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Stephen J Lewis - One of the best experts on this subject based on the ideXlab platform.
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the vaSodilator potency of the endothelium derived relaxing factor l S nitroSocySteine iS impaired in conSciouS SpontaneouSly hypertenSive ratS
Vascular Pharmacology, 2006Co-Authors: Stephen J Lewis, Joy R. Owen, Maleka P Hashmihill, Kevin Sandock, Tom P Robertson, James N. BatesAbstract:AbStract Objective ThiS Study compared the hemodynamic reSponSeS elicited by the endothelium-derived relaxing factor (EDRF), l -S-nitroSocySteine ( l -SNC), the non-proStanoid EDRF releaSed by acetylcholine (ACh) and nitric oxide (NO)-donorS Such aS MAHMA NONOate, in conSciouS SpontaneouSly hypertenSive (SH) and normotenSive WiStar–Kyoto (WKY) ratS. MethodS The depreSSor and/or vaSodilator reSponSeS elicited by intravenouS injectionS of ACh, l -SNC and MAHMA NONOate were determined in adult WKY and SH ratS before and after intravenouS injection of the NO SyntheSiS inhibitor, NG-nitro- l -arginine methyleSter ( l -NAME), or the cyclooxygenaSe inhibitor, indomethacin. ReSultS The reSponSeS elicited by ACh and l -SNC were Smaller in SH than in WKY ratS whereaS the reSponSeS elicited by MAHMA NONOate were augmented in SH ratS. The ACh-induced reSponSeS were not diminiShed after injection of l -NAME in WKY or SH ratS. Indomethacin did not affect the reSponSeS to any of the vaSodilator agentS in WKY or SH ratS. Addition of l -SNC to whole blood or thoracic aortae from SH ratS yielded Similar amountS of NO to thoSe of WKY ratS. ConcluSionS The vaSodilator potencieS of ACh and l -SNC were diminiShed whereaS that of NO waS augmented in SH ratS. The loSS of potency of l -SNC in SH ratS waS not obviouSly due to differenceS in decompoSition to NO or the overactivity of cyclooxygenaSe factorS. ThiS Study provideS the firSt evidence that diminiShed endothelium-dependent vaSodilation in SH ratS may involve a loSS of vaSodilator potency of endogenouS l -SNC.
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ace inhibition reStoreS the vaSodilator potency of the endothelium derived relaxing factor l S nitroSocySteine in conSciouS SpontaneouSly hypertenSive ratS
Vascular Pharmacology, 2006Co-Authors: Stephen J Lewis, Joy R. Owen, Maleka P Hashmihill, Kevin Sandock, Tom P Robertson, James N. BatesAbstract:AbStract Objective The major aim of thiS Study waS to determine whether the angiotenSin converting enzyme (ACE) inhibitorS, captopril or enalapril, reStore the diminiShed vaSodilator potency of the endothelium-dependent agoniSt, acetylcholine (ACh), and the endothelium-derived relaxing factor (EDRF), l - S -nitroSocySteine (L-SNC), in conSciouS SpontaneouSly HypertenSive (SH) ratS. MethodS The hemodynamic reSponSeS elicited by i.v. injectionS of ACh, L-SNC, and nitric oxide donorS Such aS MAHMA NONOate, were determined in SH ratS treated for 7 dayS with captopril, enalapril, or the direct vaSodilator hydralazine. The effectS of captopril, enalapril or hydralazine on oxidant StreSS levelS in blood Serum and aorta of WKY and SH ratS were alSo determined. ReSultS Captopril, enalapril and hydralazine elicited equivalent fallS in mean arterial preSSure and SyStemic vaScular reSiStanceS in SH ratS. ACh- and L-SNC-induced vaSodilation were increaSed in captopril- or enalapril-treated SH ratS Such that the reSponSeS were equal to thoSe in normotenSive WiStar Kyoto ratS. The attenuated reSponSeS of ACh and L-SNC in SH ratS were not improved by hydralazine. The vaSodilator effectS of MAHMA NONOate, which were SubStantially augmented in SH ratS, were not affected by captopril, enalapril or hydralazine. The levelS of oxidant StreSS were markedly reduced in captopril- or enalapril-treated but not hydralazine-treated SH ratS. ConcluSionS The finding that the ACE inhibitorS improved the vaSodilator potencieS of L-SNC and the EDRF releaSed by ACh in SH ratS, SuggeStS that the diminiShed vaSodilator potency of theSe compoundS waS due to augmented ACE activity, which increaSed oxidant StreSS levelS. ThiS Study provideS the firSt evidence to Support the concept that ACE inhibition lowerS arterial preSSure in SH ratS, at leaSt in part, by reStoring the vaSodilator potency of endothelium-derived L-SNC.
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S-nitroSocySteine elicitS hemodynamic reSponSeS Similar to thoSe of the Bezold-JariSch reflex via activation of StereoSelective recognition SiteS.
European Journal of Pharmacology, 2006Co-Authors: Stephen J Lewis, Joy R. Owen, James N. BatesAbstract:We report here that L-S-nitroSocySteine elicitS rapid doSe-dependent reductionS in mean arterial blood preSSure, heart rate, cardiac output and renal Sympathetic nerve activity in conSciouS ratS whereaS D-S-nitroSocySteine elicitS minor reSponSeS. The reductionS in mean arterial blood preSSure, heart rate and cardiac output elicited by L- and D-S-nitroSocySteine were markedly diminiShed after blockade of cardiac muScarinic cholinergic receptorS whereaS the reductionS in renal Sympathetic nerve activity were not affected. The Bezold-JariSch reflex-like pattern of reSponSeS elicited by the StereoiSomerS, SuggeStS that (i) L- and L-S-nitroSocySteine may activate StereoSelective recognition SiteS on vagal cardiopulmonary afferentS, which elicit hemodynamic reSponSeS via increaSeS in cardiovagal efferent nerve activity and decreaSeS in Sympathetic nerve activity, and (ii) L-S-nitroSocySteine iS a more potent agoniSt of theSe recognition SiteS than iS D-S-nitroSocySteine.
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vaSodilator actionS of the endothelium derived relaxing factor l S nitroSocySteine in anaeSthetized ratS are markedly diminiShed by peroxynitrite
Clinical and Experimental Pharmacology and Physiology, 2005Co-Authors: Jonathan E Graves, James N. Bates, Neil W Kooy, Stephen J LewisAbstract:Summary 1. The aim of the preSent Study waS to aSSeSS the effectS of the potent oxidant and nitrating agent peroxynitrite on the haemodynamic actionS of the endothelium-derived S-nitroSothiol l-S-nitroSocySteine. 2. The haemodynamic actionS of l-S-nitroSocySteine (12.5–100 nmol/kg, i.v.) were determined in pentobarbital-anaeSthetized ratS before and after the induction of tachyphylaxiS to peroxynitrite achieved by giving 10 intravenouS injectionS of a 10 µmol/kg doSe. 3. l-S-NitroSocySteine elicited doSe-dependent reductionS in mean arterial blood preSSure and in hindquarter and meSenteric vaScular reSiStance. 4. The l-S-nitroSocySteine-induced reSponSeS were SubStantially attenuated after adminiStration of peroxynitrite. 5. We have reported previouSly that nitric oxide-mediated vaSodilation iS not diminiShed after the induction of tachyphylaxiS to peroxynitrite. Taken together, theSe findingS Support the concept that peroxynitrite reduceS the vaSodilator actionS of l-S-nitroSocySteine via oxidation and/or nitration of putative membrane-bound S-nitroSothiol recognition SiteS.
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peroxynitrite elicitS dySfunction of StereoSelective S nitroSocySteine recognition SiteS
Journal of Cardiovascular Pharmacology, 2005Co-Authors: Stephen J Lewis, James N. Bates, Jonathan E Graves, Neil W KooyAbstract:The aim of thiS Study waS to determine whether induction of tachyphylaxiS to peroxynitrite (induced by giving 10 intravenouS injectionS of a 10-micromol/kg doSe) differentially affectS the vaSodilator reSponSeS elicited by SyStemic injectionS of the L- and D-iSomerS of S-nitroSocySteine (L-SNC and D-SNC), in pentobarbital-aneSthetized ratS. L- and D-SNC (12.5-200 nmol/kg, iv) elicited doSe-dependent reductionS in hindquarter, meSenteric, and renal vaScular reSiStanceS. The L-SNC-induced vaSodilator reSponSeS in the hindquarter and renal vaScular bedS were virtually aboliShed whereaS the vaSodilator reSponSeS in meSenteric bed were markedly diminiShed after adminiStration of peroxynitrite. The D-SNC-induced vaSodilator reSponSeS in the hindquarter and renal bedS were Slightly attenuated whereaS the vaSodilator reSponSeS in the meSenteric bed were not diminiShed after adminiStration of peroxynitrite. The vaSodilator reSponSeS elicited by the nitric oxide donor, MAHMA NONOate (5-50 nmol/kg, iv), were not attenuated by peroxynitrite. The finding that induction of tachyphylaxiS to peroxynitrite diminiSheS the effectS of L- and D-SNC but not MAHMA NONOate SuggeStS that the StereoiSomerS exert their vaSodilator effectS by mechaniSmS other than their decompoSition to nitric oxide. Moreover, the finding that induction of tachyphylaxiS to peroxynitrite cauSeS a more pronounced attenuation of the vaSodilator effectS of L- than D-SNC SupportS evidence that the StereoiSomerS differentially interact with StereoSelective S-nitroSothiol recognition SiteS in the vaSculature. Taken together, theSe novel reSultS Support the poSSibility that peroxynitrite diminiSheS the vaSodilator potencieS of L- and D-SNC by oxidation and/or nitration of amino acidS in theSe recognition SiteS.
James N. Bates - One of the best experts on this subject based on the ideXlab platform.
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ace inhibition reStoreS the vaSodilator potency of the endothelium derived relaxing factor l S nitroSocySteine in conSciouS SpontaneouSly hypertenSive ratS
Vascular Pharmacology, 2006Co-Authors: Stephen J Lewis, Joy R. Owen, Maleka P Hashmihill, Kevin Sandock, Tom P Robertson, James N. BatesAbstract:AbStract Objective The major aim of thiS Study waS to determine whether the angiotenSin converting enzyme (ACE) inhibitorS, captopril or enalapril, reStore the diminiShed vaSodilator potency of the endothelium-dependent agoniSt, acetylcholine (ACh), and the endothelium-derived relaxing factor (EDRF), l - S -nitroSocySteine (L-SNC), in conSciouS SpontaneouSly HypertenSive (SH) ratS. MethodS The hemodynamic reSponSeS elicited by i.v. injectionS of ACh, L-SNC, and nitric oxide donorS Such aS MAHMA NONOate, were determined in SH ratS treated for 7 dayS with captopril, enalapril, or the direct vaSodilator hydralazine. The effectS of captopril, enalapril or hydralazine on oxidant StreSS levelS in blood Serum and aorta of WKY and SH ratS were alSo determined. ReSultS Captopril, enalapril and hydralazine elicited equivalent fallS in mean arterial preSSure and SyStemic vaScular reSiStanceS in SH ratS. ACh- and L-SNC-induced vaSodilation were increaSed in captopril- or enalapril-treated SH ratS Such that the reSponSeS were equal to thoSe in normotenSive WiStar Kyoto ratS. The attenuated reSponSeS of ACh and L-SNC in SH ratS were not improved by hydralazine. The vaSodilator effectS of MAHMA NONOate, which were SubStantially augmented in SH ratS, were not affected by captopril, enalapril or hydralazine. The levelS of oxidant StreSS were markedly reduced in captopril- or enalapril-treated but not hydralazine-treated SH ratS. ConcluSionS The finding that the ACE inhibitorS improved the vaSodilator potencieS of L-SNC and the EDRF releaSed by ACh in SH ratS, SuggeStS that the diminiShed vaSodilator potency of theSe compoundS waS due to augmented ACE activity, which increaSed oxidant StreSS levelS. ThiS Study provideS the firSt evidence to Support the concept that ACE inhibition lowerS arterial preSSure in SH ratS, at leaSt in part, by reStoring the vaSodilator potency of endothelium-derived L-SNC.
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the vaSodilator potency of the endothelium derived relaxing factor l S nitroSocySteine iS impaired in conSciouS SpontaneouSly hypertenSive ratS
Vascular Pharmacology, 2006Co-Authors: Stephen J Lewis, Joy R. Owen, Maleka P Hashmihill, Kevin Sandock, Tom P Robertson, James N. BatesAbstract:AbStract Objective ThiS Study compared the hemodynamic reSponSeS elicited by the endothelium-derived relaxing factor (EDRF), l -S-nitroSocySteine ( l -SNC), the non-proStanoid EDRF releaSed by acetylcholine (ACh) and nitric oxide (NO)-donorS Such aS MAHMA NONOate, in conSciouS SpontaneouSly hypertenSive (SH) and normotenSive WiStar–Kyoto (WKY) ratS. MethodS The depreSSor and/or vaSodilator reSponSeS elicited by intravenouS injectionS of ACh, l -SNC and MAHMA NONOate were determined in adult WKY and SH ratS before and after intravenouS injection of the NO SyntheSiS inhibitor, NG-nitro- l -arginine methyleSter ( l -NAME), or the cyclooxygenaSe inhibitor, indomethacin. ReSultS The reSponSeS elicited by ACh and l -SNC were Smaller in SH than in WKY ratS whereaS the reSponSeS elicited by MAHMA NONOate were augmented in SH ratS. The ACh-induced reSponSeS were not diminiShed after injection of l -NAME in WKY or SH ratS. Indomethacin did not affect the reSponSeS to any of the vaSodilator agentS in WKY or SH ratS. Addition of l -SNC to whole blood or thoracic aortae from SH ratS yielded Similar amountS of NO to thoSe of WKY ratS. ConcluSionS The vaSodilator potencieS of ACh and l -SNC were diminiShed whereaS that of NO waS augmented in SH ratS. The loSS of potency of l -SNC in SH ratS waS not obviouSly due to differenceS in decompoSition to NO or the overactivity of cyclooxygenaSe factorS. ThiS Study provideS the firSt evidence that diminiShed endothelium-dependent vaSodilation in SH ratS may involve a loSS of vaSodilator potency of endogenouS l -SNC.
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S-nitroSocySteine elicitS hemodynamic reSponSeS Similar to thoSe of the Bezold-JariSch reflex via activation of StereoSelective recognition SiteS.
European Journal of Pharmacology, 2006Co-Authors: Stephen J Lewis, Joy R. Owen, James N. BatesAbstract:We report here that L-S-nitroSocySteine elicitS rapid doSe-dependent reductionS in mean arterial blood preSSure, heart rate, cardiac output and renal Sympathetic nerve activity in conSciouS ratS whereaS D-S-nitroSocySteine elicitS minor reSponSeS. The reductionS in mean arterial blood preSSure, heart rate and cardiac output elicited by L- and D-S-nitroSocySteine were markedly diminiShed after blockade of cardiac muScarinic cholinergic receptorS whereaS the reductionS in renal Sympathetic nerve activity were not affected. The Bezold-JariSch reflex-like pattern of reSponSeS elicited by the StereoiSomerS, SuggeStS that (i) L- and L-S-nitroSocySteine may activate StereoSelective recognition SiteS on vagal cardiopulmonary afferentS, which elicit hemodynamic reSponSeS via increaSeS in cardiovagal efferent nerve activity and decreaSeS in Sympathetic nerve activity, and (ii) L-S-nitroSocySteine iS a more potent agoniSt of theSe recognition SiteS than iS D-S-nitroSocySteine.
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vaSodilator actionS of the endothelium derived relaxing factor l S nitroSocySteine in anaeSthetized ratS are markedly diminiShed by peroxynitrite
Clinical and Experimental Pharmacology and Physiology, 2005Co-Authors: Jonathan E Graves, James N. Bates, Neil W Kooy, Stephen J LewisAbstract:Summary 1. The aim of the preSent Study waS to aSSeSS the effectS of the potent oxidant and nitrating agent peroxynitrite on the haemodynamic actionS of the endothelium-derived S-nitroSothiol l-S-nitroSocySteine. 2. The haemodynamic actionS of l-S-nitroSocySteine (12.5–100 nmol/kg, i.v.) were determined in pentobarbital-anaeSthetized ratS before and after the induction of tachyphylaxiS to peroxynitrite achieved by giving 10 intravenouS injectionS of a 10 µmol/kg doSe. 3. l-S-NitroSocySteine elicited doSe-dependent reductionS in mean arterial blood preSSure and in hindquarter and meSenteric vaScular reSiStance. 4. The l-S-nitroSocySteine-induced reSponSeS were SubStantially attenuated after adminiStration of peroxynitrite. 5. We have reported previouSly that nitric oxide-mediated vaSodilation iS not diminiShed after the induction of tachyphylaxiS to peroxynitrite. Taken together, theSe findingS Support the concept that peroxynitrite reduceS the vaSodilator actionS of l-S-nitroSocySteine via oxidation and/or nitration of putative membrane-bound S-nitroSothiol recognition SiteS.
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peroxynitrite elicitS dySfunction of StereoSelective S nitroSocySteine recognition SiteS
Journal of Cardiovascular Pharmacology, 2005Co-Authors: Stephen J Lewis, James N. Bates, Jonathan E Graves, Neil W KooyAbstract:The aim of thiS Study waS to determine whether induction of tachyphylaxiS to peroxynitrite (induced by giving 10 intravenouS injectionS of a 10-micromol/kg doSe) differentially affectS the vaSodilator reSponSeS elicited by SyStemic injectionS of the L- and D-iSomerS of S-nitroSocySteine (L-SNC and D-SNC), in pentobarbital-aneSthetized ratS. L- and D-SNC (12.5-200 nmol/kg, iv) elicited doSe-dependent reductionS in hindquarter, meSenteric, and renal vaScular reSiStanceS. The L-SNC-induced vaSodilator reSponSeS in the hindquarter and renal vaScular bedS were virtually aboliShed whereaS the vaSodilator reSponSeS in meSenteric bed were markedly diminiShed after adminiStration of peroxynitrite. The D-SNC-induced vaSodilator reSponSeS in the hindquarter and renal bedS were Slightly attenuated whereaS the vaSodilator reSponSeS in the meSenteric bed were not diminiShed after adminiStration of peroxynitrite. The vaSodilator reSponSeS elicited by the nitric oxide donor, MAHMA NONOate (5-50 nmol/kg, iv), were not attenuated by peroxynitrite. The finding that induction of tachyphylaxiS to peroxynitrite diminiSheS the effectS of L- and D-SNC but not MAHMA NONOate SuggeStS that the StereoiSomerS exert their vaSodilator effectS by mechaniSmS other than their decompoSition to nitric oxide. Moreover, the finding that induction of tachyphylaxiS to peroxynitrite cauSeS a more pronounced attenuation of the vaSodilator effectS of L- than D-SNC SupportS evidence that the StereoiSomerS differentially interact with StereoSelective S-nitroSothiol recognition SiteS in the vaSculature. Taken together, theSe novel reSultS Support the poSSibility that peroxynitrite diminiSheS the vaSodilator potencieS of L- and D-SNC by oxidation and/or nitration of amino acidS in theSe recognition SiteS.
Moran Benhar - One of the best experts on this subject based on the ideXlab platform.
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S nitroSocySteine and glutathione depletion Synergize to induce cell death in human tumor cellS inSightS into the redox and cytotoxic mechaniSmS
Free Radical Biology and Medicine, 2020Co-Authors: Alaa Knany, Rotem Engelman, Hiba Abu Hariri, Shyam Biswal, Haguy Wolfenson, Moran BenharAbstract:AbStract Nitric oxide (NO)-dependent Signaling and cytotoxic effectS are mediated in part via protein S-nitroSylation. The magnitude and duration of S-nitroSylation are governed by the two main thiol reducing SyStemS, the glutathione (GSH) and thioredoxin (Trx) antioxidant SyStemS. In recent yearS, approacheS have been developed to harneSS the cytotoxic potential of NO/nitroSylation to inhibit tumor cell growth. However, progreSS in thiS area haS been hindered by inSufficient underStanding of the balance and interplay between cellular nitroSylation, other oxidative proceSSeS and the GSH/Trx SyStemS. In addition, the mechaniStic relationShip between thiol redox imbalance and cancer cell death iS not fully underStood. Herein, we explored the redox and cellular effectS induced by the S-nitroSylating agent, S-nitroSocySteine (CySNO), in GSH-Sufficient and -deficient human tumor cellS. We uSed l -buthionine-Sulfoximine (BSO) to induce GSH deficiency, and employed redox, biochemical and cellular aSSayS to interrogate molecular mechaniSmS. We found that, under GSH-Sufficient conditionS, a CySNO challenge (100–500 μM) reSultS in a marked yet reverSible increaSe in protein S-nitroSylation in the abSence of appreciable S-oxidation. In contraSt, under GSH-deficient conditionS, CySNO induceS elevated and SuStained levelS of both S-nitroSylation and S-oxidation. ExperimentS in variouS cancer cell lineS Showed that adminiStration of CySNO or BSO alone commonly induce minimal cytotoxicity whereaS BSO/CySNO combination therapy leadS to extenSive cell death. StudieS in HeLa cancer cellS revealed that treatment with BSO/CySNO reSultS in dual inhibition of the GSH and Trx SyStemS, thereby amplifying redox StreSS and cauSing cellular dySfunction. In particular, BSO/CySNO induced rapid oxidation and collapSe of the actin cytoSkeletal network, followed by loSS of mitochondrial function, leading to profound and irreverSible decreaSe in ATP levelS. Further obServationS indicated that BSO/CySNO-induced cell death occurS via a caSpaSe-independent mechaniSm that involveS multiple StreSS-induced pathwayS. The preSent findingS provide new inSightS into the relationShip between cellular nitroSylation/oxidation, thiol antioxidant defenSeS and cell death. TheSe reSultS may aid future effortS to develop NO/redox-baSed anticancer approacheS.
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NitroSothiol-Trapping-BaSed Proteomic AnalySiS of S-NitroSylation in Human Lung Carcinoma CellS.
PLOS ONE, 2017Co-Authors: Shani Ben-lulu, Tamar Ziv, Pnina Weisman-shomer, Moran BenharAbstract:NitroSylation of cySteineS reSidueS (S-nitroSylation) mediateS many of the cellular effectS of nitric oxide in normal and diSeaSed cellS. Recent reSearch indicateS that S-nitroSylation of certain proteinS could play a role in tumor progreSSion and reSponSiveneSS to therapy. However, the protein targetS of S-nitroSylation in cancer cellS remain largely unidentified. In thiS Study, we uSed our recently developed nitroSothiol trapping approach to explore the nitroSoproteome of human A549 lung carcinoma cellS treated with S-nitroSocySteine or pro-inflammatory cytokineS. USing thiS approach, we identified about 300 putative nitroSylation targetS in S-nitroSocySteine-treated A549 cellS and approximately 400 targetS in cytokine-Stimulated cellS. Among the more than 500 proteinS identified in the two ScreenS, the majority repreSent novel targetS of S-nitroSylation, aS revealed by compariSon with publicly available nitroSoproteomic data. By coupling the trapping procedure with differential thiol labeling, we identified nearly 300 potential nitroSylation SiteS in about 150 proteinS. The proteomic reSultS were validated for Several proteinS by an independent approach. Bioinformatic analySiS highlighted important cellular pathwayS that are targeted by S-nitroSylation, notably, cell cycle and inflammatory Signaling. Taken together, our reSultS identify new molecular targetS of nitric oxide in lung cancer cellS and SuggeSt that S-nitroSylation may regulate Signaling pathwayS that are critically involved in lung cancer progreSSion.
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A SubStrate Trapping Approach IdentifieS ProteinS Regulated by ReverSible S-nitroSylation*□S
2016Co-Authors: Shani Ben-lulu, Tamar Ziv, Pnina Weisman-shomer, Arie Admon, Moran BenharAbstract:tranSlational modification of cySteine reSidueS, haS emerged aS an important regulatory mechaniSm in di-verSe cellular proceSSeS. Yet, knowledge about the S-nitroSoproteome in different cell typeS and cellular con-textS iS Still limited and many queStionS remain regarding the preciSe roleS of protein S-nitroSylation and denitroSy-lation. Here we preSent a novel Strategy to identify reverS-ibly nitroSylated proteinS. Our approach iS baSed on nitro-Sothiol capture and enrichment uSing a thioredoxin trap mutant, followed by protein identification by maSS Spec-trometry. Employing thiS approach, we identified more than 400 putative nitroSo-proteinS in S-nitroSocySteine-treated human monocyteS and about 200 nitroSylation SubStrateS in endotoxin and cytokine-Stimulated mouSe macrophageS. The large majority of theSe repreSent nove
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NO inhibitS the adheSion of E. hiStolytica to HeLa cellS.
2014Co-Authors: Rivka Hertz, Moran Benhar, Shani Ben Lulu, Preeti Shahi, Meirav Trebicz-geffen, Serge AnkriAbstract:A. E.hiStolytica trophozoiteS Strain HM-1:IMSS were grown in Diamond'S TYI-S-33 medium and expoSed to 250 µM or 500 µM S-nitroSocySteine (CySNO) for 20 minuteS before their incubation with a fixed HeLa cell monolayer for 30 minuteS. The reSpective value of the control waS taken aS 100%. Data are expreSSed aS the mean and Standard deviation of three independent experimentS that were performed in triplicate. The adheSion of the untreated control and CySNO-treated trophozoiteS waS Significantly different (p
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AnalySiS of S-nitroSylated proteinS in E. hiStolytica after reSin-aSSiSted capture.
2014Co-Authors: Rivka Hertz, Moran Benhar, Shani Ben Lulu, Preeti Shahi, Meirav Trebicz-geffen, Serge AnkriAbstract:A. Viability of E.hiStolytica trophozoiteS which were expoSed to different concentrationS of S-nitroSocySteine (CySNO) for 20 minuteS. Data are expreSSed aS the mean and Standard deviation of three independent experimentS that were repeated twice. E.hiStolytica trophozoiteS Strain HM-1:IMSS were treated with 500 µM CySNO for 20 minuteS. The protein S-nitroSothiolS (SNO) in the cell lySateS waS Subjected to reSin-aSSiSted capture (RAC) in the preSence of 40 mM aScorbate (+ASC) or the abSence of aScorbate (–ASC). B. CoomaSSie blue Staining of S-nitrolySated proteinS. C. Functional categorieS of all S-nitroSylated proteinS. S-nitroSylated proteinS in E.hiStolytica were claSSified according to their biological role. D. Confirmation of S-nitroSylation of three proteinS, enolaSe, glyceraldehyde-3-phoSphate dehydrogenaSe, and the heavy Subunit of Gal/GalNAc lectin after reSin-aSSiSted capture by weStern blotting. ThiS figure diSplayS a repreSentative reSult from two independent experimentS.
Harry Ischiropoulos - One of the best experts on this subject based on the ideXlab platform.
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Site Specific identification of endogenouS S nitroSocySteine proteomeS
Journal of Proteomics, 2013Co-Authors: Paschalis-Thomas Doulias, Margarita Tenopoulou, Karthik Raju, Steve H Seeholzer, Harry Ischiropoulos, Lynn A SpruceAbstract:AbStract CySteine S-nitroSylation iS a poSt-tranSlational modification regulating protein function and nitric oxide Signaling. Herein the Selectivity, reproducibility, and SenSitivity of a maSS Spectrometry-baSed proteomic method for the identification of endogenouS S-nitroSylated proteinS are outlined. The method enricheS for either S-nitroSylated proteinS or peptideS through covalent binding of the cySteine Sulfur with phenylmercury at pH = 6.0. Phenylmercury reactS Selectively and efficiently with S-nitroSocySteine Since no reactivity can be documented for diSulfideS, Sulfinic or Sulfonic acidS, S-glutathionylated, S-alkylated or S-Sulfhydrylated cySteine reSidueS. A Specificity of 97 ± 1% for the identification of S-nitroSocySteine peptideS in mouSe liver tiSSue iS achieved by the incluSion of negative controlS. The method enableS the detection of 36 S-nitroSocySteine peptideS Starting with 5 pmol S-nitroSocySteine/mg of total tiSSue protein. Both the percentage of protein moleculeS modified aS well aS the occupancy by S-nitroSylation can be determined. Overall, Selective, SenSitive and reproducible enrichment of S-nitroSylated proteinS and peptideS iS achieved by the uSe of phenylmercury. The incluSion of appropriate negative controlS SecureS the preciSe identification of endogenouS S-nitroSylated SiteS and proteinS in biological SampleS. Biological Significance The current Study deScribeS a Selective, SenSitive and reproducible method for the acquiSition of endogenouSly S-nitroSylated proteinS and peptideS. The acquiSition of endogenouS S-nitroSoproteomeS provideS robuSt data that iS neceSSary for inveStigating the mechaniSm(S) of S-nitroSylation in vivo, the factorS that govern itS Selectivity, the dependency of the modification on different iSoformS of nitric oxide SynthaSeS (NOS), aS well aS the phySiological functionS of thiS protein modification. ThiS article iS part of a Special ISSue entitled: PoSttranSlational Protein modificationS in biology and Medicine.
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regulation of protein function and Signaling by reverSible cySteine S nitroSylation
Journal of Biological Chemistry, 2013Co-Authors: Neal S Gould, Paschalis-Thomas Doulias, Margarita Tenopoulou, Karthik Raju, Harry IschiropoulosAbstract:NO iS a verSatile free radical that mediateS numerouS biological functionS within every major organ SyStem. A molecular pathway by which NO accompliSheS functional diverSity iS the Selective modification of protein cySteine reSidueS to form S-nitroSocySteine. ThiS poSt-tranSlational modification, S-nitroSylation, impactS protein function, Stability, and location. DeSpite conSiderable advanceS with individual proteinS, the in vivo biological chemiStry, the Structural elementS that govern the Selective S-nitroSylation of cySteine reSidueS, and the potential overlap with other redox modificationS are unknown. In thiS minireview, we explore the functional featureS of S-nitroSylation at the proteome level and the Structural diverSity of endogenouSly modified reSidueS, and we diScuSS the potential overlap and complementation that may exiSt with other cySteine modificationS.
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maSS Spectrometry baSed identification of S nitroSocySteine in vivo uSing organic mercury aSSiSted enrichment
Methods, 2013Co-Authors: Paschalis-Thomas Doulias, Margarita Tenopoulou, Jennifer L Greene, Karthik Raju, Harry IschiropoulosAbstract:Protein S-nitroSylation iS conSidered aS one of the molecular mechaniSmS by which nitric oxide regulateS Signaling eventS and protein function. The preSent review preSentS an updated method which allowS for the Site-Specific detection of S-nitroSylated proteinS in vivo. The method iS baSed on enrichment of S-nitroSylated proteinS or peptideS uSing organomercury compoundS followed by LC–MS/MS detection. Technical aSpectS for determining the reaction and binding efficiency of the mercury reSin that aSSiStS enrichment of S-nitroSylated proteinS are preSented and diScuSSed. In addition, emphaSiS iS given to the Specificity of the method by providing technical detailS for the generation of four chemically diStinct negative controlS. Finally it iS provided an overview of the key StepS for generation and evaluation of maSS Spectrometry derived data.
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functional and Structural analySiS of the mouSe S nitroSocySteine proteome
Nitric Oxide, 2012Co-Authors: Harry IschiropoulosAbstract:CySteine S-nitroSation iS a reverSible poSttranSlational modification by which nitric oxide regulateS protein function and Signaling. While StudieS of individual proteinS have elucidated Specific functional roleS for S-nitroSylation, our knowledge of in vivo S-nitroSylation SiteS and the global appreciation of S-nitroSylation utility remainS limited. We uSed maSS Spectrometry baSed methodologieS to identify 787 S-nitroSocySteine reSidueS on 468 mouSe proteinS in vivo . Selective S-nitroSation of enzymeS participating in glycolySiS, gluconeogeneSiS, pyruvate metaboliSm, TCA cycle and oxidative phoSphorylation indicateS a prominent functional regulation in mitochondrial bioenergeticS and metaboliSm. In the liver, a novel regulation of very long acyl-CoA dehydrogenaSe (VLCAD) by endothelial nitric oxide SynthaSe-dependent, Site Specific S-nitroSation, waS documented. The modification of VLCAD at cySteine238 lowered the km and effectively reverSed hepatic SteatoSiS in leptin deficient mice. TheSe data implicate protein S-nitroSylation in the regulation of mitochondrial β -oxidation of fatty acidS and aS a potential mediator of nonalcoholic fatty liver diSeaSe.
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identification of S nitroSylation motifS by Site Specific mapping of the S nitroSocySteine proteome in human vaScular Smooth muScle cellS
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Todd M Greco, Roberto Hodara, Ioannis Parastatidis, Harry F G Heijnen, Michelle K Dennehy, D C Liebler, Harry IschiropoulosAbstract:S-nitroSylation, the Selective modification of cySteine reSidueS in proteinS to form S-nitroSocySteine, iS a major emerging mechaniSm by which nitric oxide actS aS a Signaling molecule. Even though nitric oxide iS intimately involved in the regulation of vaScular Smooth muScle cell functionS, the potential protein targetS for nitric oxide modification aS well aS Structural featureS that underlie the Specificity of protein S-nitroSocySteine formation in theSe cellS remain unknown. Therefore, we uSed a proteomic approach uSing Selective peptide capturing and Site-Specific adduct mapping to identify the targetS of S-nitroSylation in human aortic Smooth muScle cellS upon expoSure to S-nitroSocySteine and propylamine propylamine NONOate. ThiS Strategy identified 20 unique S-nitroSocySteine-containing peptideS belonging to 18 proteinS including cytoSkeletal proteinS, chaperoneS, proteinS of the tranSlational machinery, veSicular tranSport, and Signaling. Sequence analySiS of the S-nitroSocySteine-containing peptideS revealed the preSence of acid/baSe motifS, aS well aS hydrophobic motifS Surrounding the identified cySteine reSidueS. High-reSolution immunogold electron microScopy Supported the cellular localization of Several of theSe proteinS. IntereStingly, Seven of the 18 proteinS identified are localized within the ER/Golgi complex, SuggeSting a role for S-nitroSylation in membrane trafficking and ER StreSS reSponSe in vaScular Smooth muScle.
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S-NitroSocySteine Inhibition of Human Platelet Secretion IS Correlated With IncreaSeS in Platelet cGMP LevelS
2015Co-Authors: E H Lieberman, M E MendelsohnAbstract:platelet cGMP levelS. S-nitroSocySteine inhibition of human platelet Secretion iS correlated with increaSeS i
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S nitroSocySteine inhibition of human platelet Secretion iS correlated with increaSeS in platelet cgmp levelS
Circulation Research, 1991Co-Authors: E H Lieberman, Sarah Oneill, M E MendelsohnAbstract:Platelet inhibition by exogenouS and endogenouS nitrovaSodilatorS haS been Shown to be aSSociated with increaSeS in cGMP, but proof of a role for cGMP in thiS proceSS iS lacking. We therefore Studied the effectS of cGMP and guanylate cyclaSe Stimulation on human platelet Secretion by pharmacologically modulating intraplatelet cGMP levelS. The endothelium-derived relaxing factor (EDRF)-like activator of guanylate cyclaSe, S-nitroSocySteine (SNOC), led to a doSe-dependent inhibition of Secretion in intact human plateletS (IC50 = 10(-6) M). The cGMP phoSphodieSteraSe inhibitor M&B 22,948 augmented SNOC-induced inhibition of Secretion through elevationS in cGMP without affecting cAMP levelS (from 50% to 81% inhibition verSuS control, p = 0.02). Methylene blue reverSed the inhibitory effectS of SNOC on platelet Secretion (p = 0.03). Dibutyryl-cGMP and 8-bromo-cGMP alSo Significantly inhibited Secretion in thiS SyStem. Incubation of plateletS with exogenouS cGMP to achieve intraplatelet cGMP levelS comparable to thoSe after SNOC treatment reSulted in Similar degreeS of inhibition of Secretion (32% inhibition verSuS control, p = 0.01) and waS alSo potentiated by M&B 22,948 (from 32% to 68% inhibition, p = 0.003). In addition, a highly Significant correlation between intraplatelet cGMP levelS and the degree of inhibition of Secretion waS demonStrable in theSe StudieS (r = 0.94, p = 0.016). TheSe data demonStrate that elevation of intraplatelet cGMP levelS by the EDRF-like compound SNOC iS correlated with inhibition of human platelet Secretion.
