RESUMO
BACKGROUND: Calotropis procera is a laticiferous plant (Apocynaceae) found in tropical regions all over the world. The ultrastructural characteristics of laticifers, their restricted distribution among different taxonomic groups, and in some species in each clade, as peptidases from latex, make them very attractive for biological analysis. OBJECTIVE: The study aims to investigate the effects of LP-PII-IAA (laticifer protein (LP) sub-fraction II (PII) of C. procera presenting an iodoacetamide-inhibited cysteine proteinase activity) on irinotecan-induced intestinal mucositis, a serious adverse effect of this medicine for the treatment of cancer. METHODS: LP-PII-IAA is composed of closely related isoforms (90%) of peptidases derived from catalysis and an osmotin protein (5%). Animals receiving co-administration of LP-PII-IAA presented a significant decrease in mortality, absence of diarrhea, histological preservation, and normalization of intestinal functions. RESULTS: Clinical homeostasis was accompanied by a reduction in MPO activity and declined levels of IL-1ß, IL-6 and KC, while the IL-10 level increased in LP-PII-IAA-treated animals. COX-2 and NF-kB immunostaining was reduced and the levels of oxidative markers (GSH, MDA) were normalized in animals that received LP-PII-IAA. CONCLUSION: We suggest that peptidases from the latex of Calotropis procera were instrumental in the suppression of the adverse clinical and physiological effects of irinotecan.
Assuntos
Calotropis , Cisteína Proteases , Animais , Calotropis/química , Ciclo-Oxigenase 2 , Interleucina-10 , Interleucina-6 , Iodoacetamida , Irinotecano/farmacologia , Látex/química , Látex/farmacologia , NF-kappa B , Proteínas de Plantas/farmacologia , Proteínas de Plantas/uso terapêuticoRESUMO
Metabolic control analysis (MCA) is a promising approach in biochemistry aimed at understanding processes in a quantitative fashion. Here the contribution of enzymes and transporters to the control of a given pathway flux and metabolite concentrations is determined and expressed quantitatively by means of numerical coefficients. Metabolic flux can be influenced by a wide variety of modulators acting on one or more metabolic steps along the pathway. We describe a laboratory exercise to study metabolic regulation of human erythrocytes (RBCs). Within the framework of MCA, students use these cells to determine the sensitivity of the glycolytic flux to two inhibitors (iodoacetic acid: IA, and iodoacetamide: IAA) known to act on the enzyme glyceraldehyde-3-phosphate-dehydrogenase. Glycolytic flux was estimated by determining the concentration of extracellular lactate, the end product of RBC glycolysis. A low-cost colorimetric assay was implemented, that takes advantage of the straightforward quantification of the absorbance signal from the photographic image of the multi-well plate taken with a standard digital camera. Students estimate flux response coefficients for each inhibitor by fitting an empirical function to the experimental data, followed by analytical derivation of this function. IA and IAA exhibit qualitatively different patterns, which are thoroughly analyzed in terms of the physicochemical properties influencing their action on the target enzyme. IA causes highest glycolytic flux inhibition at lower concentration than IAA. This work illustrates the feasibility of using the MCA approach to study key variables of a simple metabolic system, in the context of an upper level biochemistry course. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):502-515, 2018.
Assuntos
Bioquímica/educação , Eritrócitos/metabolismo , Glicólise , Colorimetria , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Eritrócitos/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Iodoacetamida/química , Iodoacetamida/farmacologia , Ácido Iodoacético/química , Ácido Iodoacético/farmacologia , EstudantesRESUMO
Collapse of the mitochondrial membrane potential (MMP) is often considered the initiation of regulated cell death (RCD). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is an uncoupler of the electron transport chain (ETC) that facilitates the translocation of protons into the mitochondrial matrix leading to the collapse of the MMP. Several cell stress responses such as mitophagy, mitochondrial biogenesis and the ubiquitin proteasome system may differentially contribute to restrain the initiation of RCD depending on the extent of mitochondrial damage. We induced graded mitochondrial damage after collapse of MMP with the mitochondrial uncoupler CCCP in Burkitt's lymphoma cells, and we evaluated the effect of several drugs targeting cell stress responses over RCD at 72 hr, using a multiparametric flow cytometry approach. CCCP caused collapse of MMP after 30 min., massive mitochondrial fission, oxidative stress and increased mitophagy within the 5-15 µM low-dose range (LDR) of CCCP. Within the 20-50 µM high-dose range (HDR), CCCP caused lysosomal destabilization and rupture, thus precluding mitophagy and autophagy. Cell death after 72 hr was below 20%, with increased mitochondrial mass (MM). The inhibitors of mitophagy 3-(2,4-dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1H)-quinazolinone (Mdivi-1) and vincristine (VCR) increased cell death from CCCP within the LDR, while valproic acid (an inducer of mitochondrial biogenesis) also increased MM and cell death within the LDR. The proteasome inhibitor, MG132, increased cell death only in the HDR. Doxycycline, an antibiotic that disrupts mitochondrial biogenesis, had no effect on cell survival, while iodoacetamide, an inhibitor of glycolysis, increased cell death at the HDR. We conclude that mitophagy influenced RCD of lymphoma cells after MMP collapse by CCCP only within the LDR, while proteasome activity and glycolysis contributed to survival in the HDR under extensive mitochondria and lysosome damage.
Assuntos
Linfoma de Burkitt/tratamento farmacológico , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Desacopladores/farmacologia , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/patologia , Autofagia/efeitos dos fármacos , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Iodoacetamida/farmacologia , Leupeptinas/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Quinazolinonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Vincristina/farmacologiaRESUMO
Este artículo fue concebido para analizar la función de la Escuela de Salud Pública de México (ESPM) desde el año 2000 hasta el presente. Uno de sus puntos centrales es el análisis del proceso de reorientación de la labor educativa de la escuela con la finalidad de responder a los retos en materia de salud y educación surgidos a finales del siglo XX. Para exponer cómo ha evolucionado dicho proceso, retomamos tres ejes rectores que caracterizan la labor de la escuela en la actualidad: el cambio de modelo pedagógico, la incorporación de las tecnologías de la información y las comunicaciones, y la profesionalización de la docencia. Con la exposición de este tema, y a través del contraste entre el pasado y el presente, buscamos completar la historia de trabajo ininterrumpido de la Escuela durante sus 92 años de existencia, que ha trascendido los confines del país.
This article was conceived to analyze the work of the School of Public Health of Mexico (ESPM for is acronym in Spanish) from the year 2000 to the present day. One of the highlights that we will examine is the reorientation of the educational work of the school in order to meet the challenges in health and education that emerged during the end of the twentieth century. In order to explain the evolution of this process, we will describe the three main guiding principles that characterize the present work of the school: the pedagogical model's change, the incorporation of the information and communication technologies, and the professionalization in teaching. The purpose of this work is to define those guiding principles, and to expose, through the contrast between past and present, the complete history of uninterrupted work of the School of Public Health of Mexico during its ninety-two years of existence, that has gone beyond the boundaries of the country.
Assuntos
Animais , Feminino , Humanos , Camundongos , Cisteína Endopeptidases/metabolismo , Mengovirus/enzimologia , Proteínas Virais , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Capsídeo/metabolismo , Cloretos/farmacologia , Cisteína Endopeptidases/genética , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Células HeLa , Iodoacetamida/farmacologia , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Compostos de Zinco/farmacologiaRESUMO
The lattices of Carica candamarcensis and Carica papaya, members of the Caricaceae family, contain isoforms of cysteine proteinases that help protect these plants against injury. In a prior study, we fractionated 14 discrete proteinaceous components from C. candamarcensis, two of them displaying mitogenic activity in mammalian cells. In this study, we compared the kinetic parameters of one of the mitogenic proteinases (CMS2MS2) with one of the isoforms displaying the highest enzyme activity of this group (CMS1MS2). Both enzymes display a similar Km value with either BAPNA (Benzoyl-Arg-pNA) or PFLPNA (Pyr-Phe-Leu-pNA), but the kcat of CMS1MS2 is about 14-fold higher for BAPNA and 129-fold higher with PFLPNA. While both enzymes are inhibited by E-64 and iodoacetamide, chicken cystatin fully inhibits CMS1MS2, but scarcely affects activity of CMS2MS2. Based on the structure of these proteins and other enzymes from the Caricaceae family whose structures have been resolved, it is proposed that Arg(180) located in the cleft at the active site in CMS2MS2 is responsible for its resistance to cystatin.
Assuntos
Caricaceae/enzimologia , Cistatinas , Cisteína Proteases/química , Inibidores de Cisteína Proteinase , Mitógenos/química , Peptídeo Hidrolases/química , Proteínas de Plantas/química , Animais , Galinhas , Cisteína Proteases/metabolismo , Iodoacetamida , Mitógenos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Isoformas de Proteínas , Relação Estrutura-AtividadeRESUMO
The effects of the alkylaminoalkanethiosulfuric acids (AAATs), new schistosomicidal drugs, on Schistosoma mansoni ATP diphosphohydrolase isoforms, members of the NTPDase family, were analyzed. Pre-incubation of worm adult tegument with AAATs derivatives, with small apolar alkyl groups and an apolar alkane portion of 6 or 8 carbon atoms linked to the amino group, inhibited ATPase activity with a Ki 100-1000 microM. Little inhibition (20%) was observed when ADP was the substrate. The 2-[(tert-butyl)amino]-1-ethanethiosulfuric acid (100 microM) which has a less lipophilic structure, inhibited 28% ATPase and 12% ADPase activities, suggesting that the lipophilicity, although important, is not the only requisite for enzyme activity inhibition. The N-(sec-butyl)-2-bromo-1-octanaminium bromide, which contains a bromide atom instead of thiosulphate, inhibited <10% of the enzyme activity, suggesting the involvement of cysteine residue(s) from S. mansoni ATP diphosphohydrolase isoforms in a mixed disulfide formation. Treatment of parasite tegument with 5 mM iodoacetamide or 1 mM dithiothreitol protected ATPase and ADPase activities against inhibition by AAATs, corroborating the participation of disulfide interchange in the AAATs mechanism. Since S. mansoni ATP diphosphohydrolase isoforms and potato apyrase share structural similarities, the latter enzyme was also tested. ADPase activity from potato apyrase was inhibited by 55%, showing a higher sensitivity to 1 mM AAATs than that shown by ADPase activity from the tegument, while the ATPase activities from both samples showed similar inhibition levels. Furthermore, sulfhydryl reagents protected potato apyrase activity. Therefore, it is possible that both soluble S. mansoni ATP diphosphohydrolase and membrane-associated isoforms are targets for the AAATs.
Assuntos
Apirase/antagonistas & inibidores , Schistosoma mansoni/efeitos dos fármacos , Esquistossomicidas/farmacologia , Ésteres do Ácido Sulfúrico/farmacologia , Adenosina Trifosfatases/metabolismo , Animais , Apirase/metabolismo , Ditiotreitol/farmacologia , Glutationa/farmacologia , Iodoacetamida/farmacologia , Masculino , Camundongos , Schistosoma mansoni/enzimologia , Solanum tuberosum/enzimologia , Reagentes de Sulfidrila/farmacologiaRESUMO
There have been numerous methods proposed to measure the circulating blood volume (CBV). Nevertheless, none of them have been massively and routinely accepted in clinical diagnosis. This study describes a simple and rapid method, on a rabbit model, using the dilution of autologous red cells labeled with a nitroxide radical (Iodoacetamide-TEMPO), which can be detected by electronic spin resonance (ESR) spectroscopy. Blood samples were withdrawn and re-injected using the ears' marginal veins. The average CBV measured by the new method/body weight (CBV(IAT)/BW) was 59 +/- 7 mL/kg (n = 33). Simultaneously, blood volume determinations using the nitroxide radical and (51)Cr (CBV(Cr)) were performed. In the plot of the difference between the methods (CBV(IAT) - CBV(Cr)) against the average (CBV(IAT) + CBV(Cr))/2, the mean of the bias was -1.1 +/- 6.9 mL and the limits of agreement (mean difference +/-2 SD) were -14.9 and 12.7 mL. Lin's concordance correlation coefficient p(c) = 0.988. Thus, both methods are in close agreement. The development of a new method that allows a correct estimation of the CBV without using radioactivity, avoiding blood manipulation, and decreasing the possibility of blood contamination with similar accuracy and precision of that of the "gold standard method" is an innovative proposal.
Assuntos
Circulação Sanguínea , Determinação do Volume Sanguíneo/métodos , Animais , Óxidos N-Cíclicos , Espectroscopia de Ressonância de Spin Eletrônica , Iodoacetamida , Coelhos , Marcadores de SpinRESUMO
In this work we identified specific bovine leukocytes that were bound by the Mannheimia haemolytica adhesin molecule (MhA) and the biological effect on the leukocytes. Histochemical staining and flow cytometry showed that MhA bind neutrophils (90%) and monocytes (5%). MhA induced an oxidative response in purified neutrophils; this effect was 1.5-fold higher than the effect observed with control cells activated with Zymosan. Cellular binding by MhA was inhibited with GlcNAc and its oligomers, as well as by glycoproteins containing tri- and tetra-antennary N-glycosydically linked glycans. MhA-induced oxidative burst was significantly inhibited by GlcNAc, iodoacetamide, superoxide dismutase, and piroxicam (p<0.05). Our findings suggest that among bovine leukocytes, neutrophils are the main target for MhA, inducing production of oxidative radicals by non-opsonic mechanism that seem to play an important role in tissue damage during mannheimiosis.
Assuntos
Adesinas Bacterianas/farmacologia , Doenças dos Bovinos/microbiologia , Bovinos/sangue , Mannheimia haemolytica/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Pasteurelose Pneumônica/imunologia , Explosão Respiratória/efeitos dos fármacos , Acetilglucosamina/imunologia , Adesinas Bacterianas/imunologia , Adesinas Bacterianas/isolamento & purificação , Animais , Doenças dos Bovinos/sangue , Doenças dos Bovinos/imunologia , Citometria de Fluxo/veterinária , Imuno-Histoquímica/veterinária , Iodoacetamida/farmacologia , Mannheimia haemolytica/isolamento & purificação , Ativação de Neutrófilo/imunologia , Neutrófilos/microbiologia , Pasteurelose Pneumônica/microbiologia , Piroxicam/farmacologia , Explosão Respiratória/imunologia , Superóxido Dismutase/farmacologiaRESUMO
In this study, we report the ultrastructural and growth alterations caused by cysteine peptidase inhibitors on the plant trypanosomatid Phytomonas serpens. We showed that the cysteine peptidase inhibitors at 10 microM were able to arrest cellular growth as well as promote alterations in the cell morphology, including the parasites becoming short and round. Additionally, iodoacetamide induced ultrastructural alterations, such as disintegration of cytoplasmic organelles, swelling of the nucleus and kinetoplast-mitochondrion complex, which culminated in parasite death. Leupeptin and antipain induced the appearance of microvillar extensions and blebs on the cytoplasmic membrane, resembling a shedding process. A 40 kDa cysteine peptidase was detected in hydrophobic and hydrophilic phases of P. serpens cells after Triton X-114 extraction. Additionally, we have shown through immunoblotting that anti-cruzipain polyclonal antibodies recognised two major polypeptides in P. serpens, including a 40 kDa component. Flow cytometry analysis confirmed that this cruzipain-like protein has a location on the cell surface. Ultrastructural immunocytochemical analysis demonstrated the presence of the cruzipain-like protein on the surface and in small membrane fragments released from leupeptin-treated parasites. Furthermore, the involvement of cysteine peptidases of P. serpens in the interaction with explanted salivary glands of the phytophagous insect Oncopeltus fasciatus was also investigated. When P. serpens cells were pre-treated with either cysteine peptidase inhibitors or anti-cruzipain antibody, a significant reduction of the interaction process was observed. Collectively, these results suggest that cysteine peptidases participate in several biological processes in P. serpens including cell growth and interaction with the invertebrate vector.
Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Trypanosomatina/crescimento & desenvolvimento , Animais , Anticorpos Antiprotozoários/imunologia , Antipaína/farmacologia , Divisão Celular , Células Cultivadas , Cistatinas/farmacologia , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/metabolismo , Detergentes/farmacologia , Citometria de Fluxo/métodos , Heterópteros , Imuno-Histoquímica/métodos , Iodoacetamida/farmacologia , Leucina/análogos & derivados , Leucina/farmacologia , Leupeptinas/farmacologia , Proteínas de Membrana/metabolismo , Microscopia Eletrônica/métodos , Octoxinol , Proteínas de Plantas/metabolismo , Polietilenoglicóis/farmacologia , Proteínas de Protozoários , Glândulas Salivares/metabolismo , Trypanosomatina/efeitos dos fármacos , Trypanosomatina/ultraestruturaRESUMO
The distribution of intramembrane particles in human erythrocytes was studied by freeze-fracture on young and old cells and compared to that obtained after ATP depletion or following addition of a clustering agent. It was shown that intramembrane particles became aggregated and the mean particle density increased as the cells aged. Likewise, both particle aggregation and increased density were found in young cells after moderate ATP depletion. In contrast, mean particle density was markedly reduced in both cell types after exhaustive depletion. Paradoxically, Zn treatment led to decreased particle density in young cells, whilst producing the opposite effect in aged cells. The results suggest that their low ATP content may account for the increased particle density of senescent cells.
Assuntos
Envelhecimento Eritrocítico/fisiologia , Membrana Eritrocítica/fisiologia , Membrana Eritrocítica/ultraestrutura , Eritrócitos/fisiologia , Eritrócitos/ultraestrutura , Trifosfato de Adenosina/metabolismo , Diferenciação Celular/fisiologia , Cloretos/farmacologia , Envelhecimento Eritrocítico/efeitos dos fármacos , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Técnica de Fratura por Congelamento , Humanos , Inosina/farmacologia , Iodoacetamida/farmacologia , Sulfitos/farmacologia , Compostos de Zinco/farmacologiaRESUMO
The glycolytic enzymes of the trypanosomatids, that cause a variety of medically and agriculturally important diseases, are validated targets for drug design. Design of species-specific inhibitors is facilitated by the availability of structural data. Irreversible inhibitors, that bound covalently to the parasite enzyme alone, would be potentially particularly effective. Here we determine the crystal structure of enolase from Trypanosoma brucei and show that two cysteine residues, located in a water-filled cavity near the active-site, are modified by iodoacetamide leading to loss of catalytic activity. Since these residues are specific to the Trypanosomatidae lineage, this finding opens the way for the development of parasite-specific, irreversibly binding enolase inhibitors. In the present structure, the catalytic site is partially occupied by sulphate and two zinc ions. Surprisingly, one of these zinc ions illustrates the existence of a novel enolase-binding site for divalent metals. Evidence suggests that this is the first direct visualization of the elusive inhibitory metal site, whose existence has hitherto only been inferred from kinetic data.
Assuntos
Metais/metabolismo , Fosfopiruvato Hidratase/antagonistas & inibidores , Fosfopiruvato Hidratase/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Iodoacetamida/química , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Fosfopiruvato Hidratase/metabolismo , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray , Sulfatos/metabolismo , Zinco/metabolismoRESUMO
We describe the purification of a cysteine proteinase from latex of Carica candamarcensis, hereby designated CC23. The enzyme has been purified by ion-exchange chromatography and behaves electrophoretically as a monomer of M(r) 23,000 and optimal pH of 8.0. It displays a basic isoelectric point, has one cysteine residue in the active site by titration with E-64, confirmed by DNA sequencing, and responds to proteinase inhibitors as a classic cysteine proteinase. The K(m) and k(cat)/K(m) for CC23 using BAPNA were respectively 14.7 +/- 1.8 x 10(-4) M and 1.3 x 10(3) M(-1) s(-1). Therefore, the catalytic efficiency of CC23 is sixfold higher than that of CC-I, another proteinase from the same plant. DNA primers were designed to amplify by PCR a genomic sequence related to this enzyme. An 895-bp DNA fragment was cloned and sequenced. It shows strong homology with chymopapain isoform IV from C. papaya. The translated sequence is similar to that of chymopapain isoform II (73%) and CC-III (77%) from C. candamarcensis.
Assuntos
Carica/enzimologia , Carica/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/isolamento & purificação , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Inibidores Enzimáticos/farmacologia , Genoma de Planta , Glicoproteínas/antagonistas & inibidores , Iodoacetamida/farmacologia , Cinética , Látex/química , Compostos de Mercúrio/farmacologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/antagonistas & inibidores , Homologia de Sequência de AminoácidosRESUMO
Fractionation of the serum of the venomous snake Bothrops jararaca with (NH4)2SO4, followed by phenyl-Sepharose and C4-reversed phase chromatographies, resulted in the isolation of the anti-hemorrhagic factor BJ46a. BJ46a is a potent inhibitor of the SVMPs atrolysin C (class P-I) and jararhagin (P-III) proteolytic activities and B. jararaca venom hemorrhagic activity. The single-chain, acidic (pI 4.55) glycoprotein has a molecular mass of 46 101 atomic mass units determined by MALDI-TOF MS and 79 kDa by gel filtration and dynamic laser light scattering, suggesting a homodimeric structure. mRNA was isolated from the liver of one specimen and transcribed into cDNA. The cDNA pool was amplified by PCR, cloned into a specific vector and used to transform competent cells. Clones containing the complete coding sequence for BJ46a were isolated. The deduced protein sequence was in complete agreement with peptide sequences obtained by Edman degradation. BJ46a is a 322-amino-acid protein containing four putative N-glycosylation sites. It is homologous to the proteinase inhibitor HSF (member of the fetuin family, cystatin superfamily) isolated from the serum of the snake Trimeresurus flavoviridis, having 85% sequence identity. This is the first report of a complete cDNA sequence for an endogenous inhibitor of snake venom metalloproteinases (SVMPs). The sequence reveals that the only proteolytic processing required to obtain the mature protein is the cleavage of the signal peptide. Gel filtration analyses of the inhibitory complexes indicate that inhibition occurs by formation of a noncovalent complex between BJ46a and the proteinases at their metalloproteinase domains. Furthermore, the data shows that the stoichiometry involved in this interaction is of one inhibitor monomer to two enzyme molecules, suggesting an interesting mechanism of metalloproteinase inhibition.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Venenos de Serpentes/enzimologia , Venenos de Víboras/química , Venenos de Víboras/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bothrops/sangue , Venenos de Crotalídeos/farmacologia , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Endopeptidases/química , Indicadores e Reagentes/farmacologia , Iodoacetamida/análogos & derivados , Iodoacetamida/farmacologia , Focalização Isoelétrica , Luz , Fígado/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/farmacologia , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Espalhamento de Radiação , Análise de Sequência de DNA , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Veneno de Bothrops jararacaRESUMO
Coiled-coil domains mediate the oligomerization of many proteins. The assembly of long coiled coils, such as tropomyosin, presupposes the existence of intermediates. These intermediates are not well-known for tropomyosin. Hydrostatic pressure affects the equilibrium between denatured and native forms in the direction of the form that occupies a smaller volume. The hydrophobic core is the region more sensitive to pressure, which leads in most cases to the population of intermediates. Here, we used N-(1-pyrenyl)iodoacetamide covalently bound to cysteine residues of tropomyosin (PIATm) and high hydrostatic pressure to assess the chain interaction and the inherent instability of the coiled-coil molecule. The native and denatured states of tropomyosin were determined from the pyrene excimer fluorescence. The combination of low temperature and high pressure permitted the attainment of the full denaturation of tropomyosin without the separation of the subunits. High-temperature denaturation of Tm leads to a great exchange between labeled and unlabeled Tm subunits, indicating subunit dissociation linked to unfolding. In contrast, under high pressure, unlabeled and labeled tropomyosin molecules do not exchange, demonstrating that the denatured species are dimeric. The decrease of the concentration dependence of PIATm corroborates the idea that pressure produces subdomain denaturation and that the polypeptide chains do not separate. Substantial unfolding of tropomyosin was also verified by measurements of tyrosine fluorescence and bis-ANS binding. Our results indicate the presence of independent folding subdomains with different susceptibilities to pressure along the length of the coiled-coil structure of tropomyosin.
Assuntos
Iodoacetamida/análogos & derivados , Dobramento de Proteína , Tropomiosina/química , Animais , Temperatura Baixa , Corantes Fluorescentes/química , Temperatura Alta , Pressão Hidrostática , Iodoacetamida/química , Cinética , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Coelhos , Espectrometria de Fluorescência/métodos , TermodinâmicaRESUMO
Intragastric administration of clarithromycin, a macrolide antibiotic, macroscopically protected the rat gastric mucosa from 96% ethanol-induced lesions. This protective effect was dose-dependent, the reduction being 92.3, 81.4, 52.2, and 5.4% at doses of 400, 200, 100, and 50 mg/kg, respectively. Clarithromycin protection was not significantly modified by pretreatment with either subcutaneous indomethacin (5 mg/kg), a selective cyclooxygenase inhibitor, or iodoacetamide (100 mg/kg), a specific sulfhydryl blocker. Gastric motor activity, measured by the balloon method, was inhibited by clarithromycin in a dose-dependent fashion. The inhibited gastric motor activity induced by clarithromycin was not modified by pretreatment with either indomethacin or iodoacetamide. Ethanol (1 ml/rat, gavage needle) induced hemorrhagic bandlike lesions in the mucosa of the glandular stomach along the long axis of the greater curvature with the occurrence of a complete inhibition of gastric motor activity. This inhibition was not modified by pretreatment with clarithromycin, indomethacin, or iodoacetamide. There was an increase in the fluid volume for clarithromycin at 100, 200, and 400 mg/kg at 30 min and in the mucus volume only for clarithromycin at 400 mg/kg at 30 min. Gastric mucosal blood flow was absent in the ethanol-induced lesions but in the nonlesion areas was the same in clarithromycin-pretreated and vehicle-pretreated rats as in control (no ethanol) rats. Clarithromycin protection was significantly diminished, although not completely abolished by subcutaneous yohimbine (5 mg/kg), a selective alpha2-adrenoceptor antagonist. Yohimbine also significantly reduced both basal and clarithromycin-stimulated gastric mucus secretion. Our data support the conclusion that the protective effect of intragastric clarithromycin was not mediated by endogenous prostaglandins, sulfhydryl compounds of the gastric mucosa, or changes in the gastric contractile patterns. The protection may be the result of an increase in both the fluid volume and the mucus volume retained in the gastric lumen. alpha2-Adrenoceptors possibly are involved by the mucus-dependent mechanism.
Assuntos
Antibacterianos/administração & dosagem , Claritromicina/administração & dosagem , Etanol , Gastropatias/induzido quimicamente , Estômago/efeitos dos fármacos , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Antibacterianos/farmacologia , Água Corporal/efeitos dos fármacos , Claritromicina/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Feminino , Mucosa Gástrica/irrigação sanguínea , Mucosa Gástrica/patologia , Conteúdo Gastrointestinal/efeitos dos fármacos , Motilidade Gastrointestinal/efeitos dos fármacos , Indometacina/farmacologia , Iodoacetamida/farmacologia , Masculino , Ratos , Ratos Wistar , Fluxo Sanguíneo Regional/efeitos dos fármacos , Gastropatias/patologia , Reagentes de Sulfidrila/farmacologia , Ioimbina/farmacologiaRESUMO
The purpose of this study was to investigate the role played by endogenous prostaglandins, sulfhydryls, gastric motility, fluid volume, and mucus volume retained in the gastric lumen in the protection offered by intragastric amoxicillin against ethanol-induced gastric lesions. It has been demonstrated that intragastric administration of amoxicillin (Amx) dose-dependently protected the rat gastric mucosa from 96% ethanol-induced lesions. The inhibition of the lesions was 28, 41.4, 84.7 and 90% at doses of 50, 100, 200 and 400 mg/kg, respectively. The gastroprotective effect of Amx was significantly reversed by pretreatment with both indomethacin (5 mg/kg, subcutaneously), a cyclooxygenase inhibitors, and iodoacetamide (100 mg/kgm subcutaneously), a sulfhydryl blocker. Gastric motility was measured by a balloon method. There was not any significant differences between Amx (50-400 mg/kg)-induced and spontaneous motility with regard to both amplitudes and frequently of gastric contraction. One milliliter of 96% ethanol produced hemorrhagic bandlike lesions in the corpus mucosa with the occurrence of a complete inhibition of the amplitude and frequency of gastric contraction. This inhibition of gastric motility caused by ethanol was not modified by pretreatment of Amx (400 mg/kg) alone, indomethacin plus Amx or iodoacetamide plus Amx. In addition, there was a significant increase in the mucus volume retained in the gastric lumen for Amx (200 and 400 mg/kg) at 30 min after its administration. We conclude that the intragastric Amx prospective effect against 96% eathanol-induced mucosal lesions may be mediated by endogenous prostaglandins, sulfhydryl compounds of the gastric mucosa, an increase in mucus volume retained in the gastric lumen at the time when ethanol is administered, and is not associated with the gastric motor activity.
Assuntos
Amoxicilina/farmacologia , Etanol/toxicidade , Mucosa Gástrica/efeitos dos fármacos , Penicilinas/farmacologia , Prostaglandinas/metabolismo , Análise de Variância , Animais , Interações Medicamentosas , Feminino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Motilidade Gastrointestinal/efeitos dos fármacos , Indometacina/farmacologia , Iodoacetamida/farmacologia , Masculino , Coelhos , Ratos , Ratos WistarRESUMO
Cytosolic proteinases were assayed in both morphological phases of Paracoccidioides brasiliensis. Preparations from the mycelial phase were more active in vitro than those from the yeast cells. Optimal proteinase activities for both phases occurred at pH's between 6.0 and 9.0, and at 45 degrees C. Gelatin-SDS-PAGE electrophoresis separated several bands (58-112 kDa) in mycelial preparations; a single band (70 kDa) was seen in yeast preparations. Enzymatic activities were inhibited by antipain, phenyl methyl sulfonyl fluoride (PMSF), and chymostatin, suggestive of serine proteinases. Partial inhibition of the mycelial enzymes by ethylene diamine tetraacetic acid (EDTA), 1,10-phenanthroline, and iodoacetamide, also suggested the presence of cysteine- and metallo-proteinases. The enzymatic activity increased in preparations extracted from yeast cells transforming to mycelia, and decreased in preparations obtained from the reverse process.
Assuntos
Citosol/enzimologia , Endopeptidases/metabolismo , Paracoccidioides/enzimologia , Antipaína/farmacologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Divisão Celular/efeitos dos fármacos , Quelantes/farmacologia , Ácido Edético/farmacologia , Eletroforese em Gel de Poliacrilamida , Endopeptidases/análise , Endopeptidases/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Iodoacetamida/farmacologia , Oligopeptídeos/farmacologia , Paracoccidioides/citologia , Paracoccidioides/efeitos dos fármacos , Fluoreto de Fenilmetilsulfonil/farmacologia , Inibidores de Proteases/farmacologia , Inibidores de Serina Proteinase/farmacologia , Dodecilsulfato de Sódio , TemperaturaRESUMO
The purpose of this study was to investigate the role played by endogenous prostaglandins, sulfhydryls, gastric motility, fluid volume, and mucus volume retained in the gastric lumen in the protection offered by intragastric amoxicillin against ethanol-induced gastric lesions. It has been demonstrated that intragastric administration of amoxicillin (Amx) dose-dependently protected the rat gastric mucosa from 96 per cent ethanol-induced lesions. The inhibition of the lesions was 28, 41.4, 84.7 and 90 per cent at doses of 50, 100, 200 and 400 mg/Kg, respectively. The gastroprotective effect of Amx was significantly reversed by pretreatment with both indomethacin (5 mg/Kg, subcutaneously), a cyclooxygenase inhibitors, and iodoacetamide (100 mg/Kgm subcutaneously), a sulfhydryl blocker. Gastric motility was measured by a ballon method. There was not any significant differences between Amx (50-400 mg/Kg)-induced and spontaneous motility with regard to both amplitudes and frequently of gastric contraction. One milliliter of 96 per cent ethanol produced hemorrhagic bandlike lesions in the corpus mucosa with the occurrence of a complete inhibition of the amplitude and frequency of gastric contraction. This inhibition of gastric motility caused by ethanol was not modified by pretreatment of Amx (400 mg/Kg) alone, indomethacin plus Amx or iodoacetamide plus Amx. In addition, there was a significant increase in the mucus volume retained in the gastric lumen for Amx (200 and 400 mg/Kg) at 30 min after its adminitration. We conclude that the intragastric Amx prospective effect aginst 96 per cent athanol-induced mucosal lesions may be mediated by endogenous prostaglandins, sulfhydryl compounds of the gastric mucosa, an increase in mucus volume retained in the gastric lumen at the time when ethanol is administered, and is not associated with the gastric motor activity.
Assuntos
Animais , Feminino , Ratos , Amoxicilina/farmacologia , Etanol/toxicidade , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/patologia , Penicilinas/farmacologia , Prostaglandinas/metabolismo , Análise de Variância , Interações Medicamentosas , Mucosa Gástrica/metabolismo , Motilidade Gastrointestinal/efeitos dos fármacos , Indometacina/farmacologia , Iodoacetamida/farmacologia , Ratos WistarRESUMO
The purpose of this study was to investigate the role played by endogenous prostaglandins, sulfhydryls, gastric motility, fluid volume, and mucus volume retained in the gastric lumen in the protection offered by intragastric amoxicillin against ethanol-induced gastric lesions. It has been demonstrated that intragastric administration of amoxicillin (Amx) dose-dependently protected the rat gastric mucosa from 96 per cent ethanol-induced lesions. The inhibition of the lesions was 28, 41.4, 84.7 and 90 per cent at doses of 50, 100, 200 and 400 mg/Kg, respectively. The gastroprotective effect of Amx was significantly reversed by pretreatment with both indomethacin (5 mg/Kg, subcutaneously), a cyclooxygenase inhibitors, and iodoacetamide (100 mg/Kgm subcutaneously), a sulfhydryl blocker. Gastric motility was measured by a ballon method. There was not any significant differences between Amx (50-400 mg/Kg)-induced and spontaneous motility with regard to both amplitudes and frequently of gastric contraction. One milliliter of 96 per cent ethanol produced hemorrhagic bandlike lesions in the corpus mucosa with the occurrence of a complete inhibition of the amplitude and frequency of gastric contraction. This inhibition of gastric motility caused by ethanol was not modified by pretreatment of Amx (400 mg/Kg) alone, indomethacin plus Amx or iodoacetamide plus Amx. In addition, there was a significant increase in the mucus volume retained in the gastric lumen for Amx (200 and 400 mg/Kg) at 30 min after its adminitration. We conclude that the intragastric Amx prospective effect aginst 96 per cent athanol-induced mucosal lesions may be mediated by endogenous prostaglandins, sulfhydryl compounds of the gastric mucosa, an increase in mucus volume retained in the gastric lumen at the time when ethanol is administered, and is not associated with the gastric motor activity. (AU)
Assuntos
Animais , Feminino , Ratos , Etanol/toxicidade , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/patologia , Prostaglandinas/metabolismo , Amoxicilina/farmacologia , Penicilinas/farmacologia , Mucosa Gástrica/metabolismo , Ratos Wistar , Motilidade Gastrointestinal/efeitos dos fármacos , Interações Medicamentosas , Análise de Variância , Iodoacetamida/farmacologia , Indometacina/farmacologiaRESUMO
We describe the changes in peptide composition by SDS-PAGE analysis of latex from Carica papaya collected at various times after incision of the unripe fruit. The data show that during latex coagulation several peptides are processed in an orderly fashion.