Montreal Clinical Research Institute

Abstract The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal ( https://www.encodeproject.org ), including phase II ENCODE 1 and Roadmap Epigenomics 2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis -regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org ) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

As the major regulator of vascular homeostasis, the endothelium exerts a number of vasoprotective effects, such as vasodilation, suppression of smooth muscle cell growth, and inhibition of inflammatory responses. Many of these effects are largely mediated by nitric oxide, the most potent endogenous vasodilator. Nitric oxide opposes the effects of endothelium-derived vasoconstrictors and inhibits oxidation of low-density lipoprotein. A defect in the production or activity of nitric oxide leads to endothelial dysfunction, signaled by impaired endothelium-dependent vasodilation. Accumulating evidence suggests that endothelial dysfunction is an early marker for atherosclerosis and can be detected before structural changes to the vessel wall are apparent on angiography or ultrasound. Many of the risk factors that predispose to atherosclerosis can also cause endothelial dysfunction, and the presence of multiple risk factors has been found to predict endothelial dysfunction. A number of clinical trials have shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) improve endothelial dysfunction in patients with coronary risk factors beyond what could be attributed to their impact on plasma lipids. Studies have elucidated several possible mechanisms by which statin therapy may improve endothelial dysfunction, including upregulation of nitric oxide production or activity and reduction of oxidative stress.

Peripheral tissue damage or nerve injury often leads to pathological pain processes, such as spontaneous pain, hyperalgesia and allodynia, that persist for years or decades after all possible tissue healing has occurred. Although peripheral neural mechanisms, such as nociceptor sensitization and neuroma formation, contribute to these pathological pain processes, recent evidence indicates that changes in central neural function may also play a significant role. In this review, we examine the clinical and experimental evidence which points to a contribution of central neural plasticity to the development of pathological pain. We also assess the physiological, biochemical, cellular and molecular mechanisms that underlie plasticity induced in the central nervous system (CNS) in response to noxious peripheral stimulation. Finally, we examine theories which have been proposed to explain how injury or noxious stimulation lead to alterations in CNS function which influence subsequent pain experience.

BACKGROUND: Severe acute respiratory syndrome (SARS) is caused by a newly discovered coronavirus (SARS-CoV). No effective prophylactic or post-exposure therapy is currently available. RESULTS: We report, however, that chloroquine has strong antiviral effects on SARS-CoV infection of primate cells. These inhibitory effects are observed when the cells are treated with the drug either before or after exposure to the virus, suggesting both prophylactic and therapeutic advantage. In addition to the well-known functions of chloroquine such as elevations of endosomal pH, the drug appears to interfere with terminal glycosylation of the cellular receptor, angiotensin-converting enzyme 2. This may negatively influence the virus-receptor binding and abrogate the infection, with further ramifications by the elevation of vesicular pH, resulting in the inhibition of infection and spread of SARS CoV at clinically admissible concentrations. CONCLUSION: Chloroquine is effective in preventing the spread of SARS CoV in cell culture. Favorable inhibition of virus spread was observed when the cells were either treated with chloroquine prior to or after SARS CoV infection. In addition, the indirect immunofluorescence assay described herein represents a simple and rapid method for screening SARS-CoV antiviral compounds.

BACKGROUND: Reaching the therapeutic target of remission or low-disease activity has improved outcomes in patients with rheumatoid arthritis (RA) significantly. The treat-to-target recommendations, formulated in 2010, have provided a basis for implementation of a strategic approach towards this therapeutic goal in routine clinical practice, but these recommendations need to be re-evaluated for appropriateness and practicability in the light of new insights. OBJECTIVE: To update the 2010 treat-to-target recommendations based on systematic literature reviews (SLR) and expert opinion. METHODS: A task force of rheumatologists, patients and a nurse specialist assessed the SLR results and evaluated the individual items of the 2010 recommendations accordingly, reformulating many of the items. These were subsequently discussed, amended and voted upon by >40 experts, including 5 patients, from various regions of the world. Levels of evidence, strengths of recommendations and levels of agreement were derived. RESULTS: The update resulted in 4 overarching principles and 10 recommendations. The previous recommendations were partly adapted and their order changed as deemed appropriate in terms of importance in the view of the experts. The SLR had now provided also data for the effectiveness of targeting low-disease activity or remission in established rather than only early disease. The role of comorbidities, including their potential to preclude treatment intensification, was highlighted more strongly than before. The treatment aim was again defined as remission with low-disease activity being an alternative goal especially in patients with long-standing disease. Regular follow-up (every 1-3 months during active disease) with according therapeutic adaptations to reach the desired state was recommended. Follow-up examinations ought to employ composite measures of disease activity that include joint counts. Additional items provide further details for particular aspects of the disease, especially comorbidity and shared decision-making with the patient. Levels of evidence had increased for many items compared with the 2010 recommendations, and levels of agreement were very high for most of the individual recommendations (≥9/10). CONCLUSIONS: The 4 overarching principles and 10 recommendations are based on stronger evidence than before and are supposed to inform patients, rheumatologists and other stakeholders about strategies to reach optimal outcomes of RA.

. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.

Functional antibody genes are assembled by V-D-J joining and then diversified by somatic hypermutation. This hypermutation results from stepwise incorporation of single nucleotide substitutions into the V gene, underpinning much of antibody diversity and affinity maturation. Hypermutation is triggered by activation-induced deaminase (AID), an enzyme which catalyzes targeted deamination of deoxycytidine residues in DNA. The pathways used for processing the AID-generated U:G lesions determine the variety of base substitutions observed during somatic hypermutation. Thus, DNA replication across the uracil yields transition mutations at C:G pairs, whereas uracil excision by UNG uracil-DNA glycosylase creates abasic sites that can also yield transversions. Recognition of the U:G mismatch by MSH2/MSH6 triggers a mutagenic patch repair in which polymerase eta plays a major role and leads to mutations at A:T pairs. AID-triggered DNA deamination also underpins immunoglobulin variable (IgV) gene conversion, isotype class switching, and some oncogenic translocations in B cell tumors.

BACKGROUND: In a previous study, a single injection of inclisiran, a chemically synthesized small interfering RNA designed to target PCSK9 messenger RNA, was found to produce sustained reductions in low-density lipoprotein (LDL) cholesterol levels over the course of 84 days in healthy volunteers. METHODS: We conducted a phase 2, multicenter, double-blind, placebo-controlled, multiple-ascending-dose trial of inclisiran administered as a subcutaneous injection in patients at high risk for cardiovascular disease who had elevated LDL cholesterol levels. Patients were randomly assigned to receive a single dose of placebo or 200, 300, or 500 mg of inclisiran or two doses (at days 1 and 90) of placebo or 100, 200, or 300 mg of inclisiran. The primary end point was the change from baseline in LDL cholesterol level at 180 days. Safety data were available through day 210, and data on LDL cholesterol and proprotein convertase subtilisin-kexin type 9 (PCSK9) levels were available through day 240. RESULTS: A total of 501 patients underwent randomization. Patients who received inclisiran had dose-dependent reductions in PCSK9 and LDL cholesterol levels. At day 180, the least-squares mean reductions in LDL cholesterol levels were 27.9 to 41.9% after a single dose of inclisiran and 35.5 to 52.6% after two doses (P<0.001 for all comparisons vs. placebo). The two-dose 300-mg inclisiran regimen produced the greatest reduction in LDL cholesterol levels: 48% of the patients who received the regimen had an LDL cholesterol level below 50 mg per deciliter (1.3 mmol per liter) at day 180. At day 240, PCSK9 and LDL cholesterol levels remained significantly lower than at baseline in association with all inclisiran regimens. Serious adverse events occurred in 11% of the patients who received inclisiran and in 8% of the patients who received placebo. Injection-site reactions occurred in 5% of the patients who received injections of inclisiran. CONCLUSIONS: In our trial, inclisiran was found to lower PCSK9 and LDL cholesterol levels among patients at high cardiovascular risk who had elevated LDL cholesterol levels. (Funded by the Medicines Company; ORION-1 ClinicalTrials.gov number, NCT02597127 .).

Pleiotropic effects of a drug are actions other than those for which the agent was specifically developed. These effects may be related or unrelated to the primary mechanism of action of the drug, and they are usually unanticipated. Pleiotropic effects may be undesirable (such as side effects or toxicity), neutral, or, as is especially the case with HMG-CoA reductase inhibitors (statins), beneficial. Pleiotropic effects of statins include improvement of endothelial dysfunction, increased nitric oxide bioavailability, antioxidant properties, inhibition of inflammatory responses, and stabilization of atherosclerotic plaques. These and several other emergent properties could act in concert with the potent low-density lipoprotein cholesterol-lowering effects of statins to exert early as well as lasting cardiovascular protective effects. Understanding the pleiotropic effects of statins is important to optimize their use in treatment and prevention of cardiovascular disease.

Metabolism of oxygen by cells generates potentially deleterious reactive oxygen species (ROS). Under normal conditions the rate and magnitude of oxidant formation is balanced by the rate of oxidant elimination. However, an imbalance between prooxidants and antioxidants results in oxidative stress, which is the pathogenic outcome of oxidant overproduction that overwhelms the cellular antioxidant capacity. The kidney and vasculature are rich sources of NADPH oxidase-derived ROS, which under pathological conditions play an important role in renal dysfunction and vascular damage. Strong experimental evidence indicates that increased oxidative stress and associated oxidative damage are mediators of renovascular injury in cardiovascular pathologies. Increased production of superoxide anion and hydrogen peroxide, reduced nitric oxide synthesis, and decreased bioavailability of antioxidants have been demonstrated in experimental and human hypertension. These findings have evoked considerable interest because of the possibilities that therapies targeted against free radicals by decreasing ROS generation or by increasing nitric oxide availability and antioxidants may be useful in minimizing vascular injury and renal dysfunction and thereby prevent or regress hypertensive end-organ damage. This article highlights current developments in the field of ROS and hypertension, focusing specifically on the role of oxidative stress in hypertension-associated vascular damage. In addition, recent clinical trials investigating cardiovascular benefits of antioxidants are discussed, and some explanations for the rather disappointing results from these studies are addressed. Finally, important avenues for future research in the field of ROS, oxidative stress, and redox signaling in hypertension are considered.

Major adverse reactions to antituberculosis drugs can cause significant morbidity, and compromise treatment regimens for tuberculosis (TB). Among patients treated for active TB we estimated the incidence, and risk factors, of major side effects from first-line anti-TB drugs. Side effects, resulting in modification or discontinuation of therapy, or hospitalization, were attributed on the basis of resolution after withdrawal, and/or recurrence with rechallenge. Among 430 patients treated between 1990 and 1999, the incidence of all major adverse effects was 1.48 per 100 person-months of exposure (95% confidence interval [95% CI], 1.31 to 1.61) for pyrazinamide, compared with 0.49 (95% CI, 0.42 to 0.55) for isoniazid, 0.43 (95% CI, 0.37 to 0.49) for rifampin, and 0.07 (95% CI, 0.04 to 0.10) for ethambutol. Occurrence of any major side effect was associated with female sex (adjusted hazard ratio, 2.5; 95% CI, 1.3 to 4.7), age over 60 years (adjusted hazard ratio, 2.9; 95% CI, 1.3 to 6.3), birthplace in Asia (adjusted hazard ratio, 2.5; 95% CI, 1.3 to 5.0), and human immunodeficiency virus-positive status (adjusted hazard ratio, 3.8; 95% CI, 1.05 to 13.4). Pyrazinamide-associated adverse events were associated with age over 60 years (adjusted hazard ratio, 2.6; 95% CI, 1.01 to 6.6) and birthplace in Asia (adjusted hazard ratio, 3.4; 95% CI, 1.4 to 8.3), whereas rifampin-associated adverse events were associated with age over 60 years (adjusted hazard ratio, 3.9; 95% CI, 1.02 to 14.9) and human immunodeficiency virus-positive status (adjusted hazard ratio, 8.0; 95% CI, 1.5 to 43). The incidence of pyrazinamide-induced hepatotoxicity and rash during treatment for active TB was substantially higher than with the other first-line anti-TB drugs, and higher than previously recognized.

The blood-brain barrier (BBB) is composed of tightly bound endothelial cells (ECs) and perivascular astrocytes that regulate central nervous system (CNS) homeostasis. We showed that astrocytes secrete Sonic hedgehog and that BBB ECs express Hedgehog (Hh) receptors, which together promote BBB formation and integrity during embryonic development and adulthood. Using pharmacological inhibition and genetic inactivation of the Hh signaling pathway in ECs, we also demonstrated a critical role of the Hh pathway in promoting the immune quiescence of BBB ECs by decreasing the expression of proinflammatory mediators and the adhesion and migration of leukocytes, in vivo and in vitro. Overall, the Hh pathway provides a barrier-promoting effect and an endogenous anti-inflammatory balance to CNS-directed immune attacks, as occurs in multiple sclerosis.

Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N2A cells (EC50 in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca2+ in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress. Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N2A cells (EC50 in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca2+ in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress. The mitochondrial respiratory chain on the inner mitochondrial membrane (IMM) 1The abbreviations used are: IMM, inner mitochondrial membrane; ROS, reactive oxygen species; 3NP, 3-nitropropionic acid; OMM, outer mitochondrial membrane; MPT, mitochondrial permeability transition; tBHP, t-butylhydroperoxide; Dmt, 2′,6′-dimethyltyrosine; LDL, low density lipoprotein; FCCP, carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone; CLSM, confocal laser scanning microscopy; TMRM, tetramethylrhodamine methyl ester. is a major intracellular source of reactive oxygen species (ROS). ROS cause nonspecific damage to lipids, proteins, and DNA, leading to alteration or loss of cellular function. Mitochondria are continuously exposed to ROS and accumulate oxidative damage more rapidly than the rest of the cell, especially because ROS are highly reactive and shortlived (1Kowaltowski A.J. Vercesi A.E. Free Radic. Biol. Med. 1999; 26: 463-471Crossref PubMed Scopus (706) Google Scholar). Many studies have associated mitochondrial dysfunction caused by ROS with both necrotic and apoptotic cell death (2Zamzami N. Hirsch T. Dallaporta B. Petit P.X. Kroemer G. J. Bioenerg. Biomembr. 1997; 29: 185-193Crossref PubMed Scopus (304) Google Scholar). The rate of mitochondrial ROS production can be altered by several physiological or pathological conditions. Inhibitors of the respiratory chain such as 3-nitropropionic acid (3NP), an irreversible inhibitor of the complex II enzyme succinate dehydrogenase, tend to increase ROS production (3Beal M.F. Ferrante R.J. Henshaw R. Matthews R.T. Chan P.H. Kowall N.W. Epstein C.J. Schulz J.B. J. Neurochem. 1995; 65: 919-922Crossref PubMed Scopus (157) Google Scholar, 4Lee W.T. Yin H.S. Shen Y.Z. Neuroscience. 2002; 112: 707-716Crossref PubMed Scopus (45) Google Scholar, 5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. Neurochem. 2004; 88: 1220-1228Crossref PubMed Scopus (105) Google Scholar). The inhibition of this complex seems to be related to neuronal death similar to those occurring in Huntington's disease (6Beal M.F. Brouillet E. Jenkins B.G. Ferrante R.J. Kowall N.W. Miller J.M. Storey E. Srivastava R. Rosen B.R. Hyman B.T. J. Neurosci. 1993; 13: 4181-4192Crossref PubMed Google Scholar), and antioxidants can attenuate the neurochemical changes and some behavioral disturbances caused by 3NP in animals (5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. Neurochem. 2004; 88: 1220-1228Crossref PubMed Scopus (105) Google Scholar, 7Matthews R.T. Yang L. Browne S. Baik M. Beal M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8892-8897Crossref PubMed Scopus (515) Google Scholar). Mitochondrial Ca2+ is another powerful signal for ROS production. Calcium is taken up into mitochondria via a uniporter in the IMM, and elevation of mitochondrial Ca2+ and ROS production is thought to play an important part in cell death associated with ischemia-reperfusion as well as 3NP (4Lee W.T. Yin H.S. Shen Y.Z. Neuroscience. 2002; 112: 707-716Crossref PubMed Scopus (45) Google Scholar, 5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. Neurochem. 2004; 88: 1220-1228Crossref PubMed Scopus (105) Google Scholar). Increasing evidence suggests that ROS play a key role in promoting cytochrome c release from the mitochondria (8Petrosillo G. Ruggiero F.M. Pistolese M. Paradies G. FEBS Lett. 2001; 509: 435-438Crossref PubMed Scopus (204) Google Scholar, 9Nishimura G. Proske R.J. Doyama H. Higuchi M. FEBS Lett. 2001; 505: 399-404Crossref PubMed Scopus (39) Google Scholar, 10Petrosillo G. Ruggiero F.M. Paradies G. FASEB J. 2003; 17: 2202-2208Crossref PubMed Scopus (307) Google Scholar, 11Galindo M.F. Jordan J. Gonzalez-Garcia C. Cena V. Br. J. Pharmacol. 2003; 139: 797-804Crossref PubMed Scopus (68) Google Scholar), and cytochrome c in the cytoplasm triggers activation of the caspase cascade that ultimately leads to apoptosis (12Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4463) Google Scholar, 13Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6239) Google Scholar). The mechanism underlying ROS-mediated cytochrome c release from mitochondria is still not fully understood. Cytochrome c is normally bound to the IMM by an association with cardiolipin (14Nicholls P. Biochim. Biophys. Acta. 1974; 346: 261-310Crossref PubMed Scopus (178) Google Scholar). It is now believed that cytochrome c release from mitochondria proceeds by a two-step process: dissociation of cytochrome c from cardiolipin in the IMM, followed by release of cytochrome c through the outer mitochondrial membrane (OMM) (15Ott M. Robertson J.D. Gogvadze V. Zhivotovsky B. Orrenius S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1259-1263Crossref PubMed Scopus (792) Google Scholar). Cardiolipin is rich in unsaturated fatty acids, and peroxidation of cardiolipin induces the dissociation of cytochrome c from mitochondria into the cytosol (16Shidoji Y. Hayashi K. Komura S. Ohishi N. Yagi K. Biochem. Biophys. Res. Commun. 1999; 264: 343-347Crossref PubMed Scopus (222) Google Scholar). However, the mechanism by which cytochrome c is released through the OMM is not clear. One mechanism may involve ROS-induced promotion of Ca2+-dependent mitochondrial permeability transition (MPT), with swelling of the mitochondrial matrix and rupture of the OMM (17Byrne A.M. Lemasters J.J. 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This mechanism seems likely in 3NP toxicity and ischemia-reperfusion injury, where increased intracellular Ca2+ and ROS are both present (4Lee W.T. Yin H.S. Shen Y.Z. Neuroscience. 2002; 112: 707-716Crossref PubMed Scopus (45) Google Scholar, 5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. Neurochem. 2004; 88: 1220-1228Crossref PubMed Scopus (105) Google Scholar, 22Crompton M. Andreeva L. Basic Res. Cardiol. 1993; 88: 513-523Crossref PubMed Scopus (80) Google Scholar, 23Paradies G. Petrosillo G. Pistolese M. Di Venosa N. Serena D. Ruggiero F.M. Free Radic. Biol. Med. 1999; 27: 42-50Crossref PubMed Scopus (205) Google Scholar). However, there is also evidence showing that cytochrome c can be released through the OMM in an MPT-independent manner (24Jurgensmeier J.M. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1373) Google Scholar, 25Doran E. Halestrap A.P. Biochem. 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Biol. 2001; PubMed Scopus Google Scholar). the by which cytochrome c may be released through the OMM, the to inhibit ROS-induced cytochrome c release and cell death be of peroxidation of the IMM. of the antioxidants the IMM. In addition, of the antioxidants are in of to prevent oxidative cell One used to target antioxidants such as and to mitochondria of these to such as which are rapidly taken up into the mitochondrial matrix because of the the IMM Biochem. 1999; PubMed Scopus Google Scholar, G. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The of into the mitochondrial leads to of IMM and this in isolated mitochondria with of antioxidants than Biochem. 1999; PubMed Scopus Google Scholar, G. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). of the IMM ultimately We have developed a of peptide antioxidants that are taken up by mitochondria and concentrate in the IMM. These peptide antioxidants are cell-permeable and are very intracellular ROS and cell death caused by the t-butylhydroperoxide We have used these IMM-targeted antioxidants to investigate the role of ROS in mitochondrial dysfunction in cells exposed to 3NP. investigate the by which these peptide antioxidants mitochondrial we used isolated mitochondria to to prevent MPT and cytochrome c release caused by Ca2+ and 3NP. In addition, because ROS have been implicated in contractile dysfunction associated with reperfusion of ischemic hearts, we the of these peptide antioxidants in myocardial stunning in an ex vivo heart model. to that the of these peptide antioxidants are caused by to ROS, we a peptide that properties. results that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT and rupture of the These results also a major role for ROS in mitochondrial dysfunction and reperfusion and demonstrate the of these peptide antioxidants in ischemia-reperfusion and and peptides are with alternating aromatic and basic amino and as I. S. G. C. J. Med. Chem. 2000; PubMed Scopus Google Scholar, X. J. Pharmacol. 2003; PubMed Scopus Google Scholar). acid in of the in for mitochondrial and cellular studies I. C. B. 2003; PubMed Scopus Google Scholar). from of of in of peptides to Y. H. M.A. Biochim. Biophys. 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Pharmacol. 2003; PubMed Scopus Google Scholar). with for and in the and in cell intracellular peptide cells with for and confocal laser scanning with cells a with and and demonstrate of to cells with and tetramethylrhodamine methyl for and then by ROS and cells in a density of and to for treatment with tBHP or for and with or of or for ROS by death by a cell Mitochondrial cells treated with 3NP in the or of for and then with and as Mitochondrial of mitochondrial to of and of and continuously for The of by the Mitochondrial to of and with a oxygen in the of and with the of Mitochondrial mitochondria to of and and by of the Mitochondrial mitochondria to of and and swelling by in nm a Mitochondrial Cytochrome c mitochondria in the or of for of Ca2+ to induce by to induce swelling, and the of swelling induced by Ca2+ as a of for the mitochondrial by Cytochrome c in the and a cytochrome c of the isolated heart have been D. Y. J. 2002; PubMed Scopus Google Scholar). continuously with or peptides and to for force with a into the of the and the to a then induced by of for for of of peptides by to and inhibit the oxidation of acid and in The reduced the by in the of also inhibited the oxidation of fatty and in The of not on the of the amino in that similar and However, of by and of demonstrate that the peptides are we cells with for and the of in cell taken up into The of in cell and and on a cell of T. K. H. Y. K. J. 1999; 276: Google and of the intracellular of can be to be than of used to cellular and intracellular by The confocal of into cells in the cytoplasm of the peptide from the The mitochondrial as by The of and suggests of to mitochondria cellular Mitochondrial of of peptides isolated mitochondria. of isolated mitochondria to in of the signal that the of by mitochondria not an of the we mitochondria with and in the mitochondrial of by mitochondria with by in the mitochondrial and in the mitochondrial of S.A. M. Halestrap A.P. J. 2002; PubMed Scopus Google Scholar), can be that in mitochondria. of mitochondria with reduced or by and that of this peptide into the mitochondrial matrix in a mitochondria with for and the mitochondrial to of in the membrane of both IMM and OMM of the mitochondrial with to the OMM to that of the in the and These results that the peptides are to the IMM. ROS and by that peptides are to neuronal N2A cells treated with tBHP or for and then with or or for with tBHP in increase in intracellular ROS and in cell of these cells with or reduced intracellular ROS and increased cell and with in the 3NP-induced Mitochondrial in demonstrate that in ROS can mitochondrial we the of on mitochondrial depolarization caused by treatment of cells with 3NP. cells treated with 3NP in the or of and mitochondrial by confocal In cells the mitochondria are clearly as the In cells treated with 3NP the In treatment with mitochondrial depolarization caused by 3NP ROS by mitochondria then used to the of by these peptide reduced generation of in isolated mitochondria results with In to ROS and to inhibit ROS production induced by which not have on or induced ROS production by isolated mitochondria not of on Mitochondrial in of or in mitochondria did not mitochondrial function. mitochondria with or did not rate of oxygen or not Mitochondrial membrane as by TMRM, also not by or In the of caused and Ca2+ in loss of mitochondrial of MPT MPT in of isolated mitochondria with or of Ca2+ in depolarization of the mitochondria without the of MPT and of mitochondria with also the of MPT induced by 3NP However, which mitochondrial ROS production, on or 3NP-induced MPT not Mitochondrial in of isolated mitochondria with inhibited swelling as by in nm Mitochondrial swelling also inhibited by not by Ca2+ in the of cytochrome c from the mitochondrial to the The release of mitochondrial cytochrome c inhibited by and not in the of the isolated heart of is associated with loss of contractile force D. Y. J. 2002; PubMed Scopus Google Scholar). of contractile force with of the heart with the peptides or improved contractile force of reperfusion with In not to prevent the contractile dysfunction from providing for a major role for ROS in reperfusion These peptides and are the antioxidants that target and concentrate in the IMM, scavenging of ROS the of production. these peptide we to that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP. studies with isolated mitochondria also that ROS mediate cytochrome c release via MPT and rupture of the ROS production, these peptide antioxidants to prevent mitochondrial depolarization in cells exposed to 3NP. these peptide antioxidants to prevent myocardial stunning associated with reperfusion in the ischemic heart in an ex vivo model. The of which not have to prevent mitochondrial swelling or reperfusion that the of these peptides are via The structural motif of these peptides centers on alternating aromatic and basic amino The of peptides can be to the can which can be followed by to M. I. S. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. K.F. Biochem. Pharmacol. PubMed Scopus Google Scholar), or scavenging by H. D. Biochem. Biophys. 1995; PubMed Scopus Google Scholar, J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). peptides have been to B. C. Mol. Pharmacol. 2002; PubMed Scopus Google Scholar, M. L. M.A. Biochem. Pharmacol. 2001; PubMed Scopus Google Scholar). We have that of the as in Dmt, structural to both have the of the amino acid on the scavenging of the of in with in loss of the peptides are and cell in a manner K. G. J. 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Bernassola F. Cohen I. Jacotot E. Ferri K.F. El Hamel C. Bartle L.M. Melino G. Brenner C. Goldmacher V. Kroemer G. Oncogene. 2001; 20: 4305-4316Crossref PubMed Scopus (228) Google Scholar, 21Kanno T. Sato E.E. Muranaka S. Fujita H. Fujiwara T. Utsumi T. Inoue M. Utsumi K. Free Radic. Res. 2004; 38: 27-35Crossref PubMed Scopus (114) Google Scholar). The of peptides to prevent MPT ROS and oxidative damage on mitochondria C. X. E. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). ROS production also a role in cell death caused by 3NP, an irreversible inhibitor of complex II of the respiratory The production of ROS seems to be from elevation of mitochondrial which can then to MPT, mitochondrial and cell death (4Lee W.T. Yin H.S. Shen Y.Z. Neuroscience. 2002; 112: 707-716Crossref PubMed Scopus (45) Google Scholar, 5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. 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However, the of oxygen to the treatment of the been because of the of antioxidants to cell B. D. N. E. PubMed Scopus Google Scholar). results the myocardial stunning. and to prevent stunning in the ex vivo heart reperfusion In which to the ischemic heart reperfusion stunning. We that can prevent myocardial stunning reperfusion D. Y. J. 2002; PubMed Scopus Google Scholar), with as the mechanism of The of to prevent stunning been in in M. K. and H. H. and may have myocardial and are reperfusion In we have cell-permeable peptide antioxidants that target the of ROS generation and mitochondrial function. results demonstrate that ROS play a major role in mitochondrial dysfunction induced by tBHP, and 3NP. These peptides may be beneficial in the treatment of aging and diseases associated with oxidative damage such as ischemia-reperfusion and We and for We for with the confocal and and for with the mitochondria by of of The N2A cells by of of The studies in the the and the of

The search for natriuretic hormones or factors by studies of negative pressure breathing, atrial distension experiments, head-out water immersion, expansion of blood volume, Na+/K+-ATPase inhibitors and parabiosis experiments in Dahl rats has led to the finding that the atria are a peptide-secreting endocrine gland. This new natriuretic hormone has now been purified, sequenced and synthetized, and its cDNA and gene have been cloned. The native and synthetic hormones exert identical wide ranging effects (possibly through particulate guanylate cyclase stimulation and adenylate cyclase inhibition) on the kidney, blood vessels, adrenal cortex, and pituitary. Physiopathologic implications of the hormone in experimental hypertension, congestive heart failure, and expansion of blood volume are beginning to emerge.

The Notch receptor, which is involved in numerous cell fate decisions in invertebrates and vertebrates, is synthesized as a 300-kDa precursor molecule (p300). We show here that proteolytic processing of p300 is an essential step in the formation of the biologically active receptor because only the cleaved fragments are present at the cell surface. Our results confirm and extend recent reports indicating that the Notch receptor exists at the plasma membrane as a heterodimeric molecule, but disagree as to the nature of the protease that is responsible for the cleavage that takes place in the extracellular region. We report here that constitutive processing of murine Notch1 involves a furin-like convertase. We show that the calcium ionophore A23187 and the alpha1-antitrypsin variant, alpha 1-PDX, a known inhibitor of furin-like convertases, inhibit p300 processing. When expressed in the furin-deficient Lovo cell line, p300 is not processed. In vitro digestion of a recombinant Notch-derived substrate with purified furin allowed mapping of the processing site to the carboxyl side of the sequence RQRR (amino acids 1651-1654). Mutation of these four amino acids (and of two secondary dibasic furin sites located nearby) completely abolished processing of the Notch1 receptor.

Asp299Gly and the TLR4 Thr399Ile alleles was at least 6% in all populations tested. This frequency is sufficiently high to screen at-risk patients routinely for these mutations.

An enormous number of studies in the last two decades have been devoted to investigating the role of the endothelium in cardiovascular diseases. Nonetheless, the optimal methodology for investigating the multifaceted aspects of endothelial dysfunction is still under debate. Biochemical markers, molecular genetic tests and invasive and non-invasive tools with and without pharmacological and physiological stimuli have been introduced. Furthermore newer pharmacological tools have been proposed. However, the application of these methodologies should fulfil a number of requirements in order to provide conclusive answers in this area of research. Thus, the most relevant methodological issues in the research on endothelial function and dysfunction are summarized in this paper.

The discovery of autosomal dominant hypercholesterolemic patients with mutations in the PCSK9 gene, encoding the proprotein convertase NARC-1, resulting in the missense mutations suggested a role in low density lipoprotein (LDL) metabolism. We show that the endoplasmic reticulum-localized proNARC-1 to NARC-1 zymogen conversion is Ca2+-independent and that within the zymogen autocatalytic processing site SSVFAQ [downward arrow]SIP Val at P4 and Pro at P3' are critical. The S127R and D374Y mutations result in approximately 50-60% and > or =98% decrease in zymogen processing, respectively. In contrast, the double [D374Y + N157K], F216L, and R218S natural mutants resulted in normal zymogen processing. The cell surface LDL receptor (LDLR) levels are reduced by 35% in lymphoblasts of S127R patients. The LDLR levels are also reduced in stable HepG2 cells overexpressing NARC-1 or its natural mutant S127R, and this reduction is abrogated in the presence of 5 mm ammonium chloride, suggesting that overexpression of NARC-1 increases the turnover rate of the LDLR. Adenoviral expression of wild type human NARC-1 in mice resulted in a maximal approximately 9-fold increase in circulating LDL cholesterol, while in LDLR-/- mice a delayed approximately 2-fold increase in LDL cholesterol was observed. In conclusion, NARC-1 seems to affect both the level of LDLR and that of circulating apoB-containing lipoproteins in an LDLR-dependent and -independent fashion.

OBJECTIVE: Neural apoptosis-regulated convertase (NARC)-1 is the newest member of the proprotein convertase family implicated in the cleavage of a variety of protein precursors. The NARC-1 gene, PCSK9, has been identified recently as the third locus implicated in autosomal dominant hypercholesterolemia (ADH). The 2 other known genes implicated in ADH encode the low-density lipoprotein receptor and apolipoprotein B. As an approach toward the elucidation of the physiological role(s) of NARC-1, we studied its transcriptional regulation. METHODS AND RESULTS: Using quantitative RT-PCR, we assessed NARC-1 regulation under conditions known to regulate genes involved in cholesterol metabolism in HepG2 cells and in human primary hepatocytes. We found that NARC-1 expression was strongly induced by statins in a dose-dependent manner and that this induction was efficiently reversed by mevalonate. NARC-1 mRNA level was increased by cholesterol depletion but insensitive to liver X receptor activation. Human, mouse, and rat PCSK9 promoters contain 2 typical conserved motifs for cholesterol regulation: a sterol regulatory element (SRE) and an Sp1 site. CONCLUSIONS: PCSK9 regulation is typical of that of the genes implicated in lipoprotein metabolism. In vivo, PCSK9 is probably a target of SRE-binding protein (SREBP)-2.

A recombinant vaccinia virus vector was used to coexpress the two candidate mouse prohormone convertases, PC1 and PC2, together with mouse proopiomelanocortin (POMC) in the constitutively secreting cell line BSC-40 and in the endocrine tissue-derived cell lines PC12 and AtT-20, which exhibit regulated secretion. Monitoring of POMC processing demonstrated the distinct cleavage specificities of PC1 and PC2, since in the cell lines analyzed (i) PC1 cleaves POMC into corticotropin and beta-lipotropin, (ii) PC2 cleaves POMC into beta-endorphin, an N-terminally extended corticotropin containing the joining peptide, and either alpha MSH or desacetyl-alpha MSH, and (iii) PC2 cleaves POMC at the five pairs of basic residues analyzed, whereas PC1 cleaves two of them preferentially, suggesting that PC2 has a broader spectrum of activity than PC1. These data are consistent with our hypothesis on the physiological role of PC1 and PC2 as distinct proprotein convertases acting alone or together to produce a set of tissue-specific maturation products in the brain and in peripheral tissues.