RESUMO
IFN alfacon-1 (Infergen) is a synthetic form of Interferon (IFN)-α2b. Infergen has immunomodulatory activity and is effective against hepatitis C virus. However, the effect of Infergen (IFG) on Mycobacterium tuberculosis (Mtb) has not yet been reported. Therefore, for the first time, we have studied the influence of IFG in constraining the survival of Mtb in human macrophages. We observed that IFG significantly enhanced the maturation and activation of macrophages. Further, it substantially augmented the secretion of IL-6, nitric oxide (NO) and antigen uptake. Moreover, macrophages exhibited remarkably higher bactericidal activity, as evidenced by reduction in the Mtb growth. Infergen-mediated mechanism was different from the type-1 interferons; since it worked through the activation of NF-κB, phosphorylation of STAT-3 and Akt-PI3K that improved the bactericidal activity through autophagy and NO release. In future, IFG immunotherapy can be a novel strategy for treating patients and controlling TB.
Assuntos
Autofagia , Interferon-alfa/farmacologia , Macrófagos/citologia , Óxido Nítrico/metabolismo , Tuberculose/imunologia , Tuberculose/terapia , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Linhagem Celular , Proliferação de Células , Citocinas/metabolismo , Antígenos HLA-DR/metabolismo , Humanos , Interleucina-6/metabolismo , Leucócitos Mononucleares/metabolismo , Microscopia Confocal , Mycobacterium tuberculosis , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação , Proteínas Recombinantes/farmacologia , Linfócitos T/citologia , Células THP-1 , ômega-N-Metilarginina/químicaRESUMO
Arginine methyltransferases (RMTs) catalyze the methylation of arginine residues on proteins. We examined the effects of log-phase growth, stationary-phase growth, and heat shock on the formation of methylarginines on yeast proteins to determine if the conditions favor a particular type of methylation. Utilizing linear ion trap mass spectrometry, we identify methylarginines in wild-type and RMT deletion yeast strains using secondary product ion scans (MS(3)), and quantify the methylarginines using multiple reaction monitoring (MRM). Employing MS(3) and isotopic incorporation, we demonstrate for the first time that Nη1, Nη2-dimethylarginine (sDMA) is present on yeast proteins, and make a detailed structural determination of the fragment ions from the spectra. Nη-monomethylarginine (ηMMA), Nδ-monomethylarginine (δMMA), Nη1, Nη1-dimethylarginine (aDMA), and sDMA were detected in RMT deletion yeast using MS(3) and MRM with and without isotopic incorporation, suggesting that additional RMT enzymes remain to be discovered in yeast. The concentrations of ηMMA and δMMA decreased by half during heat shock and stationary phase compared to log-phase growth of wild-type yeast, whereas sDMA increased by as much as sevenfold and aDMA decreased by 11-fold. Therefore, upon entering stressful conditions like heat shock or stationary-phase growth, there is a net increase in sDMA and decreases in aDMA, ηMMA, and δMMA on yeast proteins.
Assuntos
Arginina/química , Resposta ao Choque Térmico , Proteína-Arginina N-Metiltransferases/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Arginina/análogos & derivados , Deleção de Genes , Temperatura Alta , Espectrometria de Massas , Metilação , ômega-N-Metilarginina/químicaRESUMO
Nitric oxide synthase (NOS) isoforms are hemoenzymes that are only active as homodimers. We have examined the effect of the substrate-analogue inhibitors, N(G)-monomethyl-L-arginine (L-NMA), N(G)-nitro-L-arginine (L-NNA), N(G)-nitro-L-arginine methyl ester (L-NAME), N(5)-(1-iminoethyl)-L-ornithine (L-NIO), and N(6)-(1-iminoethyl)-L-lysine (L-NIL), the guanidine-containing inhibitor aminoguanidine (AG), and the amidine moiety-containing iNOS-specific inhibitor 1400W, on the formation of NOS dimer. Of these inhibitors, L-NMA effectively not only inhibited iNOS dimerization, but also destabilized its dimeric form in RAW264.7 cells stimulated with lipopolysaccharide plus interferon-γ, but not eNOS dimerization in endothelial cells. Importantly, this inhibition was highly correlated with NO production. These inhibitory effects were significantly reversed by addition of L-arginine. However, L-NNA, L-NAME, and AG in part or significantly increased dimerization of iNOS and eNOS in intact cells, and the other inhibitors assessed did not alter dimerization of iNOS and eNOS. These data taken together suggest that substituted groups of an arginine guanidino moiety play an important role in NOS dimerization as well as its catalytic activity. Our results indicate that l-NMA can inhibit iNOS-dependent NO production by preventing iNOS dimerization and destabilizing its dimeric form.
Assuntos
Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , ômega-N-Metilarginina/farmacologia , Animais , Linhagem Celular , Inibidores Enzimáticos/química , Guanidinas/química , Guanidinas/farmacologia , Lisina/análogos & derivados , Lisina/química , Lisina/farmacologia , Camundongos , NG-Nitroarginina Metil Éster/química , Nitroarginina/química , Nitroarginina/farmacologia , Ornitina/análogos & derivados , Ornitina/química , Ornitina/farmacologia , Multimerização Proteica/efeitos dos fármacos , Especificidade por Substrato , ômega-N-Metilarginina/químicaRESUMO
Infusion of NG-monomethyl-L-arginine (L-NMMA; 6.4 µmol/min) into hand veins can cause a 20% increase in vein size in specific subjects. This study explored potential underlying mechanisms in healthy male participants. Ten healthy male participants received in phenylephrine (PE)-preconstricted veins a dose-response curve (DRC) to L-NMMA (0.2-6.4 µmol/min) without and with coinfusion of the endothelium-dependent dilator histamine, a DRC to L-arginine with and without coinfusion of L-NMMA, a DRC to NG-monomethyl-D-arginine (D-NMMA), and a DRC to L-NMMA in prostaglandin F(2α)-(PGF(2α))-preconstricted veins. Participants were classified as L-NMMA responders (R) and nonresponders (NR). Infusion of L-NMMA resulted in a maximum venodilation of 38% ± 11% (R) versus 10% ± 5% (NR; P = .005). In PGF(2α)-preconstricted veins, L-NMMA caused venodilation to 26% ± 34% (NS) in responders. Results suggest that endothelial nitric oxide synthase-mediated formation of nitric oxide (NO) from L-NMMA in doses >3.2 µmol/min and continuous PE-induced α-adrenergic stimulation resulting in release of very small amounts of NO from L-NMMA contribute to the observed L-NMMA-induced increase in vein size. Venous reactivity to L-NMMA resulting in a phenotype as R or NR is most likely genetically predetermined, which requires further study.
Assuntos
Mãos/irrigação sanguínea , Óxido Nítrico/antagonistas & inibidores , Vasodilatadores/farmacologia , Veias/efeitos dos fármacos , ômega-N-Metilarginina/farmacologia , Adulto , Arginina/metabolismo , Autacoides/metabolismo , Estudos Cross-Over , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Infusões Intravenosas , Masculino , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Estereoisomerismo , Vasoconstritores/antagonistas & inibidores , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/administração & dosagem , Vasodilatadores/química , Veias/metabolismo , Adulto Jovem , ômega-N-Metilarginina/administração & dosagem , ômega-N-Metilarginina/químicaRESUMO
Arginine analogs were incorporated site-specifically into proteins using an in vitro translation system. In this system, mRNAs containing a CGGG codon were translated by an aminoacyl-tRNA(CCCG), which was charged with arginine analogs using yeast arginyl-tRNA synthetase. N(G)-monomethyl-L-arginine, L-citrulline and L-homoarginine were incorporated successfully into proteins using this method. The influence of arginine monomethylation in histone H3 on the acetylation of lysine residues by histone acetyltransferase hGCN5 was investigated, and the results demonstrated that K9 acetylation was suppressed by the methylation of R8 and R17 but not by R26 methylation. K18 acetylation was not affected by the methylation of R8, R17 and R26. This site-specific modification strategy provides a way to explore the roles of post-translational modifications in the absence of heterogeneity due to other modifications.
Assuntos
Arginina-tRNA Ligase/química , Arginina/análogos & derivados , Biossíntese de Proteínas , Sequência de Aminoácidos , Aminoacilação , Arginina/genética , Catálise , Citrulina/química , Citrulina/genética , Homoarginina/química , Homoarginina/genética , Metilação , Dados de Sequência Molecular , ômega-N-Metilarginina/química , ômega-N-Metilarginina/genéticaRESUMO
A CE ion trap tandem MS method was optimised for the analysis of arginine, monomethyl- and (symmetric and asymmetric) dimethylarginines in human plasma after a very reduced sample pretreatment step involving a simple protein precipitation with ACN. Several parameters affecting the analytes MS ionization and the capillary electrophoretic separation were carefully studied and optimised. The complete separation of arginine, monomethylarginine and symmetric and asymmetric dimethylarginine was obtained in formic acid BGE in short analysis time with high specificity due to MS(2) detection of specific analytes fragments. In order to achieve the detection sensitivity suitable for the analysis of asymmetric and symmetric dimethylarginine in human plasma, the field amplified sample injection was applied. Due to stacking effects, this methodology allowed to operate a consistent on-line preconcentration of the analytes before running the electrophoresis. The method was validated for linearity, repeatability, recovery and accuracy and applied to the quantitative analysis of arginine, monomethyl- and dimethylarginines in human plasma of healthy subjects.
Assuntos
Acetonitrilas/sangue , Arginina/análogos & derivados , Arginina/sangue , Eletroforese Capilar/métodos , Análise de Injeção de Fluxo/métodos , Espectrometria de Massas em Tandem/métodos , ômega-N-Metilarginina/sangue , Acetonitrilas/química , Arginina/química , Humanos , Modelos Lineares , Metilação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , ômega-N-Metilarginina/químicaAssuntos
Heme/química , Mioglobina/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Animais , Citoproteção , Humanos , Hipóxia/metabolismo , Raios Infravermelhos , Modelos Biológicos , Óxido Nítrico Sintase/metabolismo , Nitrogênio/química , Oxigênio/química , ômega-N-Metilarginina/químicaRESUMO
Photobiomodulation with near infrared light (NIR) provides cellular protection in various disease models. Previously, infrared light emitted by a low-energy laser has been shown to significantly improve recovery from ischemic injury of the canine heart. The goal of this investigation was to test the hypothesis that NIR (670 nm) from light emitting diodes produces cellular protection against hypoxia and reoxygenation-induced cardiomyocyte injury. Additionally, nitric oxide (NO) was investigated as a potential cellular mediator of NIR. Our results demonstrate that exposure to NIR at the time of reoxygenation protects neonatal rat cardiomyocytes and HL-1 cells from injury, as assessed by lactate dehydrogenase release and MTT assay. Similarly, indices of apoptosis, including caspase 3 activity, annexin binding and the release of cytochrome c from mitochondria into the cytosol, were decreased after NIR treatment. NIR increased NO in cardiomyocytes, and the protective effect of NIR was completely reversed by the NO scavengers carboxy-PTIO and oxyhemoglobin, but only partially blocked by the NO synthase (NOS) inhibitor L-NMMA. Mitochondrial metabolism, measured by ATP synthase activity, was increased by NIR, and NO-induced inhibition of oxygen consumption with substrates for complex I or complex IV was reversed by exposure to NIR. Taken together these data provide evidence for protection against hypoxia and reoxygenation injury in cardiomyocytes by NIR in a manner that is dependent upon NO derived from NOS and non-NOS sources.
Assuntos
Hipóxia , Raios Infravermelhos , Miócitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Oxigênio/química , Animais , Apoptose , Caspase 3/metabolismo , Citosol/metabolismo , Radicais Livres/metabolismo , Luz , Mitocôndrias/metabolismo , Miócitos Cardíacos/patologia , Oxiemoglobinas/química , Ratos , Ratos Sprague-Dawley , ômega-N-Metilarginina/químicaRESUMO
The suppressor activity of splenic macrophages induced by Mycobacterium intracellulare infection (MI-M phi s) against T cell concanavalin A (Con A) mitogenesis is mediated by MI-M phi's mediators, such as reactive nitrogen intermediates (RNIs), phosphatidylserine, free fatty acids, prostaglandin E(2) and to a minor extent TGF-beta. Here, we have compared the roles of RNIs and TGF-beta in the expression of MI-M phi's suppressor activity against Con A mitogenesis and anti-CD3 monoclonal antibody (mAb)- and anti-CD28 mAb-induced mitogenesis (TCR signal-induced mitogenesis) of the target T cells, and have found the following. First, N(G)-monomethyl-L-arginine (NMMA) inhibited MI-M phi's suppressor activity against TCR signal-induced mitogenesis as well as Con A mitogenesis. Second, anti-TGF-beta mAb weakly restored the MI-M phi-mediated suppression only in the case of Con A mitogenesis, under limited conditions, such as very low cell densities of MI-M phi s. Third, the blocking effects of NMMA plus anti-TGF-beta mAb were somewhat more prominent in the case of Con A mitogenesis than in the case of TCR signal-induced mitogenesis. Fourth, Con A- or TCR signal-stimulated MI-M phi s secreted significant amounts of the latent TGF-beta but not the active one. These findings indicate that RNIs, but not TGF-beta, play important roles in the MI-M phi-mediated suppression of TCR signal-induced mitogenesis, as well as Con A mitogenesis, of the target T cells.
Assuntos
Ativação de Macrófagos , Macrófagos/imunologia , Receptores de Antígenos de Linfócitos T/química , Linfócitos T/citologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Anticorpos Monoclonais/química , Apresentação de Antígeno , Antígenos CD28/biossíntese , Complexo CD3/biossíntese , Complexo CD3/química , Concanavalina A/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Tolerância Imunológica , Imunossupressores/farmacologia , Ativação Linfocitária , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Complexo Mycobacterium avium/metabolismo , Infecção por Mycobacterium avium-intracellulare/metabolismo , Nitrogênio/metabolismo , Espécies Reativas de Nitrogênio , Transdução de Sinais , Baço/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , ômega-N-Metilarginina/químicaRESUMO
The endogenous methylarginines asymmetric dimethylarginine (ADMA) and N(G)-monomethyl-L-arginine (L-NMMA) regulate nitric oxide (NO) production from neuronal NO synthase (nNOS). Under conditions of L-arginine or tetrahydrobiopterin (BH(4)) depletion, nNOS also generates superoxide, O(2)(.); however, the effects of methylarginines on this O(2)(.) generation are poorly understood. Therefore, we measured the dose-dependent effects of ADMA and L-NMMA on the rate and amount of O(2)(.) production from nNOS under conditions of L-arginine and/or BH(4) depletion, using electron paramagnetic resonance spin trapping. In the absence of L-arginine, ADMA (1 microm) inhibited O(2)(.) generation by approximately 60% from a rate of 56 to 23 nmol/mg/min, whereas L-NMMA (0.1-100 microm) had no effect. L-Arginine markedly decreased the observed O(2)(.) adduct formation; however, O(2)(.) generation from the enzyme still occurs at a low rate (12.1 nmol/mg/min). This O(2)(.) leak is NOS-derived as it is not seen in the absence of calcium and calmodulin and demonstrates that O(2)(.) generation from NOS occurs even when normal substrate/ cofactor levels are present. Under conditions of BH(4) depletion, ADMA had no effect on O(2)(.), whereas L-NMMA increased O(2)(.) production almost 3-fold. This O(2)(.) generation was >90% inhibited by imidazole, indicating that it occurred at the heme center. Thus, methylarginines can profoundly shift the balance of NO and O(2)(.) generation from nNOS. These observations have important implications with regard to the therapeutic use of methylarginine-NOS inhibitors in the treatment of disease.
Assuntos
Arginina/análogos & derivados , Biopterinas/análogos & derivados , Óxido Nítrico Sintase/fisiologia , Superóxidos/química , ômega-N-Metilarginina/química , Animais , Arginina/química , Biopterinas/química , Cálcio/química , Calmodulina/química , Linhagem Celular , Citrulina/química , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Magnetismo , Óxido Nítrico Sintase/metabolismo , Oxigênio/química , Oxigênio/metabolismo , Ratos , Detecção de Spin , Fatores de Tempo , TransfecçãoAssuntos
Amidoidrolases/química , Espectroscopia de Ressonância Magnética/métodos , Pseudomonas aeruginosa/enzimologia , Isótopos de Carbono , Cromatografia , Hidrogênio , Ligantes , Mutação , Isótopos de Nitrogênio , Conformação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Prótons , Espectrofotometria , Temperatura , ômega-N-Metilarginina/químicaRESUMO
The measurement of nitric oxide (NO) in biological samples has normally required destructive chemical techniques. The ability to detect NO non-invasively in living animals or excised organs has great potential using specialized electron paramagnetic resonance (EPR) methods. Although NO is paramagnetic, it cannot be observed directly unless it is complexed with ferrous iron-dithiocarbamate ligand spin trap complexes. Despite the minimally invasive nature of the technique, highly sensitive localized concentrations of NO may be observed ("trapped") in vivo by both L-band EPR and magnetic resonance imaging.
Assuntos
Compostos Ferrosos/química , Óxido Nítrico/química , Detecção de Spin/métodos , Tiocarbamatos/química , Animais , Encéfalo/patologia , Espectroscopia de Ressonância de Spin Eletrônica , Lipopolissacarídeos/química , Fígado/metabolismo , Magnetismo , Modelos Químicos , Ratos , Ratos Wistar , Marcadores de Spin , Fatores de Tempo , ômega-N-Metilarginina/químicaRESUMO
This study investigated the roles of endogenous nitric oxide synthase (NOS) inhibitors and endothelin-1 (ET-1) for regulating myometrial contractions during gestation in the rat. Basal and stimulated cyclic GMP production with L-arginine as a NOS substrate or sodium nitroprusside (SNP) as a NO donor were significantly enhanced at the middle of gestation (14th day), while these were greatly decreased at term (22nd day), suggesting the accelerated NO production and/or up-regulation of guanylate cyclase at the middle of gestation. NOS within the myometrium was mainly Ca(2+)dependent and partly Ca(2 + )independent and remained unaffected by aminoguanidine as an inhibitor of inducible NOS in non-pregnant and gestational myometrium. NOS activity per se and endothelial NOS (eNOS) protein expression remained unchanged at the middle and term gestation. Neuronal NOS (nNOS) and inducible NOS (iNOS) proteins were undetectable. SNP at a high concentration of 100 micromol/l failed to modify the spontaneous and ET-1-induced rhythmic contractions in non-pregnant and gestational myometrium. Contents of N(G)-monomethyl-L-arginine (L-NMMA) plus asymmetric N(G),N(G)-dimethyl-L-arginine (ADMA) as endogenous NOS inhibitors and ET-1 within the myometrium were significantly decreased at 14th and 20th days of gestation, whereas these were significantly increased at term gestation (22nd day) and after delivery. There was a significant and positive correlation between endogenous NOS inhibitor content and ET-1. ET-1 within the myometrium was significantly increased with a concomitant decrease in cyclic GMP production after the intraperitoneal application of authentic L-NMMA for 2 weeks, suggesting that the impaired NO production with endogenous NOS inhibitors would result in increased ET-1 content. These results suggest that endogenous NOS inhibitors such as L-NMMA and ADMA play an important role for regulating NO production in rat myometrium. The impaired NO production due to accumulated endogenous NOS inhibitors possibly results in increased ET-1 content within the myometrium, thereby increasing myometrial contractions at term gestation and after delivery.
Assuntos
Endotelina-1/metabolismo , Isoenzimas/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Prenhez , Gravidez/fisiologia , Contração Uterina/fisiologia , Animais , Arginina/análogos & derivados , Arginina/metabolismo , GMP Cíclico/metabolismo , Inibidores Enzimáticos/metabolismo , Feminino , Isoenzimas/antagonistas & inibidores , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Nitroprussiato/metabolismo , Ratos , Ratos Sprague-Dawley , ômega-N-Metilarginina/química , ômega-N-Metilarginina/metabolismoRESUMO
The PLP-dependent, biosynthetic arginine decarboxylase (ADC) of Yersinia pestis was investigated using steady-state kinetics employing structural analogues of arginine as both alternative substrates and competitive inhibitors. The inhibitor analysis indicates that binding of the carboxyl and guanidinium groups of the substrate, l-arginine, provides essentially all of the free energy change realized upon substrate binding in the ground state. Furthermore, recognition of the guanidinium group is primarily responsible for substrate specificity. Comparison of the steady-state parameters for a series of alternative substrates that contained chemically modified guanidinium moieties provides evidence of a role for induced fit in ADC catalysis. ADC was also characterized by UV/vis and fluorescence spectrophotometry in the presence or absence of a number of arginine analogues. The enzyme complexes formed served as models for the adsorption complex and the external aldimine complex of the enzyme with the substrate.
Assuntos
Arginina/análogos & derivados , Arginina/química , Carboxiliases/antagonistas & inibidores , Carboxiliases/biossíntese , Inibidores Enzimáticos/química , Yersinia pestis/enzimologia , Sequência de Aminoácidos , Canavanina/química , Carboxiliases/química , Catálise , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Especificidade por Substrato , ômega-N-Metilarginina/químicaRESUMO
In dorsal root ganglia (DRG) intraganglionic communication takes place both among neurons and between neurons and satellite cells. One diffusible substance involved in this signalling is nitric oxide (NO), and acetylcholine (ACh) is a candidate for the stimulation of intraganglionic NO synthesis. DRG neurons react to ACh-receptor stimulation with NO-dependent cGMP production. Here, we investigated the role of the alpha 7-subunit containing Ca(2+)-permeable nicotinic ACh receptors (nAChR) in this process. The alpha 7-nAChR mRNA and the protein were expressed in virtually all lumbar DRG neurons as evidenced by laser-assisted cell picking and oligo cell RT-PCR, in situ hybridisation and immunohistochemistry. Strong alpha 7-nAChR immunoreactivity was present in vanilloid receptor 1-immunoreactive, i.e. nociceptive, neurons. A neuronal production of NO in response to nicotine could be demonstrated in DRG slice preparations utilising the NO-sensitive fluorescent indicator diaminofluorescein diacetate (DAF-2DA). This stimulation of NO production was sensitive to inhibition of alpha 7-nAChR by mecamylamine and alpha-bungarotoxin, to inhibition of nitric oxide synthase (NOS) with L-NAME and L-NMMA, and to the blockade of voltage-operated Ca(2+) channels by verapamil. The results show the presence of the alpha 7-nAChR subunit in nociceptive rat DRG neurons and provide evidence for its coupling to NOS activation, indicating a role of this pathway in the intraganglionic communication in sensory ganglia.
Assuntos
Gânglios Espinais/metabolismo , Neurônios/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Bungarotoxinas/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Comunicação Celular , GMP Cíclico/metabolismo , Mecamilamina/farmacologia , NG-Nitroarginina Metil Éster/química , Antagonistas Nicotínicos/farmacologia , Ratos , Receptores de Droga/metabolismo , Canais de Cátion TRPV , Verapamil/farmacologia , Receptor Nicotínico de Acetilcolina alfa7 , ômega-N-Metilarginina/químicaRESUMO
Posttranslational modification of proteins allows cells to adapt and react quickly to their environment beyond the boundaries set forth by genetic code. Arginine methylation, a protein modification discovered almost 30 years ago, has recently experienced a renewed interest as several new arginine methyltransferases have been identified and numerous proteins were found to be regulated by methylation on arginine residues. Until recently, the detection of arginine methylation required the use of chromatography and mass-spectrometrical analysis. The following protocol provides guidelines for the straightforward identification of arginine-methylated proteins, made possible by the availability of novel, commercially available reagents.
Assuntos
Arginina/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Arginina/análogos & derivados , Arginina/química , Proteínas de Ligação a DNA/química , Ativação Enzimática , Humanos , Immunoblotting , Metilação , Camundongos , Testes de Precipitina , Processamento de Proteína Pós-Traducional , Proteínas/química , Fator de Transcrição STAT1 , Transdução de Sinais , Transativadores/química , Células Tumorais Cultivadas , ômega-N-Metilarginina/química , ômega-N-Metilarginina/metabolismoRESUMO
A series of bivalent thrombin inhibitors was synthesized, consisting of a d-phenylalanyl-prolyl-N(alpha)(methyl)arginyl active site blocking segment, a fibrinogen recognition exosite inhibitor part, and a peptidic linker connecting these fragments. The methylation of the P1 amino acid led to a moderate decrease in affinity compared with the unmethylated analog. In addition, it prevented the thrombin catalyzed proteolysis, independent of the P1' amino acid used. This is a significant advantage compared to the original hirulogs, which strictly require a proline as P1' amino acid to reduce the cleavage C-terminal to the arginyl residue. Several analogs were prepared by incorporation of different P1' amino acids found in natural thrombin substrates. The most potent inhibitor was I-11 [dCha-Pro-N(Me)Arg-Thr-(Gly)5-DYEPIPEEA-Cha-dGlu] with a Ki of 37 pM. I-11 is highly selective and no inhibition of the related serine proteases trypsin, factor Xa and plasmin was observed. The stability of I-11 in human plasma in vitro was strongly improved compared to hirulog-1. In addition, a significantly reduced plasma clearance of I-11 was observed after intravenous injection in rats. Results from molecular modeling suggest that a strong reorganization of the hydrogen bonds in the active site of thrombin may result in the proteolytic stability found in this inhibitor series.
Assuntos
Antitrombinas/química , ômega-N-Metilarginina/química , Sequência de Aminoácidos , Humanos , Dados de Sequência MolecularRESUMO
OBJECTIVE: To measure concentrations of nitric oxide metabolites (nitrite-nitrate [NOt]) in cartilage, synovial membrane, and cranial cruciate ligament (CCL) in dogs and evaluate associations with osteoarthritis in dogs with CCL rupture. ANIMALS: 46 dogs with CCL rupture and 54 control dogs without joint disease. PROCEDURE: Tissue specimens for histologic examination and explant culture were harvested during surgery in the CCL group or immediately after euthanasia in the control group; NOt concentrations were measured in supernatant of explant cultures and compared among dogs with various degrees of osteoarthritis and between dogs with and without CCL rupture. RESULTS: Osteoarthritic cartilage had significantly higher NOt concentration (1,171.6 nmol/g) than did healthy cartilage (491.0 nmol/g); NOt concentration was associated with severity of macroscopic and microscopic lesions. Synovial membrane NOt concentration did not differ between dogs with and without CCL rupture. Ruptured CCL produced less NOt than did intact ligaments. In control dogs, NOt concentrations were similar for intact ligaments (568.1 nmol/g) and articular cartilage (491.0 nmol/g). Synthesis of NOt was inhibited substantially by coincubation with inhibitors. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that NOt in canine joint tissues originates from the inducible nitric oxide synthase pathway. Nitric oxide metabolite production in cartilage was greater in dogs with osteoarthritis than in healthy dogs and was associated with lesion severity, suggesting that nitric oxide inhibitors may be considered as a treatment for osteoarthritis. The CCL produces substantial concentrations of NOt; the importance of this finding is unknown.
Assuntos
Lesões do Ligamento Cruzado Anterior , Ligamento Cruzado Anterior/metabolismo , Cartilagem Articular/metabolismo , Doenças do Cão/metabolismo , Óxido Nítrico/metabolismo , Membrana Sinovial/metabolismo , Animais , Ligamento Cruzado Anterior/patologia , Cartilagem Articular/patologia , Doenças do Cão/patologia , Cães , Feminino , Lisina/análogos & derivados , Lisina/química , Masculino , Nitratos/análise , Nitritos/análise , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/veterinária , Ruptura , Estatísticas não Paramétricas , Membrana Sinovial/patologia , ômega-N-Metilarginina/químicaRESUMO
We describe here a microtiter-plate assay for measuring nitric oxide synthase (NOS) activity by utilizing the spectral shift in optical absorbence between the wavelengths 405 and 420 nm on conversion of oxyhemoglobin to methemoglobin by nitric oxide (NO). This is a high-throughput assay permitting 96 or 384 simultaneous kinetic measurements and is ideal for the study of NOS inhibitors and their time dependence. It is also possible to measure enzyme rates under different conditions simultaneously for the study of the cofactor and substrate dependence of NOS preparations. The assay requires approximately 10 pmol/min of NOS activity to achieve a-l moD/min rate.
Assuntos
Biologia Molecular/métodos , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase/metabolismo , Animais , Arginina/isolamento & purificação , Inibidores Enzimáticos/química , Humanos , Isoenzimas/análise , Isoenzimas/genética , Isoenzimas/metabolismo , Metemoglobina/metabolismo , Biologia Molecular/instrumentação , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Oxiemoglobinas/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometria/instrumentação , Espectrofotometria/métodos , ômega-N-Metilarginina/químicaRESUMO
The guanidino-methylated arginine analogue NG monomethyl-L-arginine (L-NMMA) has been the standard nitric oxide synthase inhibitor used to evaluate the role of the L-arginine:nitric oxide pathway. However, L-NMMA and other methylated arginine residues are also synthesised in vivo by the action of a family of enzymes known as protein arginine methyltransferases. Proteolysis of proteins containing methylated arginine residues releases free methylarginine residues into the cytosol from where they may pass out of the cell into plasma. Of the three known methylarginine residues produced in mammals only asymmetrically methylated forms (L-NMMA and asymmetric dimethylarginine (ADMA)) but not symmetrically methylated arginine (symmetric dimethylarginine (SDMA)) inhibit nitric oxide synthase (NOS). We and others have proposed that endogenously produced asymmetrically methylated arginines may modulate NO production and that the accumulation of these residues in disease states may contribute to pathology. The activity of the enzyme dimethylarginine dimethylaminohydrolase that metabolises asymmetric methylarginines may be of critical importance in affecting NO pathways in health or disease.