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Biomedical subjects

Ferid Murad

Publications and source records attributed to Ferid Murad.

33 records · Page 2Linked to original sources
PubMed2004

Regulation of nitric oxide and soluble guanylyl cyclase.

Since the discoveries that have verified nitric oxide (NO) as an endogenously produced cell signaling molecule, research surrounding its production and mechanisms of action have been studied at an exponentially increasing rate. NO is produced by a family of enzymes termed the NO synthases (NOS), which are regulated independently by various stimuli. Once produced, NO can solicit numerous biological events by reacting with various metals, thiols, and oxygen species to modify proteins, DNA and lipids. One of the most biologically relevant actions of NO is its binding to the heme moiety in the heterodimeric enzyme, soluble guanylyl cyclase (sGC). Activation of sGC by NO results in the production of the second messenger molecule, 3',5'-cyclic guanosine monophosphate (cGMP), which can regulate numerous physiological events such as vasodilatation and neurotransmission. Here we will review the synthesis and fate of NO, and discuss the activation and regulation of the NO receptor, sGC.

Animals↗
PubMed2003

CCAAT-binding factor regulates expression of the beta1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line.

Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. We investigated human beta1 sGC transcriptional regulation in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, our study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression.

Binding Sites↗
PubMed2003

A constitutively activated mutant of human soluble guanylyl cyclase (sGC): implication for the mechanism of sGC activation.

Heterodimeric alphabeta soluble guanylyl cyclase (sGC) is a recognized receptor for nitric oxide (NO) and mediates many of its physiological functions. Although it has been clear that the heme moiety coordinated by His-105 of the beta subunit is crucial for mediating the activation of the enzyme by NO, it is not understood whether the heme moiety plays any role in the function of the enzyme in the absence of NO. Here we analyze the effects of biochemical and genetic removal of heme and its reconstitution on the activity of the enzyme. Detergent-induced loss of heme from the wild-type alphabeta enzyme resulted in several-fold activation of the enzyme. This activation was inhibited after hemin reconstitution. A heme-deficient mutant alphabetaCys-105 with Cys substituted for His-105 was constitutively active with specific activity approaching the activity of the wild-type enzyme activated by NO. However, reconstitution of mutant enzyme with heme and/or DTT treatment significantly inhibited the enzyme. Mutant enzyme reconstituted with ferrous heme was activated by NO and CO alone and showed additive effects between gaseous effectors and the allosteric activator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine. We propose that the heme moiety through its coordination with His-105 of the beta subunit acts as an endogenous inhibitor of sGC. Disruption of the heme-coordinating bond induced by binding of NO releases the restrictions imposed by this bond and allows the formation of an optimally organized catalytic center in the heterodimer.

Animals↗
PubMed2003

Generation and role of angiostatin in human platelets.

Platelets regulate new blood vessel growth, because they contain a number of angiogenesis promoters and inhibitors. Additionally, platelets contain matrix metalloproteinases (MMPs), which when released mediate platelet adhesion and aggregation, and plasminogen, a fibrinolytic system enzyme that serves to limit blood clot formation. Enzymatic cleavage of plasminogen by MMPs generates angiostatin, an angiogenesis inhibitor. Therefore, we examined whether platelets generate angiostatin during aggregation in vitro. Platelets were isolated from healthy human donors and then aggregated with collagen, thrombin, or HT-1080 fibrosarcoma cells. Angiostatin was detected by Western blot analysis in the platelet releasates of all blood donors irrespective of the aggregating agent used. Platelet pellet homogenates showed the presence of angiostatin in all donors, which was released upon aggregation. Furthermore, platelet-derived angiostatin was isolated and purified by lysine-Sepharose affinity chromatography from collagen-aggregated platelet releasates. Bioassay of platelet-derived angiostatin showed that it inhibited the formation of capillary structures by human umbilical vein endothelial cells (HUV-EC-Cs) in an in vitro angiogenesis model. Inhibition of angiostatin in platelet releasates promoted the formation of capillary structures by HUV-EC-Cs. We conclude that healthy human platelets contain angiostatin, which is released in active form during platelet aggregation, and platelet-derived angiostatin has the capacity to inhibit angiogenesis.

Angiogenesis Inhibitors↗
PubMed2003

Quantitative protein profiling in heart mitochondria from diabetic rats.

Quantitative protein profiling based on in vitro stable isotope labeling, two-dimensional polyacrylamide gel electrophoresis, and mass spectrometry is an accurate and reliable approach to measure simultaneously the relative abundance of many individual proteins within two different samples. In the present study, it was used to define a set of alterations caused by diabetes in heart mitochondria from streptozotocin-treated rats. We demonstrated that the expression of proteins from the myocardial tricarboxylic acid cycle was not altered in diabetes. However, up-regulation of the fatty acid beta-oxidation favored fatty acids over glucose as a source of acetyl CoA for the tricarboxylic acid cycle. Protein levels for several proteins involved in electron transport were modestly decreased. Whether this may depress overall ATP production remains to be established, since the protein level of ATP synthase seems to be unchanged. Other changes include down-regulation of protein levels for creatine kinase, voltage-dependent anion channel 1 (VDAC-1), HSP60, and Grp75. The mitochondria-associated level of albumin was decreased, while the level of catalase was substantially increased. All of the changes were evident as early as 1 week after streptozotocin administration. Taken together, these data point to a rapid and highly coordinated regulation of mitochondrial protein expression that occurs during the heart adaptation to diabetes.

Amino Acid Sequence↗
PubMed2003

Protein tyrosine nitration in the mitochondria from diabetic mouse heart. Implications to dysfunctional mitochondria in diabetes.

Oxidative stress has been implicated in dysfunctional mitochondria in diabetes. Tyrosine nitration of mitochondrial proteins was observed under conditions of oxidative stress. We hypothesize that nitration of mitochondrial proteins is a common mechanism by which oxidative stress causes dysfunctional mitochondria. The putative mechanism of nitration in a diabetic model of oxidative stress and functional changes of nitrated proteins were studied in this work. As a source of mitochondria, alloxan-susceptible and alloxan-resistant mice were used. These inbred strains are distinguished by the differential ability to detoxify free radicals. A proteomic approach revealed significant similarity between patterns of tyrosine-nitrated proteins generated in the heart mitochondria under different in vitro and in vivo conditions of oxidative stress. This observation points to a common nitrating species, which may derive from different nitrating pathways in vivo and may be responsible for the majority of nitrotyrosine formed. Functional studies show that protein nitration has an adverse effect on protein function and that protection against nitration protects functional properties of proteins. Because proteins that undergo nitration are involved in major mitochondrial functions, such as energy production, antioxidant defense, and apoptosis, we concluded that tyrosine nitration of mitochondrial proteins may lead to dysfunctional mitochondria in diabetes.

Alloxan↗
PubMed2003

Histone H1.2 is a substrate for denitrase, an activity that reduces nitrotyrosine immunoreactivity in proteins.

Several reports have described an activity that modifies nitrotyrosine-containing proteins and their immunoreactivity to nitrotyrosine Abs. Without knowing the product of the reaction, this new activity has been called a "denitrase." In those studies, some nonspecific proteins, which have multiple tyrosine residues, e.g., albumin, were used as a substrate. Therefore, the studies were based on an unknown mechanism of reaction and potentially a high background. To solve these problems, one of the most important things is to find a more suitable substrate for assay of the enzyme. We developed an assay strategy for determining the substrate for denitrase combining 2D-gel electrophoresis and an on-blot enzyme assay. The resulting substrate from RAW 264.7 cells was Histone H1.2, an isoform protein of linker histone. Histone H1.2 has only one tyrosine residue in the entire molecule, which ensures the exact position of the substrate to be involved. It has been reported that Histones are the most prominent nitrated proteins in cancer tissues. It was also demonstrated that tyrosine nitration of Histone H1 occurs in vivo. These findings lead us to the idea that Histone H1.2 might be an intrinsic substrate for denitrase. We nitrated recombinant and purified Histone H1.2 chemically and subjected it to an on-blot enzyme assay to characterize the activity. Denitrase activity behaved as an enzymatic activity because the reaction was time dependent and was destroyed by heat or trypsin treatment. The activity was shown to be specific for Histone H1.2, to differ from proteasome activity, and to require no additional cofactors.

Animals↗
PubMed2003

The nature of heme/iron-induced protein tyrosine nitration.

Recently, substantial evidence has emerged that revealed a very close association between the formation of nitrotyrosine and the presence of activated granulocytes containing peroxidases, such as myeloperoxidase. Peroxidases share heme-containing homology and can use H(2)O(2) to oxidize substrates. Heme is a complex of iron with protoporphyrin IX, and the iron-containing structure of heme has been shown to be an oxidant in several model systems where the prooxidant effects of free iron, heme, and hemoproteins may be attributed to the formation of hypervalent states of the heme iron. In the current study, we have tested the hypothesis that free heme and iron play a crucial role in NO(2)-Tyr formation. The data from our study indicate that: (i) hemeiron catalyzes nitration of tyrosine residues by using hydrogen peroxide and nitrite, a reaction that revealed the mechanism underlying the protein nitration by peroxidase, H(2)O(2), and NO(2)(-); (ii) H(2)O(2) plays a key role in the protein oxidation that forms the basis for the protein nitration, whereas nitrite is an essential element that facilitates nitration by the heme(Fe), H(2)O(2), and the NO(2)(-) system; (iii) the formation of a Fe(IV) hypervalent compound may be essential for heme(Fe)-catalyzed nitration, whereas O(2)(*-) (ONOO(-) formation), (*)OH (Fenton reaction), and compound III are unlikely to contribute to the reaction; and (iv) hemoprotein-rich tissues such as cardiac muscle are vulnerable to protein nitration in pathological conditions characterized by the overproduction of H(2)O(2) and NO(2)(-), or nitric oxide.

Animals↗
PubMed2003

Nitric oxide (NO)--biogeneration, regulation, and relevance to human diseases.

On October 12, 1998, the Nobel Assembly awarded the Nobel Prize in Medicine and Physiology to scientists Robert Furchgott, Louis Ignarro, and Ferid Murad for their discoveries concerning nitric oxide as a signalling molecule in the cardiovascular system. In contrast with the short research history of the enzymatic synthesis of NO, the introduction of nitrate-containing compounds for medicinal purposes marked its 150th anniversary in 1997. Glyceryl trinitrate (nitroglycerin; GTN) is the first compound of this category. Alfred Nobel (the founder of Nobel Prize) himself had suffered from angina pectoris and was prescribed nitroglycerin for his chest pain. Almost a century later, research in the NO field has dramatically extended and the role of NO in physiology and pathology has been extensively studied. The steady-state concentration and the biological effects of NO are critically determined not only by its rate of formation, but also by its rate of decomposition. Biotransformation of NO and its related N-oxides occurs via different metabolic routes within the body and presents another attractive field for our research as well as for the venture of drug discovery.

Animals↗
PubMed2003

Mechanism of vasculitis and aneurysms in Kawasaki disease: role of nitric oxide.

NO in vivo has both beneficial and nonbeneficial effects depending on site and concentration. Peroxynitrite, resulting from the reaction of NO with superoxide radical, causes cellular damage. Nitrotyrosine, end product of NO's toxic effects on cellular proteins, is a stable compound that can be used to detect evidence of harmful quantities of NO. We sought to detect nitrotyrosine in coronary arterioles of DBA/2 mice injected intraperitoneally with Lactobacillus casei cell wall. The inflammatory response induced occurred in perivascular fashion and involved mainly macrophages. It was variable according to time points, being severe on days 10 and 14 and mild to moderate on days 3 and 7. Few basal inflammatory cells appeared in controls injected with phosphate-buffered saline. Western immunoblots of homogenized hearts on days 10 and 14 demonstrated specific nitrated proteins. Immunohistochemistry of frozen sections of diseased hearts showed positive immunoreactivity for nitrotyrosine in coronary arterioles at the same time points. These findings were absent in the controls. We also determined the expression of inducible nitric oxide synthase (iNOS) in controls on days 10 and 14. iNOS colocalized with nitrotyrosine in perivascular macrophages and coronary arterioles of treated mice. Additionally, aneurysms were found on day 10 and intracardiac hemorrhage with consequent death on day 14. These observations supply evidence that NO through its reactive product, peroxynitrite, and its antigen/tissue marker, nitrotyrosine, is directly involved in coronary arteritis and aneurysm development in mice models of Kawasaki disease (KD). This article shows that macrophages are central to this and bolsters the likelihood of L. casei being the cause of KD.

Aneurysm↗
PubMed2002

Protein nitration in cardiovascular diseases.

There is growing evidence that cardiovascular disease is associated with progressive changes in the production of free radicals and radical-derived reactive species. These intermediates react with all major cellular constituents and may serve several physiological and pathophysiological functions. The nitration of protein tyrosine residues has been used as a footprint for in vivo production of radical and nonradical reactive species. Tyrosine nitration may alter protein function and metabolism and therefore, provides for further dysfunctional changes. This review focuses on an appearance of tyrosine nitrated proteins in cardiovascular tissues under different settings of cardiovascular disease. Sources of reactive species, putative mechanisms of protein nitration in vivo, as well as protein nitration under normal physiological conditions, are also described. The goal of this review is to attract more attention to identification of specific proteins, which undergo tyrosine nitration and to study a correlation between their altered function and pathology. Understanding how protein nitration affects disease progression may offer a unique option for design of antioxidant therapy for the treatment of cardiovascular complications. At the same time, protein nitration can be a biological marker of efficiency of antioxidant therapy.

Cardiovascular Diseases↗
PubMed2001

Nitric oxide synthesis and oxidative stress in the renal cortex of rats with diabetes mellitus.

Experiments were performed to test the hypothesis that diabetes mellitus disrupts the balance between synthesis and degradation of nitric oxide (NO) in the renal cortex. Diabetes was induced by injection of streptozotocin, and sufficient insulin was provided to maintain moderate hyperglycemia for the ensuing 2 wk. Despite an 80% increase in total NO synthase activity measured by L-citrulline assay, nicotinamide adenine dinucleotide phosphate-diaphorase staining was unaltered, and no changes in NO synthase isoform protein levels or their distribution were evident in renal cortex from diabetic rats. Superoxide anion production was accelerated twofold in renal cortical slices from diabetic rats, with an associated 50% increase in superoxide dismutase activity. Western blots prepared by use of a monoclonal antinitrotyrosine antibody revealed an approximately 70-kD protein in renal cortex from sham rats, the nitrotyrosine content of which was threefold greater in cortical samples from diabetic rats. These observations indicate that the early stage of diabetes mellitus provokes accelerated renal cortical superoxide anion production in a setting of normal or increased NO production. This situation can be expected to promote peroxynitrite formation, resulting in the tyrosine nitration of a single protein of unknown identity, as well as a decline in the bioavailability of NO. These events are consistent with the postulate that oxidative stress promotes NO degradation in the renal cortex during the early stage of diabetes mellitus.

Animals↗
PubMedDate not supplied

Discovery of some of the biological effects of nitric oxide and its role in cell signaling.

The role of nitric oxide in cellular signaling in the past 22 years has become one of the most rapidly growing areas in biology with more than 20,000 publications to date. Nitric oxide is a gas and free radical with an unshared electron that can regulate an ever-growing list of biological processes. In many instances nitric oxide mediates its biological effects by activating guanylyl cyclase and increasing cyclic GMP synthesis from GTP. However, the list of effects of nitric oxide that are independent of cyclic GMP is also growing at a rapid rate. For example, nitric oxide can interact with transition metals such as iron, thiol groups, other free radicals, oxygen, superoxide anion, unsaturated fatty acids and other molecules. Some of these reactions result in the oxidation of nitric oxide to nitrite and nitrate to terminate its effect, while other reactions can lead to altered protein structure, function, and/or catalytic capacity. These diverse effects of nitric oxide that are either cyclic GMP dependent or independent can alter and regulate important physiological and biochemical events in cell regulation and function. Nitric oxide can function as an intracellular messenger, an autacoid, a paracrine substance, a neurotransmitter, or as a hormone that can be carried to distant sites for effects. Thus, it is a unique simple molecule with an array of signaling functions. However, as with any messenger molecule, there can be too little or too much of the substance and pathological events result. Some of the methods to regulate either nitric oxide formation, metabolism, or function have been in clinical use for more than a century as with the use of organic nitrates and nitroglycerin in angina pectoris that was initiated in the 1870's. Current and future research with nitric oxide and cyclic GMP will undoubtedly expand the clinicians' therapeutic armamentarium to manage a number of important diseases by perturbing nitric oxide and cyclic GMP formation and metabolism. Such promise and expectations have obviously fueled the interests in these signaling molecules for a growing list of potential therapeutic applications.

Animals↗
PubMedDate not supplied

Engrafted bone marrow-derived flk-(1+) mesenchymal stem cells regenerate skin tissue.

Stem cell plasticity has created great interest because of its potential therapeutic application in degenerative or inherited diseases. Transplantation of bone marrow-derived stem cells was shown to give rise to cells of muscle, liver, nerve, endothelium, epithelium, and so on. But there are still disputes about stem cell plasticity, especially concerning the contribution of bone marrow-derived cells to skin cells. In this study, CM-DiI fluorescence-labeled Flk-(1+) bone marrow mesenchymal stem cells (bMSCs) of BALB/c mice (H-2Kd, white) were transplanted into lethally irradiated C57BL/6 mice (H-2Kb, black). By fluorescence tracing, we found that donor cells could migrate and take residency at the skin, which was confirmed by Y chromosome-specific PCR and Southern blot. The recipient mice grew white hairs about 40 days later and white hairs could spread over the body. Immunochemistry staining and RT-PCR demonstrated that skin tissue within the white hair regions was largely composed of donor-derived H-2Kd cells, including stem cells and committed cells. Furthermore, most skin cells cultured from white hair skin originated from the donor. Thus, our findings provide direct evidence that bone marrow-derived cells can give rise to functional skin cells and regenerate skin tissue. These may have important scientific implications in stem cell biology and transplantation therapy for skin tissue injury.

Animals↗