacterial pathogens, injured tissues or chronic inflammation continuing few months to years after unresolved acute responses such as non-degradable pathogens, viral infection, antigens or any  foreignmaterials, or autoimmune responses.

As the system responses arise from plasma fluid, blood vessels, blood plasma through vasciular changes, differentiation in plasma cascade systems like coagulation system, fibrinolysis, complement system and kinin system.  Some of the various mediators include bradykinin produced by kinin system, C3, C5, membrane attack system (endothelial cell activation or endothelial coagulation activation mechanism) created by the complement system; factor XII that can activate kinin, fibrinolysys and coagulation systems at the same time produced in liver; plasmin from fibrinolysis system to inactivate factor Xii and C3 formation, and thrombin of coagulation system with a reaction through protein activated receptor 1 (PAR1), which is a seven spanning membrane protein-GPCR.   This system is quite fragile and well regulated.  For example activation of inactive Factor XII by collagen, platelets, trauma such as cut, wound, surgery that results in basement membrane changes since it usually circulate in inactive form in plasma automatically initiates and alerts kinin, fibrinolysis and coagulation systems.

Furthermore, the changes reflected through receptors and create gene activation by cellular mediators to establish system wide unified mechanisms. These factors (such as IFN-gamma, IL-1, IL-8, prostaglandins, leukotrene B4,  nitric oxide, histamines,TNFa) target immune cells and redesign their responses, mast cells, macrophages, granulocytes, leukocytes, B cells, T cells) platelets, some neuron cells and endothelial cells.  Therefore, immune system can react with non-specific or specific mechanisms either for a short or a long term.

As a result, controlling of mechanisms in blood and prevention of angiogenesis answer to cure/treat many diseases  Description of angiogenesis is simply formation of new blood vessels without using or changing pre-existing capillaries.  This involves serial numbers of events play a central role during physiologic and pathologic processes such as normal tissue growth, such as in embryonic development, wound healing, and the menstrual cycle.  However this system requires three main elements:  oxygen, nutrients and getting rid of waste or end products.

Genome Wide Gene Association Studies, Genomics and Metabolomics, on the other hand, development of new technologies for diagnostics and non-invasive technologies provided better targeting systems.

In this token recent genomewide association studies showed a clear view on a disease mechanism, or that suggest a new diagnostic or therapeutic approach particularly these disorders are related to  genes within the major histocompatibility complex (MHC) that predisposes the most significant genetic effect.  Presumably, these genes are reflecting the immunoregulatory effects of the HLA molecules themselves. As a result, the working mechanism of pathological conditions are revisited or created new assumptions to develop new targets for diagnosis and treatments.

Even though B and T cells are reactive to initiate responses there are several level of mechanisms control the cell differentiation for designing rules during health or diseases. These regulators are in check for both T and B cells.  For example, during Type 1 diabetes there are presence of more limited defects in selection against reactivity with self-antigens like insulin, thus, T cell differentiation is in jeopardy.  In addition, B cells have many active checkpoints to modulate the immune responses like  pre-B cells in the bone marrow are highly autoreactive yet they prefer to stay  in naïve-B cell forms in the periphery through tyrosine phosphatase nonreceptor type 22 (PTPN22) along with many genes play a role in autoimmunity.  In a nut shell this is just peeling the first layer of the onion at the level of Mendelian Genetics.

There is a great work to be done but if one can harness the blood and immune responses many complex diseases patients may have a big relief and have a quality of life.  When we look at the picture 90% of main fluid of life, blood, carried by cardiovascular system with two main pumping mechanisms, lung with gas exchange and systemic with complex scavenger actions, collection of waste, distribution of nutrition and clean gases.  Yet, without lymphatic system body can’t make up the 100% fluid.  Therefore, 10% balance is completed by lymphatic system as a counter clockwise direction so that not only the fluid balance but also mass balance is  maintained. Finally, the immune system patches the  remaining mechanism by providing cellular support to protect the body because it contains 99% of white cells to fight against any kinds of invasion, attack, trauma.

FURTHER READINGS AND REFERENCES:

Arap W, Pasqualini R, Ruoslahti E (1998) Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science (Wash DC)279:377380.

 Brouty BD, Zetter BR (1980) Inhibition of cell motility by interferon.Science (Wash DC) 208:516518.

Ferrara N, Alitalo K (1999) Clinical Applications of angiogenic growth factors and their inhibitorsNat Med 5:13591364.

 

Ferrara N (1999) Role of vascular endothelial growth factor in the regulation of angiogenesisKidney Int 56:794814.

 

Ferrara N (1995) Leukocyte adhesion: Missing link in angiogenesisNature (Lond) 376:467.

 

Kohn EC, Alessandro R, Spoonster J, Wersto RP, Liotta LA (1995) Angiogenesis: Role of calcium-mediated signal transduction. Proc Natl Acad Sci U S A 92:13071311

Meijer DKF, Molema G (1995) Targeting of drugs to the liverSemin Liver Dis 15:202256.

Sidky YA, Borden EC (1987) Inhibition of angiogenesis by interferons: Effects on tumor- and lymphocyte-induced vascular responsesCancer Res47:51555161.

Anonymous (1999a) Genentech takes VEGF back to lab. SCRIP 2493:24.

Ziche M, Morbidelli L, Choudhuri R, Zhang HT, Donnini S, Granger HJ,Bicknell R (1997) Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest 99:26252634.

 

Yoshida S, Ono M, Shono T, Izumi H, Ishibashi T, Suzuki H, Kuwano M(1997) Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor α-dependent angiogenesis. Mol Cell Biol 17:40154023.

 

Vittet D, Prandini MH, Berthier R, Schweitzer A, Martin SH, Uzan G,Dejana E (1996) Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation stepsBlood 88:34243431.

 

Ruegg C, Yilmaz A, Bieler G, Bamat J, Chaubert P, Lejeune FJ (1998) Evidence for the involvement of endothelial cell integrin αvβ3 in the disruption of the tumor vasculature induced by TNF and IFNNat Med4:408414

Patey N, Vazeux R, Canioni D, Potter T, Gallatin WM, Brousse N (1996) Intercellular adhesion molecule-3 on endothelial cells. Expression in tumors but not in inflammatory responses. Am J Pathol 148:465472.

Oliver SJ, Banquerigo ML, Brahn E (1994) Supression of collagen-induced arthritis using an angiogenesis inhibitor AGM-1470 and microtubule stabilizer taxol. Cell Immunol 157:291299

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