Assuntos
Adaptação Ocular/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Inibidores da Síntese de Proteínas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Folhas de Planta/genética , Folhas de Planta/metabolismoRESUMO
Ribosome Inactivating Proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of the 28S rRNA. The occurrence of RIP genes has been described in a wide range of plant taxa, as well as in several species of bacteria and fungi. A remarkable case is the presence of these genes in metazoans belonging to the Culicinae subfamily. We reported that these genes are derived from a single horizontal gene transfer event, most likely from a bacterial donor species. Moreover, we have shown evidence that mosquito RIP genes are evolving under purifying selection, suggesting that these toxins have acquired a functional role in these organisms. In the present work, we characterized the intra-specific sequence variability of Aedes aegypti RIP genes (RIPAe1, RIPAe2, and RIPAe3) and tested their expression at the mRNA level. Our results show that RIPAe2 and RIPAe3 are transcribed and polyadenylated, and their expression levels are modulated across the developmental stages. Varibility among genes was observed, including the existence of null alleles for RIPAe1 and RIPAe2, with variants showing partial deletions. These results further support the existence of a physiological function for these foreign genes in mosquitoes. The possible nature of this functionality is discussed.
Assuntos
Aedes/genética , Inibidores da Síntese de Proteínas/metabolismo , Proteínas Inativadoras de Ribossomos/metabolismo , Ribossomos/metabolismo , Toxinas Biológicas/metabolismo , Aedes/fisiologia , Animais , Sequência de Bases , Proteínas Inativadoras de Ribossomos/genética , Homologia de Sequência , Toxinas Biológicas/genéticaAssuntos
Antibacterianos/farmacologia , Azitromicina/farmacologia , Cisteína/metabolismo , Farmacorresistência Bacteriana , Eritromicina/farmacologia , Lactobacillus/efeitos dos fármacos , Inibidores da Síntese de Proteínas/metabolismo , Testes de Sensibilidade Microbiana , Ativação Transcricional/efeitos dos fármacosRESUMO
Objective: To explore the effect of erythromycin on hyperoxia-induced lung injury. Methods: One-day-old preterm offspring Sprague-Dawley (SD) rats were randomly divided into four groups: group 1, air + sodium chloride; group 2, air + erythromycin;group 3, hyperoxia + sodium chloride; and group 4, hyperoxia + erythromycin. At one, seven, and 14 days of exposure, glutathione (GSH) and interleukin-1 beta (IL-1 beta) were detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), and bicinchoninic acid (BCA) was used to detect GSH protein. γ-glutamine-cysteine synthetase (γ-GCS) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results: Compared with group 1, expressions of GSH and γ-GCS mRNA in group 3 were significantly increased at one and seven days of exposure (p < 0.05), but expression of γ-GCS mRNA was significantly reduced at 14 days; expression of IL-1 beta in group 3 was significantly increased at seven days of exposure (p < 0.05), and was significantly reduced at 14 days. Compared with group 3, expressions of GSH and γ-GCS mRNA in group 4 were significantly increased at one, seven, and 14 days of exposure (p < 0.05), but expressions of GSH showed a downward trend at 14 days; expression of IL-1 beta in group 4 was significantly reduced at one and seven days of exposure (p < 0.05). Conclusions: Changes in oxidant-mediated IL-1 beta and GSH are involved in the development of hyperoxia-induced lung injury. Erythromycin may up-regulate the activity of γ-GCS, increasing the expression of GSH, inhibiting the levels of oxidant-mediated IL-1 beta and alleviating hyperoxia-induced lung injury via an antioxidant effect. .
Objetivo: Explorar o efeito da eritromicina sobre lesões pulmonares induzidas por hiperóxia. Métodos: Uma prole de ratos Sprague-Dawley (SD) prematuros com um dia de vida foi dividida aleatoriamente em quatro grupos: grupo 1 ar + cloreto de sódio, grupo 2 ar + eritromicina, grupo 3 hiperóxia + cloreto de sódio e grupo 4 hiperóxia + eritromicina. Com um, sete e 14 dias de exposição, foram detectadas Glutationa (GSH) e Interleucina-1 beta (IL-1 beta) pelo ensaio imunossorvente ligado à enzima (ELISA), e o ácido bicinconinico (BCA) foi utilizado para detectar a proteína GSH. O mRNA da γ-glutamil-cisteina-sintetase (γ-GCS) foi detectado por reação em cadeia da polimerase via transcriptase reversa (RT-PCR). Resultados: Comparadas ao grupo 1, as expressões do mRNA da GSH e da γ-GCS no grupo 3 aumentaram significativamente com um e sete dias de exposição (p < 0,05), porém a expressão de mRNA da γ-GCS diminuiu significativamente aos 14 dias; a expressão de IL-1 beta no grupo 3 aumentou significativamente aos 7 dias de exposição (p < 0,05) e diminuiu significativamente aos 14 dias. Comparadas ao grupo 3, as expressões do mRNA da GSH e da γ-GCS no grupo 4 aumentaram significativamente com um, sete e 14 dias de exposição (p < 0,05), porém as expressões de GSH mostraram uma tendência de queda aos 14 dias; a expressão de IL-1 beta no grupo 4 foi reduzida significativamente com um e sete dias de exposição (p < 0,05). Conclusões: As variações de IL-1 beta e GSH mediadas por oxidantes estão envolvidas no desenvolvimento de lesão pulmonar induzida por hiperóxia. A eritromicina poderá regular positivamente a atividade da γ-GCS, aumentando a expressão de GSH, inibindo os níveis de interleucina-1beta mediada por ...
Assuntos
Animais , Feminino , Masculino , Eritromicina/farmacologia , Glutamato-Cisteína Ligase/efeitos dos fármacos , Hiperóxia/metabolismo , Interleucina-1beta/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Animais Recém-Nascidos , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Glutationa/metabolismo , Interleucina-1beta/metabolismo , Lesão Pulmonar/metabolismo , Oxigênio/metabolismo , Oxigênio/farmacologia , Inibidores da Síntese de Proteínas/metabolismo , Distribuição Aleatória , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: To explore the effect of erythromycin on hyperoxia-induced lung injury. METHODS: One-day-old preterm offspring Sprague-Dawley (SD) rats were randomly divided into four groups: group 1, air + sodium chloride; group 2, air + erythromycin;group 3, hyperoxia + sodium chloride; and group 4, hyperoxia + erythromycin. At one, seven, and 14 days of exposure, glutathione (GSH) and interleukin-1 beta (IL-1 beta) were detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), and bicinchoninic acid (BCA) was used to detect GSH protein. γ-glutamine-cysteine synthetase (γ-GCS) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Compared with group 1, expressions of GSH and γ-GCS mRNA in group 3 were significantly increased at one and seven days of exposure (p < 0.05), but expression of γ-GCS mRNA was significantly reduced at 14 days; expression of IL-1 beta in group 3 was significantly increased at seven days of exposure (p < 0.05), and was significantly reduced at 14 days. Compared with group 3, expressions of GSH and γ-GCS mRNA in group 4 were significantly increased at one, seven, and 14 days of exposure (p < 0.05), but expressions of GSH showed a downward trend at 14 days; expression of IL-1 beta in group 4 was significantly reduced at one and seven days of exposure (p < 0.05). CONCLUSIONS: Changes in oxidant-mediated IL-1 beta and GSH are involved in the development of hyperoxia-induced lung injury. Erythromycin may up-regulate the activity of γ-GCS, increasing the expression of GSH, inhibiting the levels of oxidant-mediated IL-1 beta and alleviating hyperoxia-induced lung injury via an antioxidant effect.
Assuntos
Eritromicina/farmacologia , Glutamato-Cisteína Ligase/efeitos dos fármacos , Hiperóxia/metabolismo , Interleucina-1beta/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Glutationa/metabolismo , Interleucina-1beta/metabolismo , Lesão Pulmonar/metabolismo , Masculino , Oxigênio/metabolismo , Oxigênio/farmacologia , Inibidores da Síntese de Proteínas/metabolismo , Distribuição Aleatória , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Alterations in platelet aggregation in septic conditions are well established. However, little is known about the effects of lipopolysaccharide (LPS) on platelet adhesion. We have therefore investigated the effects of LPS in human platelet adhesion, using an in vitro model of platelet adhesion to fibrinogen-coated wells. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 x 10(8) platelets/ml) were allowed to adhere. Adherent platelets were quantified through measurement of acid phosphatase activity. Calcium mobilization in Fura2-AM-loaded platelets was monitored with a spectrofluorimeter. Platelet flow cytometry in thrombin-stimulated platelets was performed using monoclonal mouse anti-platelet GPIIb/IIIa antibody (PAC-1). Prior incubation of washed platelets with LPS (0.01-300 microg/ml) for 5 to 60 min concentration- and time-dependently inhibited non-activated platelet adhesion. In thrombin-activated (50 mU/ml) platelets, LPS inhibited the adhesion to a significantly lesser extent than non-activated platelets. Cyclohexamide, superoxide dismutase polyethylene glycol (PEG-SOD) or catalase polyethylene glycol did not affect the LPS responses. No alterations in cyclic GMP levels were seen after platelet incubation with LPS, except with the highest concentration employed (300 microg/ml) where an increase of 36% (P < 0.05) was observed. Thrombin increased by 7.5-fold the internal Ca(2+) platelet levels, an effect markedly inhibited by LPS. Thrombin induced concentration-dependent platelet GPIIb/IIIa activation, but LPS failed to affect the activation state of this membrane glycoprotein. In conclusion, LPS inhibits human platelet adhesion to fibrinogen by mechanisms involving blockade of external Ca(2+), independently of cGMP generation and activation of GPIIb/IIIa complex.
Assuntos
Plaquetas , Lipopolissacarídeos/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Fosfatase Ácida/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Cálcio/metabolismo , Catalase/metabolismo , Células Cultivadas , GMP Cíclico/metabolismo , Cicloeximida/metabolismo , Fosfatase 2 de Especificidade Dupla/metabolismo , Fibrinogênio/metabolismo , Humanos , Camundongos , Testes de Função Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Inibidores da Síntese de Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Although there is evidence suggesting that statins may exert an endothelial protecting effect, recent in vitro data have shown that these compounds may induce endothelial cells (EC) apoptosis. We previously reported that the Fas-death receptor may induce apoptosis of the liver sinusoid endothelial cells (LSEC), and that TNF-alpha increases the susceptibility of these cells to suffer Fas-mediated apoptosis. Based on this evidence, in this study, we investigated the effect of simvastatin on Fas-mediated LSEC apoptosis. Simvastatin induced a significant reduction in LSEC viability, in a dose dependent manner, under serum-containing or serum-free conditions. This effect was prevented by mevalonate and GGPP, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. The simvastatin effect on LSEC death was not associated with increased activation of caspase-3. We found that simvastatin increased the susceptibility of LSEC death mediated by Fas. Further, simvastatin increased LSEC-apoptosis induced by Fas and TNF-alpha. Mevalonate and GGPP partially prevented simvastatin-induced sensitization to LSEC death mediated by Jo2 and TNF-alpha, but not Jo2 alone. Simvastatin did not induce up-regulation of the Fas on the LSEC. Our results provide evidence of simvastatin in modulating Fas-mediated apoptosis in endothelial cells. These results may have clinical implications in those clinical conditions associated with high levels of FasL and TNF-alpha.
Assuntos
Anticolesterolemiantes/farmacologia , Apoptose/efeitos dos fármacos , Células Endoteliais , Fígado/citologia , Sinvastatina/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Receptor fas/metabolismo , Animais , Anticorpos Antinucleares/metabolismo , Caspase 3/metabolismo , Sobrevivência Celular , Células Cultivadas , Cicloeximida/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Ativação Enzimática/efeitos dos fármacos , Proteína Ligante Fas/metabolismo , Humanos , Ácido Mevalônico/metabolismo , Camundongos , Fosfatos de Poli-Isoprenil/metabolismo , Inibidores da Síntese de Proteínas/metabolismoRESUMO
Foram realizados três experimentos para estudar os parâmetros de degradação protéica ruminal. No primeiro, foram incubadas, em líquido ruminal de bovinos, dietas isoprotéicas contendo capim-elefante, fubá de milho e farelo de soja, em cinco níveis de concentrado (0:100, 25:75, 50:50, 75:25 e 100:0), adicionado ou não de monensina (5µM). Houve efeito linear decrescente do nível de concentrado sobre a concentração de amônia e degradabilidade da proteína bruta (DPB), e efeito cúbico sobre a concentração de proteína solúvel, com máximo valor em dieta com 25 por cento de concentrado. A monensina diminuiu a DPB e a concentração de proteína solúvel, sem afetar a produção de amônia. No segundo experimento, foram incubados cinco diferentes volumosos (silagens de milho - Zea mays L. e de capim-elefante - Pennisetum purpureum, silagem pré-secada de braquiária -Brachiaria decumbens, feno de Tifton 85 -Cynodon sp. amonizado e feno de Tifton 85). A silagem pré-secada de capim-braquiária e o feno de Tifton 85 amonizado apresentaram as maiores concentrações de amônia (8,7 e 5,3mg/dl) e proteína solúvel (5,4 e 7,0mg/dl), devido aos seus maiores teores de PB, seguidos da silagem de capim-elefante e feno de Tifton 85. A DPB variou de 29,6 a 80,6 por cento, para a silagem pré-secada de braquiária e para o feno de Tifton 85, e a degradabilidade potencial da matéria seca de 40,1 a 64,3 por cento, para a silagem de capim-elefante e silagem pré-secada de braquiária, respectivamente. A degradabilidade efetiva da proteína bruta apresentou baixos valores devido à baixa taxa de degradação da fração insolúvel. No terceiro experimento, foram incubados diferentes tipos de camas de frango (casca de café, capim-elefante seco picado, sabugo de milho ou cepilho), contendo ou não monensina (5µM). Não houve diferença nas concentrações de amônia entre as diferentes camas de frango, na ausência de monensina. Entretanto, com monensina, a cama de capim-elefante apresentou o...
Three experiments were carried out in order to study the parameters of ruminal protein degradation. In the first, isoproteic diets, constituted of elephant grass, ground corn and soybean meal, at five concentrate levels (0:100, 25:75, 50:50, 75:25 e 100:0), with or without monensin (5µM), were incubated in ruminal fluid of bovines. There was a decreasing linear effect of the concentrate level on ammonia concentration and degradability of crude protein, and cubic effect in soluble protein concentration, with the largest value in the diet with 25 percent concentrate. Monensin decreased degradability of crude protein and soluble protein concentration with no effect on ammonia production. In the second, five different roughages (corn and elephant grass silage - Pennisetum purpureum, Brachiaria haylage - Brachiaria decumbens, ammoniated Tifton 85 hay - Cynodon sp. e Tifton 85 hay). The were incubated Brachiaria haylage and the ammoniated Tifton 85 hay showed the greatest concentrations of ammonia (8.7 and 5.3mg/dl) and soluble protein (5.4 and 7.0mg/dl), due to their higher crude protein content, followed by elephant grass silage and Tifton 85 hay. The degradability of crude protein ranged from 29.6 to 80.6 percent for Brachiaria haylage and Tifton 85 hay, and the degradability of dry matter ranged from 40.1 to 64.3 percent for elephant grass silage and Brachiaria haylage, respectively. The effective degradability of crude protein showed low values due to low degradation rate of the insoluble fraction. In the third, four different poultry litter (hulls coffee, shopped dry elephant grass, corn cobs and wood shavings) were incubated, with or without monensin (5µM). No difference in ammonia concentration among the poultry litter samples, was observed in the absence of monensin. However, when monensin was present, the grass poultry litter showed the lowest ammonia level and wood poultry litter the highest. The poultry litter influenced the soluble...
Assuntos
Bovinos , Inibidores da Síntese de Proteínas/metabolismo , Proteínas/metabolismo , Rúmen/fisiologiaRESUMO
Foram realizados três experimentos para estudar os parâmetros de degradação protéica ruminal. No primeiro, foram incubadas, em líquido ruminal de bovinos, dietas isoprotéicas contendo capim-elefante, fubá de milho e farelo de soja, em cinco níveis de concentrado (0:100, 25:75, 50:50, 75:25 e 100:0), adicionado ou não de monensina (5µM). Houve efeito linear decrescente do nível de concentrado sobre a concentração de amônia e degradabilidade da proteína bruta (DPB), e efeito cúbico sobre a concentração de proteína solúvel, com máximo valor em dieta com 25 por cento de concentrado. A monensina diminuiu a DPB e a concentração de proteína solúvel, sem afetar a produção de amônia. No segundo experimento, foram incubados cinco diferentes volumosos (silagens de milho - Zea mays L. e de capim-elefante - Pennisetum purpureum, silagem pré-secada de braquiária -Brachiaria decumbens, feno de Tifton 85 -Cynodon sp. amonizado e feno de Tifton 85). A silagem pré-secada de capim-braquiária e o feno de Tifton 85 amonizado apresentaram as maiores concentrações de amônia (8,7 e 5,3mg/dl) e proteína solúvel (5,4 e 7,0mg/dl), devido aos seus maiores teores de PB, seguidos da silagem de capim-elefante e feno de Tifton 85. A DPB variou de 29,6 a 80,6 por cento, para a silagem pré-secada de braquiária e para o feno de Tifton 85, e a degradabilidade potencial da matéria seca de 40,1 a 64,3 por cento, para a silagem de capim-elefante e silagem pré-secada de braquiária, respectivamente. A degradabilidade efetiva da proteína bruta apresentou baixos valores devido à baixa taxa de degradação da fração insolúvel. No terceiro experimento, foram incubados diferentes tipos de camas de frango (casca de café, capim-elefante seco picado, sabugo de milho ou cepilho), contendo ou não monensina (5µM). Não houve diferença nas concentrações de amônia entre as diferentes camas de frango, na ausência de monensina. Entretanto, com monensina, a cama de capim-elefante apresentou o...(AU)
Three experiments were carried out in order to study the parameters of ruminal protein degradation. In the first, isoproteic diets, constituted of elephant grass, ground corn and soybean meal, at five concentrate levels (0:100, 25:75, 50:50, 75:25 e 100:0), with or without monensin (5µM), were incubated in ruminal fluid of bovines. There was a decreasing linear effect of the concentrate level on ammonia concentration and degradability of crude protein, and cubic effect in soluble protein concentration, with the largest value in the diet with 25 percent concentrate. Monensin decreased degradability of crude protein and soluble protein concentration with no effect on ammonia production. In the second, five different roughages (corn and elephant grass silage - Pennisetum purpureum, Brachiaria haylage - Brachiaria decumbens, ammoniated Tifton 85 hay - Cynodon sp. e Tifton 85 hay). The were incubated Brachiaria haylage and the ammoniated Tifton 85 hay showed the greatest concentrations of ammonia (8.7 and 5.3mg/dl) and soluble protein (5.4 and 7.0mg/dl), due to their higher crude protein content, followed by elephant grass silage and Tifton 85 hay. The degradability of crude protein ranged from 29.6 to 80.6 percent for Brachiaria haylage and Tifton 85 hay, and the degradability of dry matter ranged from 40.1 to 64.3 percent for elephant grass silage and Brachiaria haylage, respectively. The effective degradability of crude protein showed low values due to low degradation rate of the insoluble fraction. In the third, four different poultry litter (hulls coffee, shopped dry elephant grass, corn cobs and wood shavings) were incubated, with or without monensin (5µM). No difference in ammonia concentration among the poultry litter samples, was observed in the absence of monensin. However, when monensin was present, the grass poultry litter showed the lowest ammonia level and wood poultry litter the highest. The poultry litter influenced the soluble...(AU)
Assuntos
Rúmen/fisiologia , Proteínas/metabolismo , Inibidores da Síntese de Proteínas/metabolismo , BovinosRESUMO
Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule-stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil-specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30-60 min with eotaxin, regulated on activation, normal, T-cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule-stored cytokines and cationic proteins.
Assuntos
Degranulação Celular , Proteínas Granulares de Eosinófilos/metabolismo , Eosinófilos/metabolismo , Eosinófilos/ultraestrutura , Membranas Intracelulares/metabolismo , Antígenos CD/metabolismo , Brefeldina A/metabolismo , Quimiocina CCL11 , Quimiocina CCL5/metabolismo , Quimiocinas CC/metabolismo , Fatores Quimiotáticos de Eosinófilos/metabolismo , Proteína Básica Maior de Eosinófilos/metabolismo , Humanos , Imageamento Tridimensional , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Inibidores da Síntese de Proteínas/metabolismo , Tetraspanina 30RESUMO
Gangliosides, complex glycosphingolipids containing sialic acids, have been found to reside in glycosphingolipid-enriched microdomains (GEM) at the plasma membrane. They are synthesized in the lumen of the Golgi complex and appear unable to translocate from the lumenal toward the cytosolic surface of Golgi membrane to access the monomeric lipid transport. As a consequence, they can only leave the Golgi complex via the lumenal surface of transport vesicles. In this work we analyzed the exocytic transport of the disialo ganglioside GD3 from trans-Golgi network (TGN) to plasma membrane in CHO-K1 cells by immunodetection of endogenously synthesized GD3. We found that ganglioside GD3, unlike another luminal membrane-bounded lipid (glycosylphosphatidylinositol-anchored protein), did not partition into GEM domains in the Golgi complex and trafficked from TGN to plasma membrane by a brefeldin A-insensitive exocytic pathway. Moreover, a dominant negative form of Rab11, which prevents exit of vesicular stomatitis virus glycoprotein from the Golgi complex, did not influence the capacity of GD3 to reach the cell surface. Our results strongly support the notion that most ganglioside GD3 traffics from the TGN to the plasma membrane by a non-conventional vesicular pathway where lateral membrane segregation of vesicular stomatitis virus glycoprotein (non-GEM resident) and glycosylphosphatidylinositol-anchored proteins (GEM resident) from GD3 is required before exiting TGN.
Assuntos
Brefeldina A/metabolismo , Membrana Celular/metabolismo , Exocitose/fisiologia , Gangliosídeos/metabolismo , Inibidores da Síntese de Proteínas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismo , Animais , Transporte Biológico/fisiologia , Células CHO , Ceramidas/metabolismo , Cricetinae , Detergentes/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Glicoproteínas de Membrana/metabolismo , Octoxinol/metabolismo , Propanolaminas/metabolismo , Pirrolidinas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Esfingomielinas/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
AIMS: To investigate the effect of the culture supernatants from three newly isolated Streptomyces strains, 221, 235 and 606 on eukaryotic cells. METHODS AND RESULTS: Cell lines were treated with the culture filtrates and assayed for protein synthesis by metabolic labelling, followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. RNA synthesis was investigated by [5-3H]uridine incorporation. The three culture filtrates presented a strong inhibitory activity, reducing total protein synthesis of different eukaryotic cell lines by more than 85%. No effect on cellular RNA synthesis was detected. The culture filtrates did not affect the growth of the prokaryotic cells tested. CONCLUSIONS: These new Streptomyces strains, recently isolated from Brazilian tropical soils, produce molecule(s) with inhibitory activity specific to eukaryote protein synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptomyces strains 221, 235 and 606, probably representing new species, might produce new bioactive compound(s), and can be used as valuable tools to study the protein synthesis pathway in eukaryotes.
Assuntos
Inibidores da Síntese de Proteínas/metabolismo , Streptomyces/metabolismo , Células 3T3 , Animais , Brasil , Linhagem Celular , Meios de Cultura , Humanos , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/isolamento & purificação , Inibidores da Síntese de Proteínas/farmacologia , RNA/biossíntese , Microbiologia do Solo , Streptomyces/efeitos dos fármacos , Streptomyces/crescimento & desenvolvimento , Streptomyces/isolamento & purificação , Clima TropicalRESUMO
Streptomyces venezuelae synthesizes chloramphenicol (Cm), an inhibitor of ribosomal peptidyl transferase activity, thereby inhibiting bacterial growth. The producer escapes autoinhibition by its own secondary metabolite through phosphorylation of Cm by chloramphenicol phosphotransferase (CPT). In addition to active site binding, CPT binds its product 3-phosphoryl-Cm, in an alternate product binding site. To address the mechanisms of Cm tolerance of the producer, the crystal structures of CPT were determined in complex with either the nonchlorinated Cm (2-N-Ac-Cm) at 3.1 A resolution or the antibiotic's immediate precursor, the p-amino analog p-NH(2)-Cm, at 2.9 A resolution. Surprisingly, p-NH(2)-Cm binds CPT in a novel fashion. Additionally, neither 2-N-Ac-Cm nor p-NH(2)-Cm binds to the secondary product binding site.
Assuntos
Proteínas de Bactérias , Resistência ao Cloranfenicol , Cloranfenicol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Streptomyces/efeitos dos fármacos , Streptomyces/enzimologia , Sítios de Ligação , Cloranfenicol/química , Cloranfenicol/farmacologia , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/metabolismoRESUMO
In previous work we showed that apoptosis in retinal tissue from developing rats can be induced by inhibition of protein synthesis (Rehen et al. 1996, Development, 122, 1439-1448). Here we show that recent postmitotic cells are the cells sensitive to apoptosis triggered by blockade of protein synthesis. To label all proliferating cells in the retina, a series of injections of the nucleotide analogue, bromo-deoxy-uridine (BrdU, 60 mg/kg b.w.), was given in rat pups. Then, explants of the retina were incubated in vitro with the inhibitor of protein synthesis anisomycin (1.0-3.2 microg/mL) for 1 day to induce apoptosis. Detection of apoptotic bodies under differential interference contrast microscopy was combined with immunocytochemistry for BrdU, proliferating cell nuclear antigen (PCNA) or for various markers of retinal cell differentiation. Despite the large number of BrdU- and PCNA-labelled cells in the tissue, the vast majority of the cells that underwent apoptosis were postmitotic cells which have left the mitotic cycle 3-4 days before. However, these cells were not labelled with antibodies to calretinin, calbindin, rhodopsin or to a Muller glial cell marker, suggesting that these are early postmitotic neurons. We suggest that during migration and initial differentiation, the apoptotic machinery is blocked by suppressor proteins, thus allowing recent postmitotic cells to find their final positions and differentiate while protected from apoptosis.
Assuntos
Apoptose , Mitose , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas/metabolismo , Retina/citologia , Retina/metabolismo , Animais , Anisomicina/farmacologia , Bromodesoxiuridina , Calbindina 2 , Calbindinas , Diferenciação Celular , Divisão Celular , Células Cultivadas , Imuno-Histoquímica , Antígeno Nuclear de Célula em Proliferação/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Retina/efeitos dos fármacos , Rodopsina/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
An inhibitory activity blocking protein synthesis elongation in several eukaryotic systems has been detected in Leishmania mexicana extracts. This factor, which competes with aminoacylation of tRNA and also affects the subsequent polymerization step, is a strong inhibitor of polypeptide synthesis induced by poly U in wheat-germ extracts or by endogenous mRNAs in rat liver cell-free systems. The purified translational inhibitor has shown to be essentially free of proteins. Several chemical and biochemical properties of the inhibition factor have supported the conclusion that it behaves as a 200 bases RNA with a high content of secondary structure.