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Calcium Dependent NOS Induction by Sex Hormones: Estrogen

Posted in Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Nitric Oxide in Health and Disease, Personalized and Precision Medicine & Genomic Research, Pharmaceutical R&D Investment, Population Health Management, Genetics & Pharmaceutical, Reproductive Biology & Bio Instrumentation, Technology Transfer: Biotech and Pharmaceutical, tagged Calcium, Endothelium, Estradiol, Estrogen, Isozyme, Messenger RNA, nitric oxide, Nitric oxide synthase, NO, sex hormones on October 3, 2012| 5 Comments »

Calcium Dependent NOS Induction by Sex Hormones: Estrogen

Reporter and Curator: Sudipta Saha, Ph.D.

Nitric oxide (NO) synthases (NOSs) constitute a family of isozymes that catalyze the oxidation of L-arginine to NO and citrulline. First identified in the vascular endothelium, NO synthesis has subsequently been shown to play important roles in:

the regulation of vascular and gastrointestinal tone,
in cell-mediated cytotoxicity against bacteria and tumors, and
in a variety of central and peripheral nervous system activities.

NOSs can be divided into three functional classes based on their sensitivity to calcium.

The cytokine- or bacterial product-inducible isoenzyme iNOS binds calmodulin tightly at resting intracellular calcium concentrations.
The constitutive forms, isozymes eNOS (originally described in endothelial cells) and
nNOS (originally described in neuronal tissue), bind calmodulin in a reversible and calcium-dependent fashion.

The mechanisms by which their synthesis is controlled are unknown. The cDNA species encoding the rat, mouse, and human nNOS, the human and bovine eNOS, and iNOS from several species and cell types have been cloned and sequenced. The three human isozymes characterized to date are distinct, with their deduced protein sequences showing only 50-60%o amino acid identity. nNOS, which in rats and humans localizes to neurons in the central and peripheral nervous system and colocalizes with NADPHdiaphorase activity, has also been shown to be widely distributed in several non-neuronal tissues including human skeletal muscle.

It had been thought that both nNOS and eNOS were purely constitutive enzymes, although studies suggest eNOS may be induced by shear stress. Studies demonstrate that these NOSs can be induced in several tissues during pregnancy and in nonpregnant female and male animals by estradiol and that in skeletal muscle it is accompanied by an increase in NOS-specific mRNA.

Evidences emerging from various laboratories showed that there is an increase in the release of NO from the vasculature during pregnancy. Furthermore, treatment of pregnant animals at the end of gestation with tamoxifen reduced NOS activity in the cerebellum, an organ where tamoxifen acts as a pure estrogen-receptor antagonist. Thus, the increase in calcium-dependent NOS activity during pregnancy is mediated by estrogen. This conclusion is supported by the fact that treatment of nonpregnant females and male animals with estradiol also increased calcium-dependent NOS activity in all tissues studied.

Interestingly, testosterone treatment also increased cerebellar NOS activity without affecting other tissues. However, testosterone may increase brain NOS by directly binding estrogen receptors as has been reported. Furthermore, the cerebellum was the only tissue in the male to respond to a 5-day course of estradiol, suggesting that it may have a larger number and/or a greater availability of estrogen receptors than other tissues. In addition, the brain is rich in aromatase, which converts testosterone into estradiol. This, together with the observation that progesterone does not induce NOS, indicates that the induction of both nNOS and eNOS is specific for estrogen and not a characteristic of all sex steroids. These experiments do not exclude the possibility that the addition of progesterone might modify the estradiol effect.

The increases in NOS activity are the result of augmented enzyme synthesis (enzyme induction) since they are accompanied by increases in the specific mRNAs for both eNOS and nNOS. It is not, however, possible to tell whether the increases in mRNA are caused by an upregulation of mRNA synthesis (transcriptional induction) or decreased mRNA breakdown.

Although calcium-dependent NOS activity was increased by estradiol in tissues obtained from both female and male guinea pigs, a longer duration of treatment was necessary in the male. The most likely explanation for this observation is that the number or availability of estrogen receptors is initially too low in most tissues of the male and requires a period of estrogen priming. Although other factors may play a role, the duration of exposure may well explain the observation that the effect of pregnancy on NOS-specific mRNA is greater than estradiol alone.

The observation that estradiol induces calcium-dependent NOSs has several important implications:

An increase in release of NO from the endothelium would decrease vascular tone and contractility, events that are characteristic in pregnancy.
Heterogeneity among tissue endothelium regarding the effects of estrogen on basal NO release could explain the selective redistribution of maternal cardiac output to organs important for a successful pregnancy.
Consistent with this possibility is the observation that the effect of pregnancy on endothelium-derived NO is greatest in the uterine artery, followed by the mesenteric artery and then renal arteries.
An alternative hypothesis to explain the adaptation of smooth muscle to pregnancy is that it is caused by prostacyclin. Prostacyclin is increased during pregnancy and contributes to the observed reduced contractility of the ovine uterine artery to angiotensin II.

However, estradiol does not increase the synthesis of prostacyclin by the endothelium, nor does inhibition of prostacyclin synthesis prevent the effects of pregnancy on smooth muscle. In addition, both the incidence of esophageal reflux and the gastrointestinal transit time are increased during pregnancy. Although this phenomenon has previously been attributed to a direct effect of progesterone, NO is a powerful dilator of the gastrointestinal smooth muscle. If the increase in NOS activity observed in the esophagus applies to the bowel, enhanced NO might be the mechanism underlying both increased esophageal reflux and transit time.

The biological signifcance of an estradiol-dependent increase in the NOS in the central nervous system is of great interest and deserves further investigation. Furthermore, an estradiol-mediated increase in NOS in the vasculature could be the mechanism whereby premenopausal women are protected from coronary artery disease since increased NOS may slow the development of atherosclerosis and reduce the contractile response to acute thrombosis. Finally, the induction of calcium-dependent NOS enzymes by estradiol suggests that the present classification of this family of enzymes into constitutive and inducible types needs to be revised, since eNOS and nNOS enzymes at least are both constitutive and inducible.

Source References:

http://www.ncbi.nlm.nih.gov/pubmed?term=Calcium%20dependent%20NOS%20induction%20by%20sex%20hormones

Other research published on Nitric Oxide on this Scientific Web Site include the following:

Nitric Oxide in bone metabolism July 16, 2012

Author: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/07/16/nitric-oxide-in-bone-metabolism/?goback=%2Egde_4346921_member_134751669

Nitric Oxide production in Systemic sclerosis July 25, 2012

Curator: Aviral Vatsa, PhD, MBBS

http://pharmaceuticalintelligence.com/2012/07/25/nitric-oxide-production-in-systemic-sclerosis/?goback=%2Egde_4346921_member_138370383

Nitric Oxide Signalling Pathways August 22, 2012 by

Curator/ Author: Aviral Vatsa, PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/?goback=%2Egde_4346921_member_151245569

Nitric Oxide: a short historic perspective August 5, 2012

Author/Curator: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/05/nitric-oxide-a-short-historic-perspective-7/

Nitric Oxide: Chemistry and function August 10, 2012

Curator/Author: Aviral Vatsa PhD, MBBS

http://pharmaceuticalintelligence.com/2012/08/10/nitric-oxide-chemistry-and-function/?goback=%2Egde_4346921_member_145137865

Nitric Oxide and Platelet Aggregation August 16, 2012 by

Author: Dr. Venkat S. Karra, Ph.D.

http://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/?goback=%2Egde_4346921_member_147475405

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure August 20, 2012

Author: Larry Bernstein, MD

http://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad August 16, 2012

Reporter: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/08/16/nitric-oxide-the-nobel-prize-in-physiology-or-medicine-1998-robert-f-furchgott-louis-j-ignarro-ferid-murad/

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012

Author: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012

Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery

Curator: Aviva Lev-Ari, PhD, RN, July 12, 2012

http://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk/

Bone remodelling in a nutshell June 22, 2012

Author: Aviral Vatsa, Ph.D., MBBS

http://pharmaceuticalintelligence.com/2012/06/22/bone-remodelling-in-a-nutshell/

Targeted delivery of therapeutics to bone and connective tissues: current status and challenges – Part 1

AuthorL Aviral Vatsa, PhD, September 23, 2012

http://pharmaceuticalintelligence.com/2012/09/23/targeted-delivery-of-therapeutics-to-bone-and-connective-tissues-current-status-and-challenges-part-i/

Calcium dependent NOS induction by sex hormones: Estrogen

Curator: S. Saha, PhD, October 3, 2012

http://pharmaceuticalintelligence.com/2012/10/03/calcium-dependent-nos-induction-by-sex-hormones/

Nitric Oxide and Platelet Aggregation

Author V. Karra, PhD, August 16, 2012

http://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Curator: Aviva Lev-Ari, PhD, July 16, 2012

http://pharmaceuticalintelligence.com/?s=Nebivolol

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Increased risks of obesity and cancer, Decreased risk of type 2 diabetes: The role of Tumor-suppressor phosphatase and tensin homologue (PTEN)

Reporter: Aviva Lev-Ari, PhD, RN

Screen Shot 2021-07-19 at 7.13.38 PM

Word Cloud By Danielle Smolyar

PTEN Mutations as a Cause of Constitutive Insulin Sensitivity and Obesity

Aparna Pal, M.R.C.P., Thomas M. Barber, D.Phil., M.R.C.P., Martijn Van de Bunt, M.D., Simon A. Rudge, Ph.D., Qifeng Zhang, Ph.D., Katherine L. Lachlan, M.R.C.P.C.H., Nicola S. Cooper, M.R.C.P., Helen Linden, M.R.C.P., Jonathan C. Levy, M.D., F.R.C.P., Michael J.O. Wakelam, Ph.D., Lisa Walker, D.Phil., M.R.C.P.C.H., Fredrik Karpe, Ph.D., F.R.C.P., and Anna L. Gloyn, D.Phil.

N Engl J Med 2012; 367:1002-1011 September 13, 2012DOI: 10.1056/NEJMoa1113966

BACKGROUND

Epidemiologic and genetic evidence links type 2 diabetes, obesity, and cancer. The tumor-suppressor phosphatase and tensin homologue (PTEN) has roles in both cellular growth and metabolic signaling. Germline PTEN mutations cause a cancer-predisposition syndrome, providing an opportunity to study the effect of PTENhaploinsufficiency in humans.

METHODS

We measured insulin sensitivity and beta-cell function in 15 PTENmutation carriers and 15 matched controls. Insulin signaling was measured in muscle and adipose-tissue biopsy specimens from 5 mutation carriers and 5 well-matched controls. We also assessed the effect of PTEN haploinsufficiency on obesity by comparing anthropometric indexes between the 15 patients and 2097 controls from a population-based study of healthy adults. Body composition was evaluated by means of dual-emission x-ray absorptiometry and skinfold thickness.

RESULTS

Measures of insulin resistance were lower in the patients with aPTEN mutation than in controls (e.g., mean fasting plasma insulin level, 29 pmol per liter [range, 9 to 99] vs. 74 pmol per liter [range, 22 to 185]; P=0.001). This finding was confirmed with the use of hyperinsulinemic euglycemic clamping, showing a glucose infusion rate among carriers 2 times that among controls (P=0.009). The patients’ insulin sensitivity could be explained by the presence of enhanced insulin signaling through the PI3K-AKT pathway, as evidenced by increased AKT phosphorylation. The PTEN mutation carriers were obese as compared with population-based controls (mean body-mass index [the weight in kilograms divided by the square of the height in meters], 32 [range, 23 to 42] vs. 26 [range, 15 to 48]; P<0.001). This increased body mass in the patients was due to augmented adiposity without corresponding changes in fat distribution.

CONCLUSIONS

PTEN haploinsufficiency is a monogenic cause of profound constitutive insulin sensitization that is apparently obesogenic. We demonstrate an apparently divergent effect of PTEN mutations: increased risks of obesity and cancer but a decreased risk of type 2 diabetes owing to enhanced insulin sensitivity. (Funded by the Wellcome Trust and others.)

Supported by grants from the Wellcome Trust (095101/Z/10Z, to Dr. Gloyn), the Medical Research Council (G0700222, to Dr. Gloyn; and G0800467, to Drs. Pal and Gloyn), the National Institute for Health Research Oxford Biomedical Research Centre (to Drs. Pal, Karpe, and Gloyn), the Biotechnology and Biological Sciences Research Council (to Drs. Rudge, Zhang, and Wakelam), and the European Union Seventh Framework Program LipodomicNet (202272, for adipocyte signaling work, to Drs. Wakelam and Karpe).

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

We thank the clinicians Trevor R.P. Cole, Louise Izatt, Carole McKeown, Eamonn R. Maher, and Mary Porteous for referring patients for this study; the research nurses Beryl Barrow and Jane Cheeseman for assistance with collecting clinical data; Amy Barrett for analysis of PTEN expression; Sandy Humphries for analysis of apolipoprotein B; Tim James and colleagues at the John Radcliffe Hospital, Oxford, for analysis of glucose and insulin; the NIHR Cambridge Biomedical Research Centre Core Biochemical Assay Laboratory for analysis of leptin and adiponectin; Leanne Hodson and Barbara Fielding for access to control dual-emission x-ray absorptiometry scans and phenotypic data on postmenopausal controls; and Jonathan Clark and Izabella Niewczas for providing lipid standards for the mass-spectrometry analysis.

SOURCE INFORMATION

From the Oxford Centre for Diabetes Endocrinology and Metabolism, University of Oxford, Oxford (A.P., T.M.B., M.V.B., J.C.L., F.K., A.L.G.); the Oxford National Institute for Health Research Biomedical Research Centre (A.P., J.C.L., F.K., A.L.G.) and the Oxford Regional Genetics Centre (H.L., L.W.), Churchill Hospital, Oxford; the Inositide Laboratory, the Babraham Institute, Babraham, Cambridge (S.A.R., Q.Z., M.J.O.W.); Wessex Clinical Genetics Service, University Hospital Southampton, Southampton (K.L.L.); the Department of Human Genetics and Genomic Medicine, Faculty of Medicine, University of Southampton, Southampton (K.L.L.); and West Midlands Regional Clinical Genetics Service, Birmingham Women’s Hospital, Birmingham (N.S.C.) — all in the United Kingdom.

Address reprint requests to Dr. Gloyn at the Oxford Centre for Diabetes Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Headington, Oxford OX3 7LE, United Kingdom, or atanna.gloyn@drl.ox.ac.uk.

Source:

http://www.nejm.org/doi/full/10.1056/NEJMoa1113966?query=genetics

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Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation

Posted in Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cardiovascular Pharmacogenomics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Glycobiology: Biopharmaceutical Production, Pharmacodynamics and Pharmacokinetics, International Global Work in Pharmaceutical, Metabolomics, Molecular Genetics & Pharmaceutical, Nutrigenomics, Nutrition, Personalized and Precision Medicine & Genomic Research, Pharmacotherapy of Cardiovascular Disease, Population Health Management, Genetics & Pharmaceutical, Population Health Management, Nutrition and Phytochemistry, Scientist: Career considerations, Systemic Inflammatory Response Related Disorders, Technology Transfer: Biotech and Pharmaceutical, tagged Acetyl-CoA, Adenosine triphosphate, anaerobic and aerobic gycolysis, ATP, ATP demands, Cellular respiration, Citric acid cycle, fermentation, gluconeogenesis, hydrophobic interaction, N:S, overflow at branch points, oxidative stress, Pasteur effect, PFK 2, plant protein diet, protein calorie malnutrition, pyruvate dehydrogenase, Saccharomyces cerevisiae, SIRT3, synthetic small DNA ligands, TCA cycle intermediates, Warburg Effect on September 26, 2012| 26 Comments »

English: A diagram of cellular respiration including glycolysis, Krebs cycle, citric acid cycle, and the electron transport chain (Photo credit: Wikipedia)

Reporter and Curator: Larry H Bernstein, MD, FACP

Introduction

Mitochondria are essential for life, and are critical for the generation of ATP. Otto Warburg won the Nobel Prize in 1918 for his studies of respiration and he described a situation of impaired respiration in cancer cells causing them to produce lactic acid, like bacteria. This has been termed facultative anaerobic glycolysis. The metabolic explanation for mitochondrial respiration had to await the Nobel discoveries of the Krebs cycle and high energy ~P in acetyl CoA by Fritz Lippman. The Krebs cycle generates 16 ATPs I respiration compared to 2 ATPs through glycolysis. The discovery of the genetic code with the “Watson-Crick” model and the identification of DNA polymerase opened a window for contuing discovery leading to the human genome project at 20th century end that has now been followed by “ENCODE” in the 21st century. This review opens a rediscovery of the metabolic function of mitochondria and adaptive functions with respect to cancer and other diseases.

Function in aerobic and anaerobic metabolism

Two-carbon compounds – the TCA, the pentose phosphate pathway, together with gluconeogenesis and the glyoxylate cycle are essential for the provision of anabolic precursors. Yeast environmental diversity mostly leads to a vast metabolic complexity driven by carbon and the energy available in environmental habitats. This resulted in much early research on analysis of yeast metabolism associated with glucose catabolism in Saccharomyces cerevisiae, under both aerobic and anaerobic environments. Yeasts may be physiologically classified with respect to the type of energy-generating process involved in sugar metabolism, namely non-, facultative- or obligate fermentative. The nonfermentative yeasts have exclusively a respiratory metabolism and are not capable of alcoholic fermentation from glucose, while the obligate-fermentative yeasts – “natural respiratory mutants” – are only capable of metabolizing glucose through alcoholic fermentation. Most of the yeasts identified are facultative-fermentative ones, and depending on the growth conditions, the type and concentration of sugars and/or oxygen availability, may display either a fully respiratory or a fermentative metabolism or even both in a mixed respiratory-fermentative metabolism (e.g., S. cerevisiae). The sugar composition of the media and oxygen availability are the two main environmental conditions that have a strong impact on yeast metabolic physiology, and three frequently observed effects associated with the type of energy-generating processes involved in sugar metabolism and/or oxygen availability are Pasteur, Crabtree and Custer. In modern terms the Pasteur effect refers to an activation of anaerobic glycolysis in order to meet cellular ATP demands owing to the lower efficiency of ATP production by fermentation compared with respiration. In 1861 Pasteur observed that S. cerevisiae consume much more glucose in the absence of oxygen than in its presence. S. cerevisiae only shows a Pasteur at low growth rates and at resting-cell conditions, where a high contribution of respiration to sugar catabolism occurs owing to the loss of fermentative capacity. The Crabtree effect is defined as the occurrence of alcoholic fermentation under aerobic conditions, explained by a theory involving “limited respiratory capacities” in the branching point of pyruvate metabolism. The Custer effect is known as the inhibition of alcoholic fermentation by the absence of oxygen. It is thought that the Custer effect is caused by reductive stress.

Glycolysis

Once inside the cell, glucose is phosphorylated by kinases to glucose 6-phosphate and then isomerized to fructose 6-phosphate, by phosphoglucose isomerase. The next enzyme is phospho-fructokinase, which is subject to regulation by several metabolites, and further phosphorylates fructose 6-phosphate to fructose 1,6-bisphosphate. These steps of glycolysis require energy in the form of ATP. Glycolysis leads to pyruvate formation associated with a net production of energy and reducing equivalents. Approximately 50% of glucose 6-phosphate is metabolized via glycolysis and 30% via the pentose phosphate pathway in Crabtree negative yeasts. However, about 90% of the carbon going through the pentose phosphate pathway reentered glycolysis at the level of fructose 6-phosphate or glyceraldehyde 3-phosphate. The pentose phosphate pathway in Crabtree positive yeasts (S. cerevisiae) is predominantly used for NADPH production but not for biomass production or catabolic reactions.
Pyruvate branch point. At the pyruvate (the end product of glycolysis) branching point, pyruvate can follow three different metabolic fates depending on the yeast species and the environmental conditions. On the other hand, the carbon flux may be distributed between the respiratory and fermentative pathways. Pyruvate might be directly converted to acetyl–cofactor A (CoA) by the mitochondrial multienzyme complex pyruvate dehydrogenase (PDH) after its transport into the mitochondria by the mitochondrial pyruvate carrier. Alternatively, pyruvate can also be converted to acetyl–CoA in the cytosol via acetaldehyde and to acetate by the so-called PDH-bypass pathway. Compared with cytosolic pyruvate decarboxylase, the mitochondrial PDH complex has a higher affinity for pyruvate and therefore most of the pyruvate will flow through the PDH complex at low glycolytic rates. However, at increasing glucose concentrations, the glycolytic rate will increase and more pyruvate is formed, saturating the PDH bypass and shifting the carbon flux through ethanol production. In the yeast S. cerevisiae, the external glucose level controls the switch between respiration and fermentation.

Rodrigues F, Ludovico P and Leão C. Sugar Metabolism in Yeasts: an Overview of Aerobic and Anaerobic Glucose Catabolism. In Molecular and Structural Biology. Chapter 6. qxd 07/23/05 P117
Eriksson P, Andre L, Ansell R, Blomberg A, Adler L (1995) Cloning and characterization of GPD2, a second gene encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae, and its comparison with GPD1. Mol Microbiol 17:95–107.
Flikweert MT, van der Zanden L, Janssen WM, Steensma HY, van Dijken JP, Pronk JT (1996)Pyruvate decarboxylase: an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose. Yeast 12:247–257.

Biogenesis of mitochondrial structures from aerobically grown S. cerevisiae

Under aerobic conditions S. cerevisiae forms mitochondria which are classical in their properties,
but the number, morphology, and enzyme activity of these mitochondria are also affected by catabolite repression, but it cannot respire under anaerobic conditions and lacks cytochromes. These structures were isolated from anaerobically grown yeast cells and contain malate and succinate dehydrogenases, ATPase, and DNA characteristic of yeast mitochondria. These lipid-complete structures consist predominantly of double-membrane vesicles enclosing a dense matrix which contains a folded inner membrane system bordering electron-transparent regions similar to the cristae of mitochondria.

The morphology of the structures is critically dependent on their lipid composition
Their unsaturated fatty acid content is similar to that of mitochondria from aerobically grown cells
The structures from cells grown without lipid supplements have simpler morphology – a dense granular matrix surrounded by a double membrane but have no obvious folded inner membrane system within the matrix
The lipid-depleted structures are only isolated in intact form from protoplasts
The synthesis of ergosterol and unsaturated fatty acids is oxygen-dependent and anaerobically grown cells may be depleted of these lipid components
The cytology of anaerobically grown yeast cells is profoundly affected by both lipid-depletion and catabolite repression
Lipid-depleted anaerobic cells, membranous mitochondrial profiles were not demonstrable
The structures from the aerobically and anaerobically grown cells are markedly different in morphology and fatty acid composition, but both contain mitochondrial DNA and a number of mitochondrial enzymes

The phospholipid composition of various strains of Saccharomyces cerevisiae, wild type and petite (cytoplasmic respiratory deficient) yeasts and derived mitochondrial mutants grown under conditions designed to induce variations in the complement of mitochondrial were fractionated into various subcellular fractions and analyzed for cytochrome oxidase (in wild type) and phospholipid composition . 90% or more of the phospholipid, cardiolipin was found in the mitochondrial membranes of wild type and petite yeast . Cardiolipin content differed markedly under various growth conditions .

Stationary yeast grown in glucose had better developed mitochondria and more cardiolipin than repressed log phase yeast .
Aerobic yeast contained more cardiolipin than anaerobic yeast .
Respiration-deficient cytoplasmic mitochondrial mutants, both suppressive and neutral, contained less cardiolipin than corresponding wild types .
A chromosomal mutant lacking respiratory function had normal cardiolipin content .
Log phase cells grown in galactose and lactate, which do not readily repress the development of mitochondrial membranes, contained as much cardiolipin as stationary phase cells grown in glucose .
Cytoplasmic mitochondrial mutants respond to changes in the glucose concentration of the growth medium by variations in their cardiolipin content in the same way as wild type yeast does under similar growth conditions.
It is of interest that the chromosomal petite, which as far as can be ascertained has qualitatively normal mitochondrial DNA and a normal cardiolipin content when grown under maximally derepressed conditions .

Thus, the genetic defect in this case probably does not diminish the mass of inner mitochondrial membrane under appropriate conditions . This suggests the cardiolipin content of yeast is a good indicator of the state of development of mitochondrial membrane.
Jakovcic S, Getz Gs, Rabinowitz M, Jakob H, Swift H. Cardiolipin Content Of Wild Type and Mutant Yeasts in Relation to Mitochondrial Function and Development. JCB 1971. jcb.rupress.org
Jakovcic S, Haddock J, Getz GS, Rabinowitz M, Swift H. Biochem J. 1971; 121 :341 .
EPHRUSSI, B . 1953 . Nucleocytoplasmic Relations in Microorganisms . Clarendon Press, Oxford.

Mitochondria, hydrogenosomes and mitosomes

Before and after the publication of an unnoticed article in 1905 by Mereschkowsky there were many publications dealing with plant “chimera’s” and cytoplasmic inheritance in plants, which should have favoured the interpretation of plastids as “semi-autonomous” symbiotic entities in the cytoplasm of the eukaryotic plant cell. Twenty years after Mereschkowsky’s plea for an endosymbiotic origin of plastids, Wallin (1925, 1927) postulated the “bacterial nature of mitochondria”. And so it is one of the mysteries of the 20th century that an endosymbiotic origin of plastids had not been generally accepted before the 1970s, primarily because one cannot experience the consequences of mutations in the mitochondrial genome by naked eye.

Mitochondrial DNA is usually present in multiple copies in one and the same mitochondrion and those in the hundreds to thousands of mitochondria in a single cell are not necessarily identical.
The random partitioning of the mitochondria in mitosis (and meiosis) frequently results in a more or less biased distribution of the diverent mitochondria in the daughter cells, eventually causing diverent phenotypes in different tissues obscuring the maternal inheritance
It was not until the 1990s that certain diseases—which had been interpreted as being X-chromosomal with incomplete penetrance—eventually turned out to be

Lastly, the vast majority of mitochondrial proteins are encoded in the nucleus and, consequently, mutations in the corresponding genes exhibit a Mendelian, and not a cytoplasmic, maternal inheritance
In the 1970s and 1980s the unequivocal demonstration of mitochondrial DNA occurred
and mitochondrial mutations at the DNA level provided the final proof for the role of such mutations in a wealth of hereditary diseases in man.

The genomics era provided the tools to prove the endosymbiont-hypothesis for the origin of the eukaryotic cell

Since DNA does not arise de novo, the genomes of organisms and organelles provide a historical record for the evolution of the eukaryotic cell and its organelles. The DNA sequences of two to three genomes of the eukaryotic cell turned out to be a record of the evolution of the eukaryotic life on earth. The analysis of organelle genomes unequivocally revealed a cyanobacterial origin for plastids and an -proteobacterial origin for mitochondria. Both plastids and mitochondria appear to be monophyletic, i.e. plastids derived from one and the same cyanobacterial ancestor, and mitochondria from one and the same -proteobacterial ancestor.
The evolution of the eukaryotic cell appears to have involved one (in the case of animals) or two (in the case of plants) events that took place 1.5 to 2 billion years ago. However, it appears that symbioses involving one or the other eubacterium arose repeatedly during the billions of years available. For example, photosynthetic algae by phagotrophic eukaryotes, negating the hypothesis of a single eukaryotic event, rather than stringent selection shaping the diversity of present-day life. Recent hypotheses for the origin of the nucleus have postulated that introns, which could be acquired by the uptake of the -proteobacterial endosymbiont, forced the nucleus-cytosol compartmentalization. Lateral gene transfer among eukaryotes is more frequent than was assumed earlier, and “mitochondrial genes” in the nuclear genomes of amitochondrial organisms are not necessarily the consequence of a transient presence of a DNA-containing mitochondrial-like organelle.
To cope with the obvious ubiquity of “mitochondrial” genes and the chimerism of the DNA of present day eukaryotes, the hydrogen hypothesis postulates that an archaeal host took up a eubacterial symbiont that became the ancestor of mitochondria and hydrogenosomes. The hydrogen hypothesis has the potential to explain both the monophyly of the mitochondria, and the existence of “anaerobic” and “aerobic” variants of one and the same original organelle. Based on these observations we have only the terms “mitochondrion”, “hydrogenosome” and “mitosome” to classify the various variants of the mitochondrial family.
Hackstein JHP, Joachim Tjaden J , Huynen M. Mitochondria, hydrogenosomes and mitosomes: products of evolutionary tinkering! Curr Genet (2006) 50:225–245. DOI 10.1007/s00294-006-0088-8.

Lineages

A look at the phylogenetic distribution of characterized anaerobic mitochondria among animal lineages shows that these are not clustered but spread across metazoan phylogeny. The biochemistry and the enzyme equipment used in the facultatively anaerobic mitochondria of metazoans is nearly identical across lineages, strongly indicating a common origin from an archaic metazoan ancestor. The organelles look like hydrogenosomes – anaerobic forms of mitochondria that generate H2 and adenosine triphosphate (ATP) from pyruvateoxidation and which were previously found only in unicellular eukaryotes. The animals harbor structures resembling prokaryotic endosymbionts, reminiscent of the methanogenic endosymbionts found in some hydrogenosome-bearing protists; fluorescence of F420, a typical methanogen cofactor, or lack thereof, will bring more insights as to what these structures are. If we follow the anaerobic lifestyle further back into evolutionary history, beyond the origin of the metazoans, we see that the phylogenetic distribution of eukaryotes with facultative anaerobic mitochondria, eukaryotes with hydrogenosomes and eukaryotes that possess mitosomes (reduced forms of mitochondria with no direct role in ATP synthesis) the picture is similar to that seen for animals. In all six of the major lineages (or supergroups) of eukaryotes that are currently recognized, forms with anaerobic mitochondria have been found. The newest additions to the growing collection of anaerobic mitochondrial metabolisms are the denitrifying foraminiferans. A handful of about a dozen enzymes make the difference between a ‘normal’ O2-respiring mitochondrion found in mammals, and the energy metabolism of eukaryotes with anaerobic mitochondria, hydrogenosomes or mitosomes. Notably, the full complement of those enzymes, once thought to be specific to eukaryotic anaerobes, surprisingly turned up in the green alga Chlamydomonas reinhardtii , which produces O2 in the light, has typical O2-respiring mitochondria but, within about 30 min of exposure to heterotrophic, anoxic and dark conditions, expresses its anaerobic biochemistry to make H2 in the same way as trichomonads, the group in which hydrogenosomes were discovered. Chlamydomonas provides evidence which indicates that the ability to inhabit oxygen-harbouring, as well as anoxic environments, is an ancestral feature of eukaryotes and their mitochondria. The prokaryote inhabitants have existed for well over a billion years, and have reached this new habitat by dispersal, not by adaptive evolution de novo and in situ. Indeed, geochemical evidence has shown that methanogenesis and sulphate reduction, and the niches in which they occur, are truly ancient.
Mentel and Martin. Anaerobic mitochondria: more common all the time. BMC Biology 2010; 8:32. BioMed Central Ltd. http://www.biomedcentral.com/1741-7007/8/32.

Anaerobic mitochondrial enzymes

Mitochondria from the muscle of the parasitic nematode Ascaris lumbricoides var. suum function anaerobically in electron transport-associated phosphorylations under physiological conditions. These helminth organelles have been fractionated into inner and outer membrane, matrix, and inter-membrane space fractions. The distributions of enzyme systems were determined and compared with corresponding distributions reported in mammalian mitochondria. Succinate and pyruvate dehydrogenases as well as NADH oxidase, Mg++-dependent ATPase, adenylate kinase, citrate synthase, and cytochrome c reductases were determined to be distributed as in mammalian mitochondria. In contrast with the mammalian systems, fumarase and NAD-linked “malic” enzyme were isolated primarily from the intermembrane space fraction of the worm mitochondria. These enzymes are required for the anaerobic energy-generating system in Ascaris and would be expected to give rise to NADH in the intermembrane space.
Pyruvate kinase activity is barely detectable in Ascaris muscle. Therefore, rather than giving rise to cytoplasmic pyruvate, CO2 is fixed into phosphoenolpyruvate, resulting in the formation of oxalacetate which, in turn, is reduced by NADH to form malate regenerating glycolytic NAD . Ascaris muscle mitochondria utilize malate anaerobically as their major substrate by means of a dismutation reaction. The “malic” enzyme in the mitochondrion catalyzes theoxidation of malate to form pyruvate, CO2, and NADH. This reaction serves to generate intramitochondrial reducing power in the form of NADH. Concomitantly, fumarase catalyzes thedehydration of an equivalent amount of malate to form fumarate which, in turn, is reduced by an NADH-linked fumarate reductase to succinate. The flavin-linked fumarate reductase reaction results in a site I electron transport-associated phosphorylation of ADP, giving rise to ATP. This identifies a proton translocation system to obtain energy generation.
Rew RS, Saz HJ. Enzyme Localization in the Anaerobic Mitochondria Of Ascaris Lumbricoides. The Journal Of Cell Biology 1974; 63: 125-135. jcb.rupress.org

Mitochondrial redox status

Tumor cells are characterized by accelerated growth usually accompanied by up-regulated pathways that ultimately increase the rate of ATP production. These cells can suffer metabolic reprogramming, resulting in distinct bioenergetic phenotypes, generally enhancing glycolysis channeled to lactate production. These investigators showed metabolic reprogramming by means of inhibitors of histone deacetylase (HDACis), sodium butyrate and trichostatin. This treatment was able to shift energy metabolism by activating mitochondrial systems such as the respiratory chain and oxidative phosphorylation that were largely repressed in the untreated controls.
Amoêdo ND, Rodrigues MF, Pezzuto P, Galina A, et al. Energy Metabolism in H460 Lung Cancer Cells: Effects of Histone Deacetylase Inhibitors. PLoS ONE 2011; 6(7): e22264. doi:10.1371/ journal.pone.0022264
Antioxidant pathways that rely on NADPH are needed for the reduction of glutathione and maintenance of proper redox status. The mitochondrial matrix protein isocitrate dehydrogenase 2 (IDH2) is a major source of NADPH. NAD+-dependent deacetylase SIRT3 is essential for the prevention of age related hearing loss of caloric restricted mice. Oxidative stress resistance by SIRT3 was mediated through IDH2. Inserting SIRT3 Nε-acetyl-lysine into position 413 of IDH2 and has an activity loss by as much as 44-fold. Deacetylation by SIRT3 fully restored maximum IDH2 activity. The ability of SIRT3 to protect cells from oxidative stress was dependent on IDH2, and the deacetylated mimic, IDH2K413R variant was able to protect Sirt3-/- MEFs from oxidative stress through increased reduced glutathione levels. The increased SIRT3 expression protects cells from oxidative stress through IDH2 activation. Together these results uncover a previously unknown mechanism by which SIRT3 regulates IDH2 under dietary restriction. Recent findings demonstrate that IDH2 activities are a major factor in cancer, and as such, these results implicate SIRT3 as a potential regulator of IDH2-dependent functions in cancer cell metabolism.
Wei Yu, Dittenhafer-Reed KE and JM Denu. SIRT3 Deacetylates Isocitrate Dehydrogenase 2 (IDH2) and Regulates Mitochondrial Redox Status. JBC Papers in Press. Published on March 13, 2012 as Manuscript M112.355206. http://www.jbc.org
Computationally designed drug small molecules targeted for metabolic processes: a bridge from the genome to repair of dysmetabolism
New druglike small molecules with possible anticancer applications were computationally designed. The molecules formed stable complexes with antiapoptotic BCL-2, BCL-W, and BFL-1 proteins. These findings are novel because, to the best of the author’s knowledge, molecules that bind all three of these proteins are not known. A drug based on them should be more economical and better tolerated by patients than a combination of drugs, each targeting a single protein. The calculated drug-related properties of the molecules were similar to those found in most commercial drugs. The molecules were designed and evaluated following a simple, yet effective procedure. The procedure can be used efficiently in the early phases of drug discovery to evaluate promising lead compounds in time- and cost-effective ways.
Keywords: small molecule mimetics, antiapoptotic proteins, computational drug design.

Tardigrades

Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum. To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade Milnesium tardigradum were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from Hypsibius dujardini, revealing major pathways involved in resistance against extreme environmental conditions. ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for M. tardigradum are different from typical motifs known from higher animals. M. tardigradum and H. dujardini protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of M. tardigradum. These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and M. tardigradum in particular so highly stress resistant.
Keywords: RNA, expressed sequence tag, cluster, protein family, adaptation, tardigrada, transcriptome

Epicrisis

This discussion has disparate pieces that are tied together by dysfunctional changes that are

adaptations from metabolic process in the channeling of energy dependent of mitochondrial enzymes in interaction with three to 6 carbon carbohydrates, high energy phosphate, oxygen and membrane lipid structures, as well as
proteins rich or poor in sulfur linked with genome specific targets, and semisynthetic modifications, oxidative stress
leading to a new approach to pharmaceutical targeted drug design.

Determinants of Brain Cell Metabolic Phenotypes and Energy Substrate Utilization Unraveled with a Modeling Approach (ploscompbiol.org)
Mitochondria and Cancer: An overview of mechanisms (pharmaceuticalintelligence.com)
Making a molecular micromap: Imaging the yeast 26S proteasome at near-atomic resolution (phys.org)
Fermentation (ahschoolapbio2013.wordpress.com)
Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function (pharmaceuticalintelligence.com)
What Causes Aging and How Does Dietary Restriction Increase Life Span? (understandnutrition.com)
Diaz-Cano SJ. Tumor heterogeneity: mechanisms and bases for a reliable application of molecular marker design. Int J Mol Sci 2012; 13(2):1951-2011 PMID 22408433Bernstein LH. Expanding the Genetic Alphabet and linking the genome to the metabolome.PharmaIntell.wordpress.com. luly 24, 2012.
Sainani K. Meet the Skeptics: Why Some Doubt Biomedical Models – and What it Takes to Win Them Over. IMAG Futures meeting. 2009
Ritusaxena. ENCODE: the key to unlocking the secrets of complex genetic diseases. PharmaIntell.wordpress.com 2012.
Ritusaxena. Scientists use natural agents for prostate cancer bone metastasis treatment. PharmaIntell.wordpress.com. 2012
Ritusaxena. β Integrin emerges as an important player in mitochondrial dysfunction associated Gastric Cancer. PharmaIntell.wordpress.com 2012

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Nitric Oxide Covalent Modifications: A Putative Therapeutic Target?

Posted in Biomarkers & Medical Diagnostics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Frontiers in Cardiology and Cardiovascular Disorders, Health Economics and Outcomes Research, HTN, Nitric Oxide in Health and Disease, Pharmaceutical Industry Competitive Intelligence, Pharmaceutical R&D Investment, Pharmacotherapy of Cardiovascular Disease, tagged Bachelor of Medicine Bachelor of Surgery, Biology, Cell Biology, nitric oxide, Posttranslational modification, Stephen J. Williams, Therapeutic target, Vatsa on September 24, 2012| 14 Comments »

Author: Stephen J. Williams, PhD

The finding that a substance, derived from vascular endothelium, that could control vascular tone and induce smooth muscle relaxation, led to the discovery of nitric oxide (NO) as a major physiological mediator (1) in many cell types and processes. Other investigators, working with platelets, determined that nitric oxide is a potent inhibitor, via an autocrine pathway, of platelet aggregation and adhesion to the vessel wall (2). Nitric oxide is also an important regulator of neurotransmission in the nonadrenergic-noncholinergic system in gastric tissue (3,4). In addition nitric oxide is involved in macrophage-mediated cytotoxicity, (5)based on the observation the cytotoxic action of macrophages required external arginine, which summarily was converted to citrulline, releasing the nitric oxide involved in the cell-killing process. The above physiological responses represent highly regulated, short-term responses that, as seen with classical receptor-based agonists such as epinephrine, terminate once the agonist (NO) is removed. Given the short half-life of nitric oxide and these rapid physiologic responses, nitric oxide has been given the role of a second messenger within the cell.

However nitric oxide also produces some physiologically, pharmacologically, and pathologically relevant changes, lasting longer time periods, which is the main focus of this article. For example, nitric oxide is important in the development of long term potentiation (a model of learning and memory), neural plasticity, and neurite outgrowth, revealing nitric oxide can induce more permanent changes in cellular and tissue reorganization (6-9). Other pathologic and toxicological responses to nitric oxide include cell death from excitotoxic amino acids (glutamate, kainite), oxidative stress, DNA and protein damage, and disease progression in Alzheimer’s disease, epilepsy, aging, apoptosis and Huntington’s chorea (10-12). These effects persist over longer time frames than the effects which most second messenger systems occur. These cellular changes can be described by biochemical changes on protein and nucleic acid modification, metabolism (13-15), DNA synthesis and replication, and molecular and organelle reorganization. The pharmacological and toxicological implications of such cellular changes are inherent in the persistent effects of nitric oxide on biological systems. The mechanism of nitric oxide-induced physiology and toxicology had been assumed to involve the stimulation of soluble guanylate cyclase, raising intracellular cGMP levels. As discussed further, this mechanism of action does not account for all the actions of nitric oxide, especially in nitric oxide-induced pathologies. Other mechanisms of action include post-translational modifications of proteins such as S-nitrosylations, ADP-ribosylations, and a unique nonenzymatic covalent attachment of NAD⁺ to the regulatory site of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a reaction specific to this dehydrogenase. GAPDH is a true multifunctional protein involved in diverse cellular functions such as glycolysis, endocytosis, RNA processing and stability, DNA replication and repair, and involved in apoptosis. GAPDH has been implicated in trinucleotide repeat neurodegenerative disorders such as Huntington’s disease, spinocerebellar ataxia, via binding to the polyglutamated forms of huntingtin and ataxin, protein modifications only seen in these respective diseases. GAPDH has also been implicated in Alzheimer’s disease as well, in genetic linkage studies as well as a β-amyloid precursor protein binding partner (for reviews see (16-20)).

Next to phosphorylation, ADP ribosylation and NAD⁺ modifications are the second most common enzymatic protein modifications in nature and regulates many cellular processes in nervous tissue, tumoral cell growth, cytoskeletal function, cell death and apoptosis, immune function, and bacterial cytotoxicity(21,22). These include poly ADP-ribosylations such as histones in the apoptotic process, and ADP-ribosylation of G-proteins by pertussis and cholera toxin. Interestingly, nitric oxide and other oxidants promote nonenzymatic ribosylation of proteins such as GAPDH. Unlike the enzymatic reactions, this modification is covalent and generally considered irreversible and either involves nitrosylation of critical reactive cysteine residues or nitric oxide-mediated attachment of the whole NAD⁺ moiety, a reaction akin to aging of enzymes by reactive oxygen species. There have been multiple intracellular targets of nitric oxide, with the result of inhibiting activity and/or protein interactions. These include mitochondrial enzymes such as aconitase (23) and cytochrome oxidase (24), cytosolic enzymes such as cyclooxygenase and affect heme-containing proteins hemoglobin and myoglobin. Such nitric oxide mediated effects on these systems were cGMP-independent, therefore independent of nitric oxide synthase. The inhibition of GAPDH glycolytic activity by nitric oxide and NO-mediated NAD⁺ modification has been widely studied (21,25) and widely accepting to be important in nitric oxide mediated pathology (16,26-33).

So can this NO-NAD⁺ modification of GAPDH be useful as a therapeutic target for diseases such as Huntington’s, Alzheimer’s or other nitric oxide associated pathologies? This is as much an intriguing idea as one fraught with caveats and technical issues. First there is ample evidence that alterations of GAPDH structure/function exist in these neurodegenerative diseases and evidence that this type of modification may be important in the etiology of such diseases(34-41).

Second, as mentioned before, this modification is unique for GAPDH and would offer a disease-specific target(42). Third, and most interesting, is the multifunctionality of GAPDH, therefore such modification has the possibility for affecting many processes involved in the disease progression. However there is the big caveat and problem. Such NO-NAD⁺ modifications are a covalent reaction, thought to be irreversible. Studies on purified GAPDH reveal such modification is released by chemicals that can reduce the cysteine covalent bond such as HgCl₂ or NaOH treatment(17). However such treatment would be impractical for in-vivo use. The ideal situation would be the discovery of an enzyme activity comparable to phosphatases which could enzymatically release the NO-NAD⁺ modification from GAPDH. A proof of concept experiment could involve creation of a genetically engineered enzyme capable of this reaction. Therapeutic use of such an enzyme would depend of course on bioavailability. Interestingly there has been evidence of cellular NO reductase activities, capable of removing the S-nitrosylation on reactive thiols. Enzymes with denitrosylation activities include the thioredoxin system, superoxide dismutase, and xanthine oxidoreductase (34-40). Possible therapeutic strategies may include regulation of these intracellular reductase activities.

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34. Beigi, F., Gonzalez, D. R., Minhas, K. M., Sun, Q. A., Foster, M. W., Khan, S. A., Treuer, A. V., Dulce, R. A., Harrison, R. W., Saraiva, R. M., Premer, C., Schulman, I. H., Stamler, J. S., and Hare, J. M. (2012) Proceedings of the National Academy of Sciences of the United States of America 109, 4314-4319

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Other research paper on Nitric Oxide were published on this Scientific Web site as follows:

Discovery of nitric oxide and its role in vascular biology

Nitric Oxide and Platelet Aggregation

Inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

Nitric Oxide in bone metabolism

Nitric oxide and signalling pathways

Rationale of NO use in hypertension and heart failure

Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

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Junk DNA codes for valuable miRNAs: non-coding DNA controls Diabetes

Posted in Biomarkers & Medical Diagnostics, Bone Disease and Musculoskeletal Disease, Cardiovascular Pharmacogenomics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Computational Biology/Systems and Bioinformatics, Genome Biology, Genomic Testing: Methodology for Diagnosis, Glycobiology: Biopharmaceutical Production, Pharmacodynamics and Pharmacokinetics, Liver & Digestive Diseases Research, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Nutrigenomics, Nutrition, Personalized and Precision Medicine & Genomic Research, Systemic Inflammatory Response Related Disorders, Technology Transfer: Biotech and Pharmaceutical, Uncategorized, tagged DNA, ENCODE, Genome-wide association study, GWAS, Margaret Baker, Messenger RNA, MicroRNA, niRNA, RNA, T1D, T2D on September 24, 2012| 11 Comments »

Author: Margaret Baker, PhD, Registered Patent Agent

The Encyclopedia of DNA Elements (ENCODE) Project was launched in September of 2003. In 2007 the ENCODE project was expanded to study the entire human genome, Genome-wide association studies or GWAS, and published a Nature paper entitled “An integrated encyclopedia of DNA elements in the human genome,” this month also all data are available at http://genome.ucsc.edu/ENCODE/. Novel functional roles have been discovered for both transcribed and non-transcribed portions of DNA. See several articles and commentary in Science 7 September 2012: Vol. 337 no. 6099 including Maurano et al. pp. 1190-1195 DOI: 10.1126/science.1222794b

For the first time, the 3-dimensional connections that cross the genome have been mapped as long-range looping interactions between functional elements and the genes controlled. These regions of the genome, formerly referred to as “junk DNA”, have the potential to be involved in disease initiation, pathophysiology, and complications. Further, epigenetic factors may be seen to play a more direct role in the expression or silencing of protein coding genes as DNase I hot spots, nucleosomal anchor points, and DNA methylation sites are added to the map.

Non-coding transcribed DNA includes a large percentage of sequences coding for RNA. In fact, RNA encoding genes number nearly equal to the protein encoding genes- 18,400 v 20,687 – and previously unknown non-coding RNA (ncRNA) have also been characterized.

Some of the known elements that were cataloged include:

cis elements – promoters, transcription factor binding sites;
gene contiguous non-coding stretches such as introns, polyA, and UTR, splice variants;
pseudogenes (11,224);
long range gene associated elements – enhancers, insulators, suppressors, and predicted promoter flanking regions;
ribosomal RNA genes; and
sequences for 7,052 small RNAs of which 85% are small nuclear(sn)RNA, small nucleolar(sno)RNA), transfer(t)RNA, and micro(mi)RNA.

What has been found is that distinct non-coding regions, including ncRNA, can be associated with distinct disease traits. miRNA are among the non-gene encoding sequences in the genome which have already been shown to play a major post-transcriptional role in expression of multiple genes..

Most miRNA genes are intergenic or oriented antisense to neighboring genes and therefore assumed to be controlled by independent promoter units. However, in some cases a microRNA gene is transcribed together with its target gene implying coupled regulation of miRNA and protein-coding gene. About one third of miRNA genes reside in polycistronic clusters. miRNA genes can occupy the introns of protein, non-protein coding genes, or nonprotein-coding transcripts. The promoters have been shown to have some similarities in their motifs to promoters of other genes transcribed by RNA polymerase II such as protein coding genes. The ENCODE project also noted that miRNA promoters were in chromatin regions of high promiscuity. There may be up to 1000 miRNA genes in the human genome. In addition, human miRNAs show RNA editing of sequences to yield products different from those encoded by their DNA. miRNA are implicated in cellular roles as diverse as developmental timing in worms, cell death and fat metabolism in flies, haematopoiesis in mammals, and leaf development and floral patterning in plants

The final miRNA gene product is a ∼22 nt functional RNA molecule. The mature miRNA (designated miR-#) is processed from a characteristic stem–loop sequence (called a pre-mir), which in turn may be excised from a longer primary transcript (or pri-mir). It is processed by the same enzyme (DICER) that processes short hairpin RNA, forming interfering RNA, which provides and additional level of control.

MiRNA controls gene expression by binding to complementary regions of messenger transcripts in the 3’ untranslated region to repress their translation or regulate degradation. What makes the mechanism more powerful (or complicated) is the imperfect but specific binding motif associates with a large number of mRNAs in the 3’ untranslated region having the complimentary motif. Conversely then, each mRNA can potentially associate with a number miRNA. Mature processed cytosolic miRNA can act in a manner akin to small interfering(si)RNA, and form the RNA-induced silencing complex (RISC) to block translation. Computational methods have been used to identify potential gene targets based on complimentarity between the miRNA and mRNA sequences.

Gerstein et al. explored the “Architecture of the human regulatory network derived from ENCODE data” Nature 489:91-100 (06 Sep 2012) focusing on the regulation of transcription factors (TF) and association between TF and miRNAs, miRNA and miRNA, protein-protein interactions, and protein phosphorylation. Not surprisingly, not all TF are the upstream factor in each network.

These new and remarkably detailed examinations of the different elements within and transcribed from the human genome perhaps do more to aid our knowledge of why we have stumbled in attempts to eradicate diseases, initially by focusing on a single gene or constellation of coding regions. The miRNA wikipedia is also being re-written on a daily basis and new disease associations made*. As an example of a pathological state that may be linked to miRNA controlled elements, in vitro as well as in small population studies have examined miRNA species in diabetogenic conditions and patients with diabetes (Type I and Type II).

Diabetes and miRNA

In adult β-cell islets, miR-375 is low when glucose is freely available and low miR-375 induces insulin secretion. Interestingly, miR-375 is found only in brain and β-cells which share a secretion pathway.

Diabetic Complications

Organ specific miRNA have been identified in liver, skeletal muscle, kidney, vascular, and adipose tissue which are responsive to transient or sustained hyperglycemia.

miR-17-5p and miR-132 were reported to show significant differences between obese and non obese omental fat and were also abnormal in the blood of obese subjects. Altered expression of miR-17-5p and miR-132 were found to correlate significantly with BMI, fasting blood glucose and glycosylated hemoglobin. (Kloting et al. PLoS ONE 4(3), e4699 (2009).

Clinical practice related to miRNA in diabetes may be possible as one group has identified eight miRNAs (miR-144, miR-146a, miR-150, miR-182, miR-192, miR-29a, miR-30d and miR-320) as potential ‘signature miRNAs’ that could distinguish prediabetic patients from those with overt T2D (Karolina DS, Armugam A, Tavintharan S et al. MicroRNA 144 impairs insulin signaling by inhibiting the expression of insulin receptor substrate 1 in Type 2 diabetes mellitus. PLoS ONE 6(8), e22839 (2011).

Due to the autoimmune component of T1D, the constellation of miRNA would be expected to be different: upregulation of miR-510 and underexpression of miR-191 and miR-342 were observed in the Tregs (regulatory T-cells) of T1D patients (Hezova R, Slaby O, Faltejskova P et al. microRNA-342, microRNA-191 and microRNA-510 are differentially expressed in T regulatory cells of Type 1 diabetic patients. Cell. Immunol. 260(2),70–74 (2010).

Taken together with the “physical” mapping of miRNA genes in the context of the 3-dimensional genome provided by the ENCODE studies and new understanding of potential concerted regulatory mechanisms, the miRNA data for tissues and specific cell types involved in disease pathology form a new approach to either detecting or possibly correcting gene (coding or non-coding) dysregulation. miRNA mimics and anti-miRNA agents are being developed as new therapeutic modalities.

References

Bartel, DP et al. MicroRNAs: Genomics, Biogenesis, Mechanism, and Function” Cell 2004, 116:281-297.

Fernandez-Valverde, SL et al. MicroRNAs in beta-cell Biology, insulin resistance, diabetes and its complications. Diabetes July 2011 60 (7):1825-31.

Kantharidis, et al. Diabetes Complications: The MicroRNA Perspective http://diabetes.diabetesjournals.org/content/60/7/1832.short

MEDSCAPE Review article: “miRNAs and Diabetes Mellitus: miRNAs in Diabetic Complicatons” http://www.medscape.org/viewarticle/763729_6

*Based on initial studies in the worm C. elegans showing the temporal appearance of 21- and 22-nt RNAs during development, a family of highly conserved micro RNA sequences (miRNA) existing in invertebrates and vertebrates, were cataloged by Tuschl et al. at the Max-Planck-Institute and others (see Eddy, SR Non-coding RNA genes and the modern RNA world Nature Reviews Genetics, 2:920-929, 2001). The sequence-specific post-transcriptional regulatory mechanisms mediated by these miRNAs have been associated with certain disease states such as cancer miR-21) and more specifically, lung cancer (miR-124) or breast cancer (miR-7, miR-21) and new species and function continue to be found (see http://www.mirbase.org/ ).

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Expanding the Genetic Alphabet and Linking the Genome to the Metabolome

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Liver & Digestive Diseases Research, Medical and Population Genetics, Metabolomics, Molecular Genetics & Pharmaceutical, Nutrigenomics, Nutrition, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Pharmaceutical Industry Competitive Intelligence, Pharmaceutical R&D Investment, Population Health Management, Genetics & Pharmaceutical, Population Health Management, Nutrition and Phytochemistry, Proteomics, Scientist: Career considerations, Systemic Inflammatory Response Related Disorders, Technology Transfer: Biotech and Pharmaceutical, tagged adenine, ATP, biodiversity, Cancer - General, Cancer Genome Atlas, cystathionine beta synthase, cytosine, DNA, fumarate, gene modification, genetic code, genetic engineering, genetics, genome, guanine, H2S, homocysteine, inosine, malnutrition, metabolome, methionine, Nature (journal), Nature Chemical Biology, Nucleic acid double helix, nucleosides, oncometabolite, oxidative stress, proteome, regulatory pathways, S-adenosylmethionine, Scripps Research Institute, Sulfur, synthetic nucleotides, thymidine, Warburg Effect, Watson-Crick Model, xanthine on September 24, 2012| 13 Comments »

English: The citric acid cycle, also known as the tricarboxylic acid cycle (TCA cycle) or the Krebs cycle. Produced at WikiPathways. (Photo credit: Wikipedia)

Expanding the Genetic Alphabet and Linking the Genome to the Metabolome

Reporter& Curator: Larry Bernstein, MD, FCAP

Unlocking the diversity of genomic expression within tumorigenesis and “tailoring” of therapeutic options

1. Reshaping the DNA landscape between diseases and within diseases by the linking of DNA to treatments

In the NEW York Times of 9/24,2012 Gina Kolata reports on four types of breast cancer and the reshaping of breast cancer DNA treatment based on the findings of the genetically distinct types, which each have common “cluster” features that are driving many cancers. The discoveries were published online in the journal Nature on Sunday (9/23). The study is considered the first comprehensive genetic analysis of breast cancer and called a roadmap to future breast cancer treatments. I consider that if this is a landmark study in cancer genomics leading to personalized drug management of patients, it is also a fitting of the treatment to measurable “combinatorial feature sets” that tie into population biodiversity with respect to known conditions. The researchers caution that it will take years to establish transformative treatments, and this is clearly because in the genetic types, there are subsets that have a bearing on treatment “tailoring”. In addition, there is growing evidence that the Watson-Crick model of the gene is itself being modified by an expansion of the alphabet used to construct the DNA library, which itself will open opportunities to explain some of what has been considered junk DNA, and which may carry essential information with respect to metabolic pathways and pathway regulation. The breast cancer study is tied to the “Cancer Genome Atlas” Project, already reported. It is expected that this work will tie into building maps of genetic changes in common cancers, such as, breast, colon, and lung. What is not explicit I presume is a closely related concept, that the translational challenge is closely related to the suppression of key proteomic processes tied into manipulating the metabolome.

Saha S. Impact of evolutionary selection on functional regions: The imprint of evolutionary selection on ENCODE regulatory elements is manifested between species and within human populations. 9/12/2012. PharmaceuticalIntelligence.Wordpress.com

Hawrylycz MJ, Lein ES, Guillozet-Bongaarts AL, Shen EH, Ng L, et al. An anatomically comprehensive atlas of the adult human brain transcriptome. Nature Sept 14-20, 2012

Sarkar A. Prediction of Nucleosome Positioning and Occupancy Using a Statistical Mechanics Model. 9/12/2012. PharmaceuticalIntelligence.WordPress.com

Heijden et al. Connecting nucleosome positions with free energy landscapes. (Proc Natl Acad Sci U S A. 2012, Aug 20 [Epub ahead of print]). http://www.ncbi.nlm.nih.gov/pubmed/22908247

2. Fiddling with an expanded genetic alphabet – greater flexibility in design of treatment (pharmaneogenesis?)

Diagram of DNA polymerase extending a DNA strand and proof-reading. (Photo credit: Wikipedia)

A clear indication of this emerging remodeling of the genetic alphabet is a new
study led by scientists at The Scripps Research Institute appeared in the
June 3, 2012 issue of Nature Chemical Biology that indicates the genetic code as
we know it may be expanded to include synthetic and unnatural sequence pairing (Study Suggests Expanding the Genetic Alphabet May Be Easier than Previously Thought, Genome). They infer that the genetic instructions for living organisms
that is composed of four bases (C, G, A and T)— is open to unnatural letters. An expanded “DNA alphabet” could carry more information than natural DNA, potentially coding for a much wider range of molecules and enabling a variety of powerful applications. The implications of the application of this would further expand the translation of portions of DNA to new transciptional proteins that are heretofore unknown, but have metabolic relavence and therapeutic potential. The existence of such pairing in nature has been studied in Eukariotes for at least a decade, and may have a role in biodiversity. The investigators show how a previously identified pair of artificial DNA bases can go through the DNA replication process almost as efficiently as the four natural bases. This could as well be translated into human diversity, and human diseases.

The Romesberg laboratory collaborated on the new study and his lab have been trying to find a way to extend the DNA alphabet since the late 1990s. In 2008, they developed the efficiently replicating bases NaM and 5SICS, which come together as a complementary base pair within the DNA helix, much as, in normal DNA, the base adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G). It had been clear that their chemical structures lack the ability to form the hydrogen bonds that join natural base pairs in DNA. Such bonds had been thought to be an absolute requirement for successful DNA replication, but that is not the case because other bonds can be in play.

The data strongly suggested that NaM and 5SICS do not even approximate the edge-to-edge geometry of natural base pairs—termed the Watson-Crick geometry, after the co-discoverers of the DNA double-helix. Instead, they join in a looser, overlapping, “intercalated” fashion that resembles a ‘mispair.’ In test after test, the NaM-5SICS pair was efficiently replicable even though it appeared that the DNA polymerase didn’t recognize it. Their structural data showed that the NaM-5SICS pair maintain an abnormal, intercalated structure within double-helix DNA—but remarkably adopt the normal, edge-to-edge, “Watson-Crick” positioning when gripped by the polymerase during the crucial moments of DNA replication. NaM and 5SICS, lacking hydrogen bonds, are held together in the DNA double-helix by “hydrophobic” forces, which cause certain molecular structures (like those found in oil) to be repelled by water molecules, and thus to cling together in a watery medium.

The finding suggests that NaM-5SICS and potentially other, hydrophobically bound base pairs could be used to extend the DNA alphabet and that Evolution’s choice of the existing four-letter DNA alphabet—on this planet—may have been developed allowing for life based on other genetic systems.

3. Studies that consider a DNA triplet model that includes one or more NATURAL nucleosides and looks closely allied to the formation of the disulfide bond and oxidation reduction reaction.

This independent work is being conducted based on a similar concep. John Berger, founder of Triplex DNA has commented on this. He emphasizes Sulfur as the most important element for understanding evolution of metabolic pathways in the human transcriptome. It is a combination of sulfur 34 and sulphur 32 ATMU. S34 is element 16 + flourine, while S32 is element 16 + phosphorous. The cysteine-cystine bond is the bridge and controller between inorganic chemistry (flourine) and organic chemistry (phosphorous). He uses a dual spelling, using sulfphur to combine the two referring to the master catalyst of oxidation-reduction reactions. Various isotopic alleles (please note the duality principle which is natures most important pattern). Sulfphur is Methionine, S adenosylmethionine, cysteine, cystine, taurine, gluthionine, acetyl Coenzyme A, Biotin, Linoic acid, H2S, H2SO4, HSO3-, cytochromes, thioredoxin, ferredoxins, purple sulfphur anerobic bacteria prokaroytes, hydrocarbons, green sulfphur bacteria, garlic, penicillin and many antibiotics; hundreds of CSN drugs for parasites and fungi antagonists. These are but a few names which come to mind. It is at the heart of the Krebs cycle of oxidative phosphorylation, i.e. ATP. It is also a second pathway to purine metabolism and nucleic acids. It literally is the key enzymes between RNA and DNA, ie, SH thiol bond oxidized to SS (dna) cysteine through thioredoxins, ferredoxins, and nitrogenase. The immune system is founded upon sulfphur compounds and processes. Photosynthesis Fe4S4 to Fe2S3 absorbs the entire electromagnetic spectrum which is filtered by the Allen belt some 75 miles above earth. Look up chromatium vinosum or allochromatium species. There is reasonable evidence it is the first symbiotic species of sulfphur anerobic bacteria (Fe4S4) with high potential mvolts which drives photosynthesis while making glucose with H2S.
He envisions a sulfphur control map to automate human metabolism with exact timing sequences, at specific three dimensional coordinates on Bravais crystalline lattices. He proposes adding the inosine-xanthosine family to the current 5 nucleotide genetic code. Finally, he adds, the expanded genetic code is populated with “synthetic nucleosides and nucleotides” with all kinds of customized functional side groups, which often reshape nature’s allosteric and physiochemical properties. The inosine family is nature’s natural evolutionary partner with the adenosine and guanosine families in purine synthesis de novo, salvage, and catabolic degradation. Inosine has three major enzymes (IMPDH1,2&3 for purine ring closure, HPGRT for purine salvage, and xanthine oxidase and xanthine dehydrogenase.

English: DNA replication or DNA synthesis is the process of copying a double-stranded DNA molecule. This process is paramount to all life as we know it. (Photo credit: Wikipedia)

3. Nutritional regulation of gene expression, an essential role of sulfur, and metabolic control

Finally, the research carried out for decades by Yves Ingenbleek and the late Vernon Young warrants mention. According to their work, sulfur is again tagged as essential for health. Sulfur (S) is the seventh most abundant element measurable in human tissues and its provision is mainly insured by the intake of methionine (Met) found in plant and animal proteins. Met is endowed with unique functional properties as it controls the ribosomal initiation of protein syntheses, governs a myriad of major metabolic and catalytic activities and may be subjected to reversible redox processes contributing to safeguard protein integrity.

Consuming diets with inadequate amounts of methionine (Met) are characterized by overt or subclinical protein malnutrition, and it has serious morbid consequences. The result is reduction in size of their lean body mass (LBM), best identified by the serial measurement of plasma transthyretin (TTR), which is seen with unachieved replenishment (chronic malnutrition, strict veganism) or excessive losses (trauma, burns, inflammatory diseases). This status is accompanied by a rise in homocysteine, and a concomitant fall in methionine. The ratio of S to N is quite invariant, but dependent on source. The S:N ratio is typical 1:20 for plant sources and 1:14.5 for animal protein sources. The key enzyme involved with the control of Met in man is the enzyme cystathionine-b-synthase, which declines with inadequate dietary provision of S, and the loss is not compensated by cobalamine for CH3- transfer.

As a result of the disordered metabolic state from inadequate sulfur intake (the S:N ratio is lower in plants than in animals), the transsulfuration pathway is depressed at cystathionine-β-synthase (CβS) level triggering the upstream sequestration of homocysteine (Hcy) in biological fluids and promoting its conversion to Met. They both stimulate comparable remethylation reactions from homocysteine (Hcy), indicating that Met homeostasis benefits from high metabolic priority. Maintenance of beneficial Met homeostasis is counterpoised by the drop of cysteine (Cys) and glutathione (GSH) values downstream to CβS causing reducing molecules implicated in the regulation of the 3 desulfuration pathways

4. The effect on accretion of LBM of protein malnutrition and/or the inflammatory state: in closer focus

Hepatic synthesis is influenced by nutritional and inflammatory circumstances working concomitantly and liver production of TTR integrates the dietary and stressful components of any disease spectrum. Thus we have a depletion of visceral transport proteins made by the liver and fat-free weight loss secondary to protein catabolism. This is most accurately reflected by TTR, which is a rapid turnover protein, but it is involved in transport and is essential for thyroid function (thyroxine-binding prealbumin) and tied to retinol-binding protein. Furthermore, protein accretion is dependent on a sulfonation reaction with 2 ATP. Consequently, Kwashiorkor is associated with thyroid goiter, as the pituitary-thyroid axis is a major sulfonation target. With this in mind, it is not surprising why TTR is the sole plasma protein whose evolutionary patterns closely follow the shape outlined by LBM fluctuations. Serial measurement of TTR therefore provides unequaled information on the alterations affecting overall protein nutritional status. Recent advances in TTR physiopathology emphasize the detecting power and preventive role played by the protein in hyper-homocysteinemic states.

Individuals submitted to N-restricted regimens are basically able to maintain N homeostasis until very late in the starvation processes. But the N balance study only provides an overall estimate of N gains and losses but fails to identify the tissue sites and specific interorgan fluxes involved. Using vastly improved methods the LBM has been measured in its components. The LBM of the reference man contains 98% of total body potassium (TBK) and the bulk of total body sulfur (TBS). TBK and TBS reach equal intracellular amounts (140 g each) and share distribution patterns (half in SM and half in the rest of cell mass). The body content of K and S largely exceeds that of magnesium (19 g), iron (4.2 g) and zinc (2.3 g).

TBN and TBK are highly correlated in healthy subjects and both parameters manifest an age-dependent curvilinear decline with an accelerated decrease after 65 years. Sulfur Methylation (SM) undergoes a 15% reduction in size per decade, an involutive process. The trend toward sarcopenia is more marked and rapid in elderly men than in elderly women decreasing strength and functional capacity. The downward SM slope may be somewhat prevented by physical training or accelerated by supranormal cytokine status as reported in apparently healthy aged persons suffering low-grade inflammation or in critically ill patients whose muscle mass undergoes proteolysis.

5. The results of the events described are:

Declining generation of hydrogen sulfide (H2S) from enzymatic sources and in the non-enzymatic reduction of elemental S to H2S.
The biogenesis of H2S via non-enzymatic reduction is further inhibited in areas where earth’s crust is depleted in elemental sulfur (S8) and sulfate oxyanions.
Elemental S operates as co-factor of several (apo)enzymes critically involved in the control of oxidative processes.

Combination of protein and sulfur dietary deficiencies constitute a novel clinical entity threatening plant-eating population groups. They have a defective production of Cys, GSH and H2S reductants, explaining persistence of an oxidative burden.

6. The clinical entity increases the risk of developing:

cardiovascular diseases (CVD) and
stroke

in plant-eating populations regardless of Framingham criteria and vitamin-B status.
Met molecules supplied by dietary proteins are submitted to transmethylation processes resulting in the release of Hcy which:

either undergoes Hcy — Met RM pathways or
is committed to transsulfuration decay.

Impairment of CβS activity, as described in protein malnutrition, entails supranormal accumulation of Hcy in body fluids, stimulation of activity and maintenance of Met homeostasis. The data show that combined protein- and S-deficiencies work in concert to deplete Cys, GSH and H2S from their body reserves, hence impeding these reducing molecules to properly face the oxidative stress imposed by hyperhomocysteinemia.

Although unrecognized up to now, the nutritional disorder is one of the commonest worldwide, reaching top prevalence in populated regions of Southeastern Asia. Increased risk of hyperhomocysteinemia and oxidative stress may also affect individuals suffering from intestinal malabsorption or westernized communities having adopted vegan dietary lifestyles.

Ingenbleek Y. Hyperhomocysteinemia is a biomarker of sulfur-deficiency in human morbidities. Open Clin. Chem. J. 2009 ; 2 : 49-60.

7. The dysfunctional metabolism in transitional cell transformation

A third development is also important and possibly related. The transition a cell goes through in becoming cancerous tends to be driven by changes to the cell’s DNA. But that is not the whole story. Large-scale techniques to the study of metabolic processes going on in cancer cells is being carried out at Oxford, UK in collaboration with Japanese workers. This thread will extend our insight into the metabolome. Otto Warburg, the pioneer in respiration studies, pointed out in the early 1900s that most cancer cells get the energy they need predominantly through a high utilization of glucose with lower respiration (the metabolic process that breaks down glucose to release energy). It helps the cancer cells deal with the low oxygen levels that tend to be present in a tumor. The tissue reverts to a metabolic profile of anaerobiosis. Studies of the genetic basis of cancer and dysfunctional metabolism in cancer cells are complementary. Tomoyoshi Soga’s large lab in Japan has been at the forefront of developing the technology for metabolomics research over the past couple of decades (metabolomics being the ugly-sounding term used to describe research that studies all metabolic processes at once, like genomics is the study of the entire genome).

Their results have led to the idea that some metabolic compounds, or metabolites, when they accumulate in cells, can cause changes to metabolic processes and set cells off on a path towards cancer. The collaborators have published a perspective article in the journal Frontiers in Molecular and Cellular Oncology that proposes fumarate as such an ‘oncometabolite’. Fumarate is a standard compound involved in cellular metabolism. The researchers summarize that shows how accumulation of fumarate when an enzyme goes wrong affects various biological pathways in the cell. It shifts the balance of metabolic processes and disrupts the cell in ways that could favor development of cancer. This is of particular interest because “fumarate” is the intermediate in the TCA cycle that is converted to malate.

Animation of the structure of a section of DNA. The bases lie horizontally between the two spiraling strands. (Photo credit: Wikipedia)

The Keio group is able to label glucose or glutamine, basic biological sources of fuel for cells, and track the pathways cells use to burn up the fuel. As these studies proceed, they could profile the metabolites in a cohort of tumor samples and matched normal tissue. This would produce a dataset of the concentrations of hundreds of different metabolites in each group. Statistical approaches could suggest which metabolic pathways were abnormal. These would then be the subject of experiments targeting the pathways to confirm the relationship between changed metabolism and uncontrolled growth of the cancer cells.

Junk DNA codes for valuable miRNAs (pharmaceuticalintelligence.com)
Molecular milestone: scientists unravel the human genome – Fox News (foxnews.com)
The Story of You: Encode and the human genome – video | Martin Robbins (guardian.co.uk)
Science takes a giant leap in its understanding of genetics (jessiebaldwin.wordpress.com)
Critical Breakthrough: Study Overturns Theory of ‘Junk DNA’ (the2012scenario.com)
The ENCODE delusion (scienceblogs.com)

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Amplifying Information Using S-Clustering and Relationship to Kullback-Liebler Distance: An Application to Myocardial Infarction

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, Chemical Biology and its relations to Metabolic Disease, Computational Biology/Systems and Bioinformatics, Ecosystems & Industrial Concentration in the Medical Device Sector, FDA Regulatory Affairs, Health Economics and Outcomes Research, Health Law & Patient Safety, HealthCare IT, International Global Work in Pharmaceutical, Medical Devices R&D Investment, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Population Health Management, Genetics & Pharmaceutical, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Scientist: Career considerations, Statistical Methods for Research Evaluation, tagged Anomaly identification, Bernoulli trial, clustering methods, combinatorial analysis, EHR, IT validation, Kullback-Liebler Distance, learning algorithms, multivariate classification, S-clustering, Shannon-Weaver Information theory on September 22, 2012| 3 Comments »

typical changes in CK-MB and cardiac troponin in Acute Myocardial Infarction (Photo credit: Wikipedia)

Reporter and curator:

Larry H Bernstein, MD, FCAP

This posting is a followup on two previous posts covering the design and handling of HIT to improve healthcare outcomes as well as lower costs from better workflow and diagnostics, which is self-correcting over time.

The first example is a non technology method designed by Lee Goldman (Goldman Algorithm) that was later implemented at Cook County Hospital in Chicago with great success. It has been known that there is over triage of patients to intensive care beds, adding to the costs of medical care. If the differentiation between acute myocardial infarction and other causes of chest pain could be made more accurate, the quantity of scare resources used on unnecessary admissions could be reduced. The Goldman algorithm was introduced in 1982 during a training phase at Yale-New Haven Hospital based on 482 patients, and later validated at the BWH (in Boston) on 468 patients.They demonstrated improvement in sensitivity as well as specificity (67% to 77%), and positive predictive value (34% to 42%). They modified the computer derived algorithm in 1988 to achieve better results in triage of patients to the ICU of patients with chest pain based on a study group of 1379 patients. The process was tested prospectively on 4770 patients at two university and 4 community hospitals. The specificity improved by 74% vs 71% in recognizing absence of AMI by the algorithm vs physician judgement. The sensitivity was not different for admission (88%). Decisions based solely on the protocol would have decreased admissions of patients without AMI by 11.5% without adverse effects. The study was repeated by Qamar et al. with equal success.

Pain in acute myocardial infarction (front) (Photo credit: Wikipedia)

An ECG showing pardee waves indicating acute myocardial infarction in the inferior leads II, III and aVF with reciprocal changes in the anterolateral leads. (Photo credit: Wikipedia)

Acute myocardial infarction with coagulative necrosis (4) (Photo credit: Wikipedia)

Goldman L, Cook EF, Brand DA, Lee TH, Rouan GW, Weisberg MC, et al. A computer protocol to predict myocardial infarction in emergency department patients with chest pain. N Engl J Med. 1988;318:797-803.

A Qamar, C McPherson, J Babb, L Bernstein, M Werdmann, D Yasick, S Zarich. The Goldman algorithm revisited: prospective evaluation of a computer-derived algorithm versus unaided physician judgment in suspected acute myocardial infarction. Am Heart J 1999; 138(4 Pt 1):705-709. ICID: 825629

The usual accepted method for determining the decision value of a predictive variable is the Receiver Operator Characteristic Curve, which requires a mapping of each value of the variable against the percent with disease on the Y-axis. This requires a review of every case entered into the study. The ROC curve is done to validate a study to classify data on leukemia markers for research purposes as shown by Jay Magidson in his demonstation of Correlated Component Regression (2012)(Statistical Innovations, Inc.) The test for the contribution of each predictor is measured by Akaike Information Criteria and Bayes Information Criteria, which have proved to be critically essential tests over the last 20 years.

I go back 20 years and revisit the application of these principles in clinical diagnostics, but the ROC was introduced to medicine in radiology earlier. A full rendering of this matter can be found in the following:
R A Rudolph, L H Bernstein, J Babb. Information induction for predicting acute myocardial infarction.Clin Chem 1988; 34(10):2031-2038. ICID: 825568.

Rypka EW. Methods to evaluate and develop the decision process in the selection of tests. Clin Lab Med 1992; 12:355

Rypka EW. Syndromic Classification: A process for amplifying information using S-Clustering. Nutrition 1996;12(11/12):827-9.

Christianson R. Foundations of inductive reasoning. 1964. Entropy Publications. Lincoln, MA.

Inability to classify information is a major problem in deriving and validating hypotheses from PRIMARY data sets necessary to establish a measure of outcome effectiveness. When using quantitative data, decision limits have to be determined that best distinguish the populations investigated. We are concerned with accurate assignment into uniquely verifiable groups by information in test relationships. Uncertainty in assigning to a supervisory classification can only be relieved by providing suffiuciuent data.

A method for examining the endogenous information in the data is used to determine decision points. The reference or null set is defined as a class having no information. When information is present in the data, the entropy (uncertainty in the data set) is reduced by the amount of information provided. This is measureable and may be referred to as the Kullback-Liebler distance, which was extended by Akaike to include statistical theory. An approach is devised using EW Rypka’s S-Clustering has been created to find optimal decision values that separate the groups being classified. Further, it is possible to obtain PRIMARY data on-line and continually creating primary classifications (learning matrices). From the primary classifications test-minimized sets of features are determined with optimal useful and sufficient information for accurately distinguishing elements (patients). Primary classifications can be continually created from PRIMARY data. More recent and complex work in classifying hematology data using a 30,000 patient data set and 16 variables to identify the anemias, moderate SIRS, sepsis, lymphocytic and platelet disorders has been published and recently presented. Another classification for malnutrition and stress hypermetabolism is now validated and in press in the journal Nutrition (2012), Elsevier.
G David, LH Bernstein, RR Coifman. Generating Evidence Based Interpretation of Hematology Screens via Anomaly Characterization. Open Clinical Chemistry Journal 2011; 4 (1):10-16. 1874-2416/11 2011 Bentham Open. ICID: 939928

G David; LH Bernstein; RR Coifman. The Automated Malnutrition Assessment. Accepted 29 April 2012.
http://www.nutritionjrnl.com. Nutrition (2012), doi:10.1016/j.nut.2012.04.017.

Keywords: Network Algorithm; unsupervised classification; malnutrition screening; protein energy malnutrition (PEM); malnutrition risk; characteristic metric; characteristic profile; data characterization; non-linear differential diagnosis

Summary: We propose an automated nutritional assessment (ANA) algorithm that provides a method for malnutrition risk prediction with high accuracy and reliability. The problem of rapidly identifying risk and severity of malnutrition is crucial for minimizing medical and surgical complications. We characterized for each patient a unique profile and mapped similar patients into a classification. We also found that the laboratory parameters were sufficient for the automated risk prediction.
We here propose a simple, workable algorithm that provides assistance for interpreting any set of data from the screen of a blood analysis with high accuracy, reliability, and inter-operability with an electronic medical record. This has been made possible at least recently as a result of advances in mathematics, low computational costs, and rapid transmission of the necessary data for computation. In this example, acute myocardial infarction (AMI) is classified using isoenzyme CKMB activity, total LD, and isoenzyme LD-1, and repeated studies have shown the high power of laboratory features for diagnosis of AMI, especially with NSTEMI. A later study includes the scale values for chest pain and for ECG changes to create the model.

LH Bernstein, A Qamar, C McPherson, S Zarich. Evaluating a new graphical ordinal logit method (GOLDminer) in the diagnosis of myocardial infarction utilizing clinical features and laboratory data. Yale J Biol Med 1999; 72(4):259-268. ICID: 825617

The quantitative measure of information, Shannon entropy treats data as a message transmission. We are interested in classifying data with near errorless discrimination. The method assigns upper limits of normal to tests computed from Rudolph’s maximum entropy definitions of group-based normal reference. Using the Bernoulli trial to determine maximum entropy reference, we determine from the entropy in the data a probability of a positive result that is the same for each test and conditionally independent of other results by setting the binary decision level for each test. The entropy of the discrete distribution is calculated from the probabilities of the distribution. The probability distribution of the binary patterns is not flat and the entropy decreases when there is information in the data. The decrease in entropy is the Kullback-Liebler distance.

The basic principle of separatory clustering is extracting features from endogenous data that amplify or maximize structural information into disjointed or separable classes. This differs from other methods because it finds in a database a theoretic – or more – number of variables with required VARIETY that map closest to an ideal, theoretic, or structural information standard. Scaling allows using variables with different numbers of message choices (number bases) in the same matrix, binary, ternary, etc (representing yes-no; small-modest, large, largest). The ideal number of class is defined by x^n. In viewing a variable value we think of it as low, normal, high, high high, etc. A system works with related parts in harmony. This frame of reference improves the applicability of S-clustering. By definition, a unit of information is log.r r = 1.

The method of creating a syndromic classification to control variety in the system also performs a semantic function by attributing a term to a Port Royal Class. If any of the attributes are removed, the meaning of the class is made meaningless. Any significant overlap between the groups would be improved by adding requisite variety. S-clustering is an objective and most desirable way to find the shortest route to diagnosis, and is an objective way of determining practice parameters.

Multiple Test Binary Decision Patterns where CK-MB = 18 u/l, LD-1 = 36 u/l, %LD1 = 32 u/l.

No. Pattern Freq P1 Self information Weighted information

0 000 26 0.1831 2.4493 0.4485
1 001 3 0.0211 5.5648 0.1176
2 010 4 0.0282 5.1497 0.1451
3 011 2 0.0282 6.1497 0.0866
4 100 6 0.0423 4.5648 0.1929
6 110 8 0.0563 4.1497 0.2338
7 111 93 0.6549 0.6106 0.3999

Entropy: sum of weighted information (average) 1.6243 bits

The effective information values are the least-error points. Non AMI patients exhibit patterns 0, 1, 2, 3, and 4: AMI patients are 6 and 7. There is 1 fp 4, and 1 fn 6. The error rate is 1.4%.

Summary:

A major problem in using quantitative data is lack of a justifiable definition of reference (normal). Our information model consists of a population group, a set of attributes derived from observations, and basic definitions using Shannon’s information measure entropy. In this model, the population set and its values for its variables are considered to be the only information available. The finding of a flat distribution with the Bernoulli test defines the reference population that has no information. The complementary syndromic group, treated in the same way, produces a distribution that is not flat and has a less than maximum information uncertainty.

The vector of probabilities – (1/2), (1/2), …(1/2), can be related to the path calculated from the Rypka-Fletcher equation, which

Ct = 1 – 2^-k/1 -2^-n

determines the theoretical maximum comprehension from the test of n attributes. We constructed a ROC curve from theoriginal IRIS data of R Fisher from four measurements of leaf and petal with a result obtained using information-based induction principles to determine discriminant points without the classification that had to be used for the discriminant analysis. The principle of maximum entropy, as formu;ated by Jaynes and Tribus proposes that for problems of statistical inference – which as defined, are problems of induction – the probabilities should be assigned so that the entropy function is maximized. Good proposed that maximum entropy be used to define the null hypothesis and Rudolph proposed that medical reference be defined as at maximum entropy.

Rudolph RA. A general purpose information processing automation: generating Port Royal Classes with probabilistic information. Intl Proc Soc Gen systems Res 1985;2:624-30.

Jaynes ET. Information theory and statistical mechanics. Phys Rev 1956;106:620-30.

Tribus M. Where do we stand after 30 years of maximum entropy? In: Levine RD, Tribus M, eds. The maximum entropy formalism. Cambridge, Ma: MIT Press, 1978.

Good IJ. Maximum entropy for hypothesis formulation, especially for multidimensional contingency tables. Ann Math Stat 1963;34:911-34.

The most important reason for using as many tests as is practicable is derived from the prominent role of redundancy in transmitting information (Noisy Channel Theorem). The proof of this theorem does not tell how to accomplish nearly errorless discrimination, but redundancy is essential.

In conclusion, we have been using the effective information (derived from Kullback-Liebler distance) provided by more than one test to determine normal reference and locate decision values. Syndromes and patterns that are extracted are empirically verifiable.

Entropy and Syntropy (photoatelier.org)
A Software Agent for Diagnosis of ACUTE MYOCARDIAL INFARCTION (pharmaceuticalintelligence.com)
K-Nearest-Neighbors and Handwritten Digit Classification (jeremykun.wordpress.com)
Data Mining: Classification VS Clustering (cluster analysis) (parasdoshi.com)
Myocardial Infarction Algorithm Strategy 77% Effective In One Hour (guardianlv.com)
Scale‑Free Diagnosis of AMI from Clinical Laboratory Values (pharmaceuticalintelligence.com)
The great healthcare chasm: Patients want to email, access EMRs, but physicians still can’t (medcitynews.com)
Guidelines Updated for Unstable Angina/Non-ST Elevation Myocardial Infarction (pharmaceuticalintelligence.com)

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Arsenic levels in rice and rice products

Posted in Biological Networks, Gene Regulation and Evolution, Chemical Biology and its relations to Metabolic Disease, FDA Regulatory Affairs, Infectious Disease & New Antibiotic Targets, tagged Arsenic, Basmati, Cereal, FDA, food, Food and Drug Administration, Rice, toxicology on September 19, 2012| Leave a Comment »

Reporter: Prabodh Kandala, PhD

As part of an ongoing and proactive effort to monitor food safety and address contaminants in food, the U.S. Food and Drug Administration today released preliminary data on arsenic levels in certain rice and rice products. The data are part of a larger FDA data collection and analysis about arsenic levels in rice and is based on the first set of approximately 200 samples of rice and rice products collected in the U.S. marketplace.

The FDA is in the process of collecting and analyzing a total of approximately 1,200 samples to examine the issue thoroughly. This data collection will be completed by the end of 2012. Once the data collection is completed, FDA will analyze these results and determine whether or not to issue additional recommendations.

Based on the currently available data and scientific literature the FDA does not have an adequate scientific basis to recommend changes by consumers regarding their consumption of rice and rice products.

“We understand that consumers are concerned about this matter. That’s why the FDA has prioritized analyzing arsenic levels in rice. The FDA is committed to ensuring that we understand the extent to which substances such as arsenic are present in our foods, what risks they may pose, whether these risks can be minimized, and to sharing what we know,” said FDA Commissioner Margaret A. Hamburg, M.D. “Our advice right now is that consumers should continue to eat a balanced diet that includes a wide variety of grains – not only for good nutrition but also to minimize any potential consequences from consuming any one particular food.”

There are two types of arsenic compounds found in water, food, air, and soil: organic and inorganic. Together, the two types are referred to as total arsenic.

The new data show how much inorganic arsenic the FDA found in its initial samples, which include various brands of rice (non-Basmati), Basmati rice, brown rice, rice cereals (puffed, non-puffed, hot cereal, and infant cereals), rice cakes, and rice milk.

The FDA’s analysis of these initial samples found average levels of inorganic arsenic for the various rice and rice products of 3.5 to 6.7 micrograms of inorganic arsenic per serving. Serving sizes varied depending on the rice product (for example, one serving of non-Basmati rice was equal to one cup cooked). A summary of the initial 200 sample findings can be found at www.fda.gov⁴.

While the FDA data is consistent with results that Consumer Reports published today, the initial data collection is a first step in the agency’s ongoing more thorough data analysis. There are many different types of rice and rice products that are grown in different areas and under different conditions. Further analysis is needed to assess how these variations may affect the results.

“It is critical to not get ahead of the science,” said FDA Deputy Commissioner for Foods Michael Taylor. “The FDA’s ongoing data collection and other assessments will give us a solid scientific basis for determining what action levels and/or other steps are needed to reduce exposure to arsenic in rice and rice products.”

Ref:

http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm319972.htm

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Discovery of nitric oxide and its role in vascular biology

Posted in Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cardiovascular Pharmacogenomics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Frontiers in Cardiology and Cardiovascular Disorders, Glycobiology: Biopharmaceutical Production, Pharmacodynamics and Pharmacokinetics, HTN, Molecular Genetics & Pharmaceutical, Nitric Oxide in Health and Disease, Systemic Inflammatory Response Related Disorders, tagged Arginine, Aviral Vatsa, Blood vessel, Cardiovascular disease, Cardiovascular Disorders, cGMP-dependent kinases, Cytochrome c oxidase, Endothelium, history, nitric oxide, Nitric oxide synthase, NO, pathways, signalling, vascular biology, Vascular health on September 18, 2012| 14 Comments »

Curated/reported by : Aviral Vatsa PhD, MBBS

Based on : S Moncada et al

It was in 1980 that Furchgott & Zawadzki first described endothelium- dependent relaxation of the blood vessels by acetylcholine. Further studies in 1984 revealed that other factors such as bradykinin, histamine and 5-hydroxytryptamine release endothelium derived relaxing factor (EDRF), which can modulate vessel tone. EDRF was shown to stimulate soluble guanylate cyclase and was inhibited by haemoglobin. In 1986 it was demonstrated that superoxide (O₂^–) anions mediated EDRF inactivation and that the inhibitors of EDRF generated superoxide (O₂^–) anions in solution as a mean to inhibit EDRF. It was later established that all compounds that inhibit EDRF have one property in common, redox activity, which accounted for their inhibitory action on EDRF. One exception was haemoglobin, which inactivates EDRF by binding to it. In 1987 Furchgott proposed that EDRF might be nitric oxide (NO) based on a study of the transient relaxations of endothelium-denuded rings of rabbit aorta to ‘acidified’ inorganic nitrite (NO^–) solutions and the observations that superoxide dismutase (SOD, which removes O₂^–) protected EDRF. Till then NO was not known to be produced in mammalian cells. In 1988 Palmer et al could detect NO production both biologically and chemically by chemiluminescence. The following year in 1989 the enzyme responsible for NO production, NO synthase, was discovered and L-arginine:NO pathway was proposed.

Roles of L-arginine:NO pathway

By 1987 it was proposed that NO is generated in tissues other than endothelium. Hibbs et al and Marletta et al proposed that NO was generated by macrophages. Moreover release of EDRF was demonstrated in cerebellar cells following activation with N-methyl-D- aspartate (NMDA ). Both noradrenergic and cholinergic responses are ‘controlled’ by the nitrergic system so that the release of NO (e.g., during electrical field stimulation) counteracts and dominates the response to the noradrenergic or cholinergic stimulus (Cellek & Moncada, 1997). Mechanism of penile erection was unveiled by the studies on nitrergic neurotransmission that led to therapeutic intervention. Selective damage of nitrergic nerves in disease states was proposed as a potent mechanism of pathophysiology. Broadly three areas of research based on three isoforms of NOS came into being;

cardiovascular
nervous
immunology

Identification of N^G-monomethyl-L-arginine (L-NMMA) as an inhibitor of the synthesis of NO lay the basis of future research into investigating the role of NO in biological systems. In 1989 it was demonstrated that intravenous infusion of L-NMMA resulted in increase in blood pressure that was reversible by infusing L-arginine. NO was thus implicated in constantly maintaining blood vessel tone. eNOS knockout studies showed a hypertensive phenotypes in the animal models and over expression of eNOS led to lowering of the blood pressure. Furthermore, eNOS activation was attributed to phosphorylation of a specific tyrosine residue in the enzyme.

NO and Mitochondria

http://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

NO reacts with some of the complexes of the respiratory chain, and inhibits mitochondrial respiration – this is a well accepted notion. Initially it was believed that the target for NO was soluble guanylate cyclase, which in vasculature would lead to elevation of cGMP that eventually results in NO mediated vasodilatation and platelet aggregation inhibition. In 1994, another potential target, cytochrome c oxidase, for inhibitory effects of NO was discovered. This was a reversible effect, in competition with oxygen concentrations. Increases in NO production were also shown to inhibit cellular respiration irreversibly by selectively inhibiting complex I . Hence in 2002 it was proposed that this might be a mechanism through which cell pathology was initiated in certain conditions. Furthermore, NO was proposed to be implicated in the activation of the grp78-dependent stress response , via modulating calcium-related interaction between mitochondria and endoplasmic reticulum . This host defence mechanism might also have role in vasculature. Further evidence was provided in 2003 to link the role of NO in mitochondrogenesis and thus indicating that NO might be involved in the regulation of the balance between glycolysis and oxidative phosphorylation in cells.

NO and Pathophysiology

Lack of NO: By 2000, NO was established as a haemostatic regulator in the vasculature. Its absence was implicated in pathological states such as hypertension and vasospasm. These pathophysiological states share a common beginning of endothelial dysfunction, which has low NO production as one of its characterstic features. This dysfunction has been observed prior to the appearance of cardiovascular disease in predisposed subjects with family history of essential hypertension and atherosclerosis. The most likely mechanism for endothelial dysfunction is that of a reduced bioavailability of NO . The mechanism of this aspect is discussed elsewhere on this site. Protection against reduction of NO bio-availability in the vasculature is a vital therapeutic target and is extensively explored. This can be achieved by the use of antioxidants and/or augmentation of eNOS expression. In 2003 statins were shown to increase the production of endothelial NO in endothelial cell cultures and in animals by the reduction of oxidative stress or by increasing the coupling of the eNOS. It was way back in 1994 that oestrogen was shown to increase both the activity and expression of eNOS. In addition, more recently in 2003, oestrogen was shown to reduce the breakdown of available NO.

Excess of NO: In 2000 it was shown that NO produced from iNOS in vasculature is involved in extensive vasodilatation in septic shock. Later it was demonstrated that inhibition of mitochondrial respiration is an important component of the NO-induced tissue damage. This inhibition of respiration, which is initially NO-dependent and reversible, becomes persistent with time as a result of oxidative stress . Such metabolic hypoxic states where in tissues cannot utilise available oxygen due to NO, could also contribute to other inflammatory and degenerative conditions. An obvious therapeutic target for reducing NO production in such conditions would be L-NMMA. L-NMM was tested in a clinical trial for septic shock in 2004. The results were however disappointing probably due to the blanket reduction in NO production from other NOS enzymes there by having deleterious effects on the treatment group. More specific inhibitors for NOS forms are being investigated for in different disease states.

In conclusion, the L-arginine: NO pathway has had a major impact in many areas of research, specially vascular biology. A lot has been understood about this pathway and its interactions, therapeutic targets are being aggressively investigated, but further investigations are required to delineate further the role of NO in human health and disease.

Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

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Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Posted in Biological Networks, Gene Regulation and Evolution, Cardiac & Vascular Repair Tools Subsegment, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Frontiers in Cardiology and Cardiovascular Disorders, HTN, Human Immune System in Health and in Disease, International Global Work in Pharmaceutical, Metabolomics, Molecular Genetics & Pharmaceutical, Nitric Oxide in Health and Disease, Origins of Cardiovascular Disease, PCI, Pharmaceutical Analytics, Pharmaceutical Industry Competitive Intelligence, Pharmacotherapy of Cardiovascular Disease, Stem Cells for Regenerative Medicine, Systemic Inflammatory Response Related Disorders, tagged Cardiovascular disease, Coronary artery disease, Heart Failure, Hypertension, Larry H. Bernstein, nitric oxide, Nitric oxide synthase, Pulmonary hypertension, Robert F Furchgott on September 14, 2012| 17 Comments »

Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Author: Larry H. Bernstein, MD

This review identifies the complex interactions and underlying regulatory balances/imbalances between the mechanism of vasorelaxation and vasoconstriction of vascular endothelium by way of nitric oxide (NO), prostacyclin, in response to oxidative stress and intimal injury.

A series of related topics have been covered in this Scientific Web Site: http://pharmaceuticalintelligence.com on the Nitric Oxide series:
Nitric oxide: role in Cardiovascular health and disease
The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure

Imbalance of Autonomic Tone: The Promise of Intravascular Stimulation of Autonomics

Intravascular Stimulation of Autonomics: A Letter from Dr. Michael Scherlag

Will these findings impact the Clopidogrel market?

Positioning a Therapeutic Concept for Endogenous Augmentation of cEPCs — Therapeutic Indications for Macrovascular Disease: Coronary, Cerebrovascular and Peripheral

Cardiovascular Outcomes: Function of circulating Endothelial Progenitor Cells (cEPCs): Exploring Pharmaco-therapy targeted at Endogenous Augmentation of cEPCs

Endothelial Dysfunction, Diminished Availability of cEPCs, Increasing CVD Risk for Macrovascular Disease – Therapeutic Potential of cEPCs

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB

Vascular Medicine and Biology: CLASSIFICATION OF FAST ACTING THERAPY FOR PATIENTS AT HIGH RISK FOR MACROVASCULAR EVENTS Macrovascular Disease – Therapeutic Potential of cEPCs

Ethical Considerations in Studying Drug Safety — The Institute of Medicine Report

Nitric Oxide Signalling Pathways

Age-Dependent Depression in Circulating Endothelial Progenitor Cells in Coronary Artery Bypass Grafting Patients

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad

Nitric Oxide and Platelet Aggregation

Obstructive coronary artery disease diagnosed by RNA levels of 23 genes – CardioDx heart disease test wins Medicare coverage

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents

Nitric Oxide: Chemistry and function

Nitric Oxide: a short historic perspective

Human Embryonic-Derived Cardiac Progenitor Cells for Myocardial Repair

Nitric Oxide production in Systemic sclerosis

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

Endothelial Differentiation and Morphogenesis of Cardiac Precursors

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Nitric Oxide in bone metabolism

Circulating Endothelial Progenitor Cells Milestones in the research on Circulating Endothelial Progenitor Cells as diagnostic markers of cardiovascular risk have been reported in NEJM

Transcription Factor Lyl-1 Critical in Producing Early T-Cell Progenitors

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

Treatment of Refractory Hypertension via Percutaneous Renal Denervation

Triple Antihypertensive Combination Therapy Significantly Lowers Blood Pressure in Hard-to-Treat Patients with Hypertension and Diabetes

Study Finds Low Methylation Regions Prone to Structural Mutation

Atherosclerosis, studied extensively, has common features in the aorta, coronary, carotid and peripheral arteries. There is commonality in morphology of atheromatous plaques, the degree of thrombus formation and types of ensuing ischemic events, therefore, plaque disruption, the degree of thrombus formation and types of ensuing ischemic coronary syndromes of patients have been extensively studied. A concept of unstable atherosclerotic plaques that are amenable to rupture is central to acute myocardial infarction, stroke, aortic aneurysm and sequellae, and peripheral artery disease. These unstable atherosclerotic plaques: plaques with an unstable morphology are essential to the onset of unstable coronary artery disease. Plaque formation results from complex cellular interactions in the intima of arteries, which take place between resident cells of the vessel wall (smooth muscle cells and endothelial cells) and cells of the immune system (leukocytes). Local flow disturbances, extracellular matrix composed of lipids and debris are defining characteristics of the ‘atheroma’.
Inflammation and repair are key events in the natural course of atherosclerosis, and inflammatory cells have profound effects on the integrity of the plaques . Smooth muscle cells produce by far most of the extracellular matrix components of a plaque. Transforming growth factor beta (TGF-β) is one of the most potent stimulators of connective tissue production by smooth muscle cells, and large amounts of this growth factor are detected in restenosis lesions after PTCA.
Atherosclerotic plaque rupture – pathologic basis of plaque stability and instability. AC Newby, P Libby, and AC. van der Wal. Cardiovasc Res 1999; 41 (2): 334-344. doi: 10.1016/S0008-6363(98)00276-4. Vascular endothelium acts as a paracrine/endocrine organ by secreting a wide range of biologically active mediators that play a key role in regulating immune responses, vascular tone and coagulation, and act on adjacent smooth muscle cells, monocytes, macrophages, fibroblasts and organ specific cells.

The key culprits include:

VASODILITATION

vasodilators (prostacycline (PGI2),
nitric oxide (NO),
endothelium-derived relaxing factor (EDRF) ) and
cardiac specific natriuretic peptides;

The chemical nature of EDRF was not known until Palmer, Ferrige and Moncada demonstrated that nitric oxide accounted for most if not all of the biological activity of EDRF. Both EDRF and NO act through the stimulation of soluble guanylate cyclase and subsequent formation of cyclic GMP (cGMP). Cyclic GMP activates cGMP-dependent protein kinases and leads to dephosphorylation of myosin light chains and muscle relaxation.

Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987; 327:524-6.

Waldman SA, Murad F. Cyclic GMP synthesis and function. Pharma Rev 1987; 39:163-96.

Gryglewski RJ, Botting RM, Vane JR. Mediators produced by the endothelial cell. Hypertension 1988; 12:530-48.

VASOCONSTRICTION

vasoconstrictors (endothelin-1 (ET), thromboxane A2, prostaglandin H2 and components of renin-angiotensin system);
pro- and anti-thrombotic factors (tissue factor, platelet activating factor (PAF), von Willebrand factor (vWf ) );
fibrinolytic activators and inhibitors (tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1));
arachidonate metabolites (prostanoids)( Prostanoids are cyclic lipid mediators which arise from enzymic cyclooxygenation of linear polyunsaturated fatty acids, e.g. arachidonic acid (20:4 n 6, AA). Biologically active prostanoids deriving from AA include stable prostaglandins (PGs), e.g. PGE(2), PGF(2alpha), PGD(2), PGJ(2) as well as labile prostanoids, i.e. PG endoperoxides (PGG(2), PGH(2)), thromboxane A(2) (TXA(2)) and prostacyclin (PGI(2)). Stable PGs regulate smooth muscle tone and also modulate inflammatory and immune reactions in this context. PG endoperoxides are intermediates in biosynthesis of all prostanoids. Gryglewski RJ. Prostacyclin among prostanoids. Pharmacol Rep. 2008 Jan-Feb;60(1):3-11.
leukocyte adhesion molecules (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, P-selectin);
multiple cytokines transforming growth factor, proinflammatory and anti-inflammatory mediators, tumour necrosis factor, chemokines and steroids.
HU Rehman. Vascular Endothelium as an Endocrine Organ. Proc R Coll Physicians Edinb 2001; 31:149-154.

Prostanoids are cyclic lipid mediators which arise from enzymic cyclooxygenation of linear polyunsaturated fatty acids (n-6 PUFAs), e.g. arachidonic acid (20:4 n 6, AA). Labile Thromboxane A(2) (TXA(2)) ( half life of 30 s at 37 degrees C), generated by platelets has powerful vasoconstrictor, cytotoxic and thrombogenic properties.

Prostacyclin (PGI(2)) with a half life of 4 min at 37 degrees C is produced by the vascular wall (predominantly by the endothelium) and is a physiological antagonist of TXA(2), and is a powerful cytoprotective agent that exerts its action through activation of adenylate cyclase. PGI(2) acts in concert with the system consisting of NO synthase (eNOS)/nitric oxide free radical (NO)/guanylate cyclase/cyclic-GMP. Both cyclic nucleotides (c-AMP and c-GMP) act in synergy as two energetic fists which defend the cellular machinery from being destroyed by endogenous or exogenous aggressors.

Recently, a new partner has been recognized in this endogenous defensive squadron, i.e. a system involving heme oxygenase (HO-1) and carbon monoxide (CO). The expanding knowledge on the pharmacological steering of this enzymic triad (PGI(2)-S/eNOS/HO-1) is likely to contribute to the rational therapy of many systemic diseases such as

atherosclerosis,
diabetes mellitus,
arterial hypertension or
Alzheimer diseases.

The discovery of prostacyclin broadened our pathophysiological horizon, and by itself opened new therapeutic possibilities.Gryglewski RJ. Prostacyclin among prostanoids. Pharmacol Rep. 2008 Jan-Feb;60(1):3-11.

Circulating and tissue endothelin immunoreactivity correlate with the severity of atherosclerosis. In patients presenting with stable and unstable angina, circulating endothelin concentrations are elevated in the setting of acute myocardial infarction. Plasma endothelin concentrations are also elevated in patients with moderate to severe congestive heart failure and correlate with the severity of the symptoms.

The vasculature of the kidney is about ten times more sensitive than that of other organs to the vasoconstrictor effects of ET-1. Endothelin affects several aspects of renal function, including

vasoconstriction,
regulation of tubule function,
cellular proliferation and
matrix production.

Endothelin is also involved in the pathogenesis of glomerulosclerosis.
Komuro I, Kurihara H, Sugiyama T et al. Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells. FEBS Lett 1988; 238:249-52. Miyauchi T, Yanagisawa M, Tomizawa T et al. Increased plasma concentrations of endothelin-1 and big endothelin-1 in acute myocardial infarction. Lancet 1989; 2(8653):53-4.

Physical or chemical damage to the cell membrane results in the release of prostacyclin. Other stimulators of prostacyclin release are

bradykinin,
thrombin,
serotonin,
platelet-derived growth factor (PDGF),
interleukin-1 and
adenine nucleotides.

Prostacyclin acts in a paracrine manner, on the abluminal side causing relaxation of the underlying smooth muscle and in the lumen, preventing platelets clumping onto the endothelium. Vasodilator and anti-platelet actions of prostacyclin are mediated by an increase in the concentrations of cyclic adenosine monophosphate (AMP) in smooth muscle cells and platelets. Aspirin and similar substances prevent prostacyclin formation, but have little effect on normal blood pressure.

This discussion has outlined how the vascular system establishes a tight regulation over the production of these endothelium derived vasoactive factors. Its loss allows local or generalized modifications of the vascular tone. This dysregulation is involved in the pathogenesis of

hypertension,
atherosclerosis,
diabetes mellitus and
other vasospastic disorders.

HU Rehman. Vascular Endothelium as an Endocrine Organ. Proc R Coll Physicians Edinb 2001; 31:149-154.

Epicrisis

This discussiion is at the limit of comprehension in one sitting. There is much investigation into the complex issue involving interacting biological regulatory pthaways that intersect among major disease categories, which makes targeting of therapies better, but also limited in the ability to achieve meaningful success. That is a topic for further discussion.

I can say with confidence that the story here is incomplete. The final effective solution will require that we tie together the legs of a three legged stool.

The first leg is genomic, which has not reaped as much benefit as we would like, but the successes generate epectations of better.

The second stool is in the opposing balance of nucleotides involved with vascular tone, such as GMP and the action of prostacyclins.

The third led is off the ledger, but will be developed in detail. This involves the essential role of sulfur, in H2S and in the methylation process, in the interpretation of homocysteine levels in the circulation, in the action of Acetyl CoA, S-adenosyl methionine, and GSH, which requires a knowledge of palnt and protein sources of sulfur amino acids, and variable soil conditions.

There also needs to be an understanding of commonalites between

infection,
metabolic syndrome,
immune-mediated inflammatory disease,
HIV wasting, and
cancer cachexia as importantly as their separatory features.

There are a few more resources to finish the discussion of NO and prostacyclin. Atherogenesis leads to decreased bioactivity of NO and this, in turn, can precipitate enhanced cell adhesion, proliferation, vasoconstriction and accelerate the generation of atherosclerotic lesions.

It is possible that some of the detrimental effects of atherosclerosis on the NO pathway result from the generation of secondary oxidants such as peroxynitrite, a product of the reaction of NO with superoxide. The pharmacologic strategies including

the stimulation of generation of endogenous NO,
NO-replacement therapy and
decreasing oxidative stress may be useful for ameliorating the clinical course of atherosclerosis.

MW Radomski, E Salas. Nitric oxide— biological mediator, modulator and factor of injury: its role in the pathogenesis of atherosclerosis.Atherosclerosis 1995; 118: S69–S80. (Supplement) http://dx.doi.org/10.1016/0021-9150(95)90075-6,

The interactions between endothelium-derived nitric oxide (NO) and prostacyclin as inhibitors of platelet aggregation were examined.
Porcine aortic endothelial cells released NO in quantities sufficient to account for the inhibition of platelet aggregation
small amounts of prostacyclin and EDRF which synergistically inhibited platelet aggregation.
A reciprocal potentiation of both the anti- and the disaggregating activity was also observed between low concentrations of prostacyclin and authentic NO or EDRF released from endothelial cells.
It is likely that interactions between prostacyclin and NO released by the endothelium play a role in the homeostatic regulation of platelet-vessel wall interactions.

MW Radomski, RMJ Palmer & S Moncada. The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide. Br. J. Pharmac (1987), 92, 639-646.

The activity of guanylate cyclase is altered to a much larger degree than adenylate cyclase, while cyclic nucleotide phosphodiesterase activity remains unchanged. During the early phases of thrombin- and ADP-induced platelet aggregation a marked activation of the guanylate cyclase occurs whereas aggregation induced by arachidonic acid or epinephrine results in a rapid diminution of this activity.
AJ Barbera. Cyclic nucleotides and platelet aggregation effect of aggregating agents on the activity of cyclic nucleotide-metabolizing enzymes. Biochimica et Biophysica Acta (BBA) – General Subjects 1976; 444(2):579–595.
http://dx.doi.org/10.1016/0304-4165(76)90402-5,

We examined the effect of endothelin-1 (ET-1) on basal and isoprenaline-enhanced L-type Ca2P current (ICaL) in guinea-pig ventricular myocytes under nystatin-perforated patch configuration.
ET-1 at concentrations of 5 nm had little effect on basal ICa,L, but Ica,L was enhanced by isoprenaline (500 nM) and was significantly attenuated by 5 nm ET-1 by more than 50 %. This effect was reversed upon washout. ICaL enhanced by forskolin was also decreased by ET-1.
The inhibitory effect of ET-1 against isoprenaline was completely blocked by the ETA receptor antagonist BQ-123 (1 /LM).
Although ET-1 has been shown to activate specific protein kinase C (PKC) isoforms, a significant inhibitory effect of ET-1 was maintained in the presence of the PKC inhibitor bisindolylmaleimide (20 nM). The nitric oxide (NO) donor SIN-1 (10 AM) attenuated but failed to prevent the ET- 1 effect.
In summary, our results demonstrate that ET-1 has no effects on basal ica,L. However, it exerts a potent inhibitory effect against isoprenaline-enhanced Ica,L. This effect is mediated through ETA receptors coupled to PTX-sensitive G-proteins and occurs in the presence of PKC inhibition and NO generation.

GP Thomas, SM Sims, M Karmazyn. Differential effects of endothelin- 1 on basal and isoprenaline-enhanced Ca2+ current in guinea-pig ventricular myocytes. Journal of Physiology 1997;503(1):55-65

(L-arginine methyl ester) L-NAME-sensitive component of endothelium-dependent relaxation was investigated in the preconstricted femoral arteries during isometric conditions as a difference between acetylcholine-induced relaxation before and after acute NG-nitro-L-arginine methyl ester pre-treatment (L-NAME, 10-5 mol/l). Acetylcholine induced vasorelaxation of SHR was significantly greater than that in control WKY. There was a significant positive correlation between BP and L-NAME-sensitive component of relaxation of the femoral artery. There si absence of endothelial dysfunction in the femoral artery of adult borderline and spontaneously hypertensive rats and gradual elevation of L-NAME-sensitive component of vasorelaxation with increasing blood pressure.

A Puzserovap, Z Csizmadiova, I Bernatova. Effect of Blood Pressure on L-NAME-sensitive Component of Vasorelaxation in Adult Rats. Physiol. Res. 2007:56 (Suppl. 2): S77-S84.

This review concerns the role of nitric oxide (NO) in the pathogenesis of different models of experimental hypertension (NO-deficient, genetic, salt-dependent), which are characterized by a wide range of etiology. Although the contribution of NO may vary between different models of hypertension, a unifying characteristic of these models is the presence of oxidative stress that participates in the maintenance of elevated arterial pressure and seems to be a common denominator underlying endothelial dysfunction in various forms of experimental hypertension. Besides the imbalance between the endothelial production of vasorelaxing and vasoconstricting compounds as well as the relative insufficiency of vasodilator systems to compensate augmented vasoconstrictor systems, there were found numerous structural and functional abnormalities in blood vessels and heart of hypertensive animals.

J Torok. Participation of Nitric Oxide in Different Models of Experimental Hypertension. Physiol. Res. 2008; 57: 813-825.

Cardiac NO–sGC-cGMP signaling blunts cardiac stress responses, including pressure overload–induced hypertrophy. The latter itself depresses signaling through this pathway by reducing NO generation and enhancing cGMP hydrolysis. Pressure overload depresses NO/heme-dependent sGC activation in the heart, consistent with enhanced oxidation. The data reveal a novel additional mechanism for reduced NO-coupled sGC activity related to dynamic shifts in membrane microdomain localization, with Cav3-microdomains protecting sGC from heme-oxidation and facilitating NO responsiveness. Translocation of sGC out of this domain favors sGC oxidation and contributes to depressed NO-stimulated sGC activity.

EJ Tsai, Y Liu, N Koitabashi, D Bedja, et al. Pressure-Overload–Induced Subcellular
Relocalization/Oxidation of Soluble Guanylyl Cyclase in the Heart Modulates Enzyme Stimulation. Circ Res. 2012;110:295-303.

From the studies in several laboratories, we suggest the following mechanisms for the possible regulation of guanylate cyclase activity:

factors that could alter the apparent cooperative nature of the enzyme,
interactions of metal ions with the substrate or enzyme,
factors that could overcome inhibition by ATP,
mechanisms that could regulate the interconversion of latent and active forms of the enzyme,
possible translocation of particulate and soluble forms of the enzyme, and
induction or repression of the enzyme.

H Kimura, F Murada. Two forms of guanylate cyclase in mammalian tissues and possible mechanisms for their regulation. Metabolism 1975; 24(3): 439–445.

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Calcium Dependent NOS Induction by Sex Hormones: Estrogen

Calcium Dependent NOS Induction by Sex Hormones: Estrogen

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Calcium Dependent NOS Induction by Sex Hormones: Estrogen

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PTEN Mutations as a Cause of Constitutive Insulin Sensitivity and Obesity

BACKGROUND

METHODS

RESULTS

CONCLUSIONS

SOURCE INFORMATION

Source:

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Introduction

Function in aerobic and anaerobic metabolism

Glycolysis

Biogenesis of mitochondrial structures from aerobically grown S. cerevisiae

Mitochondria, hydrogenosomes and mitosomes

Lineages

Anaerobic mitochondrial enzymes

Mitochondrial redox status

Tardigrades

Epicrisis

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Diabetic Complications

References

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NO and Mitochondria

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Further Reading

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