RESUMO
Genetic variation in the IL-28B (interleukin-28B; interferon lambda 3) region has been associated with sustained virological response (SVR) rates in patients with chronic hepatitis C treated with peginterferon-α and ribavirin. However, the mechanisms by which polymorphisms in the IL-28B gene region affect host antiviral responses are not well understood. Using the HCV 1b and 2a replicon system, we compared the effects of IFN-λs and IFN-α on HCV RNA replication. The anti-HCV effect of IFN-λ3 and IFN-α in combination was also assessed. Changes in gene expression induced by IFN-λ3 and IFN-α were compared using cDNA microarray analysis. IFN-λs at concentrations of 1 ng/mL or more exhibited concentration- and time-dependent HCV inhibition. In combination, IFN-λ3 and IFN-α had a synergistic anti-HCV effect; however, no synergistic enhancement was observed for interferon-stimulated response element (ISRE) activity or upregulation of interferon-stimulated genes (ISGs). With respect to the time course of ISG upregulation, the peak of IFN-λ3-induced gene expression occurred later and lasted longer than that induced by IFN-α. In addition, although the genes upregulated by IFN-α and IFN-λ3 were similar to microarray analysis, interferon-stimulated gene expression appeared early and was prolonged by combined administration of these two IFNs. In conclusion, IFN-α and IFN-λ3 in combination showed synergistic anti-HCV activity in vitro. Differences in time-dependent upregulation of these genes might contribute to the synergistic antiviral activity.
Assuntos
Antivirais/farmacologia , Produtos Biológicos/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Interferon-alfa/farmacologia , Interleucinas/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Interferons , Análise em MicrossériesRESUMO
We isolated the gene encoding a botulinum neurotoxin (BoNT) of 1285 amino acids with a molecular weight of 147,364 from the toxigenic bacteriophage d-sA of Clostridium botulinum type D strain South African (Dsa). The BoNT of Dsa (BoNT/Dsa) is composed of three regions on the basis of the homology to BoNT types C1 (BoNT/C1) and D (BoNT/D). The N-terminal (Met-1 to Val-522) and the C-terminal regions (Trp-945 to Glu-1285) have high identity to corresponding regions of BoNT/D (96% identity) and BoNT/C1 (74% identity), respectively. The core region (Pro-523 to Lys-944) is common to three toxins (83% to 92% identity). These results suggest that neurotoxins produced from Clostridium botulinum types C and D are composed in a mosaic-like fashion.
Assuntos
Toxinas Botulínicas/genética , Clostridium botulinum/metabolismo , Mosaicismo , Sequência de Aminoácidos , Clonagem Molecular , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Anti-apoptotic members of the Bcl-2 family, such as Bcl-w, maintain cell viability by preventing the activation of the cell death effectors, the caspases. Gene targeting experiments in mice have demonstrated that Bcl-w is required for spermatogenesis and for survival of damaged epithelial cells in the gut. Bcl-w is, however, dispensable for physiological cell death in other tissues. Here we report on the analysis of Bcl-w protein expression using a panel of novel monoclonal antibodies. Bcl-w is found in a diverse range of tissues including colon, brain and testes. A survey of transformed cell lines and purified hematopoietic cells demonstrated that Bcl-w is expressed in cells of myeloid, lymphoid and epithelial origin. Subcellular fractionation and confocal laser scanning microscopy demonstrated that Bcl-w protein is associated with intracellular membranes. The implications of these results are discussed in the context of the phenotype of Bcl-w-null mice and recent data that suggest that Bcl-w may play a role in colon carcinogenesis.
Assuntos
Perfilação da Expressão Gênica , Proteínas/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Proteínas Reguladoras de Apoptose , Western Blotting , Linhagem Celular , Imunofluorescência , Humanos , Hibridomas/citologia , Hibridomas/imunologia , Membranas Intracelulares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Testes de Precipitina , Transporte Proteico , Proteínas/genética , Proteínas/imunologia , Proteínas Proto-Oncogênicas c-bcl-2 , Ratos , Ratos Wistar , TransfecçãoRESUMO
In rats, maternal exposure to restraint stress during pregnancy can induce abnormalities in the cardiovascular and central nervous systems of the offspring. These effects are mediated by long-lasting hyperactivation of the hypothalamic-pituitary-adrenal axis. However, little is known about the potential effects of stress during pregnancy on metabolic systems. We examined the effect of restraint stress in pregnant mice on the liver function of their offspring. The offspring of stressed mothers showed significantly higher lipid accumulation in the liver after weaning than did the controls; this accumulation was associated with increased expression of lipid metabolism-related proteins such as alanine aminotransferase 2 diglyceride acyltransferase 1, peroxisome proliferator-activated receptor gamma and glucocorticoid receptor. Additionally, we observed increased levels of 11ß-hydroxysteroid dehydrogenase type 1, an intercellular mediator that converts glucocorticoid from the inactive to the active form, in the foetal and postnatal periods. These results indicate that restraint stress in pregnancy in mice induces metabolic abnormalities via 11ß-hydroxysteroid dehydrogenase type 1-related pathways in the foetal liver. It is therefore possible that exposure to stress in pregnant women may be a risk factor for metabolic syndromes (e.g. fatty liver) in children.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Efeitos Tardios da Exposição Pré-Natal , Estresse Psicológico , Animais , Animais Recém-Nascidos , Feminino , Feto/metabolismo , Expressão Gênica , Camundongos Endogâmicos C57BL , GravidezRESUMO
In beta-escin-permeabilized cultured pig aortic smooth muscle cells GTP gamma S dose-dependently enhances Ca(2+)-induced, wortmannin-sensitive phosphorylation of 20 kDa myosin light chain (MLC20). GTP gamma S does not potentiate thiophosphorylation of MLC20, but does inhibit its dephosphorylation. Pretreatment with C. botulinum exotoxin C3, which specifically ADP-ribosylates and inactivates the rho family of the small molecular weight G proteins, completely abolishes the effects of GTP gamma S. These results indicate that rho is involved in the GTP gamma S-induced enhancement of Ca(2+)-dependent MLC20 phosphorylation in aortic smooth muscle cells, and strongly suggest that this effect of rho is due to inhibition of protein phosphatase activity toward MLC20.
Assuntos
Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Músculo Liso Vascular/metabolismo , Miosinas/metabolismo , Animais , Toxinas Botulínicas/farmacologia , Cálcio/farmacologia , Células Cultivadas , Escina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Técnicas In Vitro , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosforilação , SuínosRESUMO
Screening assays are the most time-consuming and labor-intensive part of generating monoclonal antibodies (MAbs). Antibodies identified by enzyme-linked immunosorbent assay (ELISA) screening often are not suitable for their intended application such as immunofluorescence staining. We describe here a rapid and efficient flow cytometric screening procedure for the identification of MAbs directed against low-abundance cytoplasmic proteins, in our case, the pro-apoptotic molecule Bim. Cells from an equal mixture of a parental cell line and a subline expressing Bim were fixed, permeabilized and incubated with hybridoma supernatants. The supernatants were derived from a fusion of Sp2/0 plasmacytoma cells and spleen cells from a rat immunized with recombinant glutathione-S-transferase (GST)-BimL fusion protein. Secondary staining with fluorochrome-labeled anti-rat Ig antibodies allowed detection of clones expressing Bim-specific antibodies. The screening procedure was rapid and efficient, and most monoclonal antibodies identified were proven to be useful for immunofluorescence staining and other applications.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Proteínas de Transporte/imunologia , Hibridomas/imunologia , Proteínas de Membrana , Proteínas Proto-Oncogênicas , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Proteínas Reguladoras de Apoptose , Proteína 11 Semelhante a Bcl-2 , Western Blotting , Proteínas de Transporte/genética , Linhagem Celular , Citometria de Fluxo , Humanos , Hibridomas/citologia , Camundongos , Testes de Precipitina , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Células Tumorais CultivadasRESUMO
1. The aim of this study was to determine whether different signal transduction mechanisms underlie the Ca2+ sensitizing effects of guanosine 5'-O-(3-thiotriphosphate) (GTP(gamma)S) and receptor agonists on beta-escin-skinned smooth muscle of rabbit mesenteric artery. 2. In the homogenate of the beta-escin-skinned arterial strip, C3 exoenzyme of Clostridium botulinum catalyzed the [32P]-ADP-ribosylation of only one protein that had the same molecular mass as the protein detected in Western blots with anti-rho p21 antibody. Pretreatment of preparations with C3 resulted in great inhibition of GTP(gamma)S-induced Ca2+ sensitization, although the effect of GTP(gamma)S at higher concentrations (> or = 30 microM) was not completely blocked by this treatment. In contrast, the enhancement by phenylephrine and histamine, in the presence of guanosine 5'-triphosphate, of the Ca2+-induced contraction was not affected by C3 pretreatment. 3. The protein kinase C (PKC) inhibitors calphostin C and staurosporine completely eliminated the enhancement by phorbol ester 12,13-dibutyrate of the Ca2+-induced contraction. However, these PKC inhibitors had no effect on GTP(gamma)S- and receptor agonist-induced Ca2+ sensitization. 4. The tyrosine kinase inhibitors genistein and tyrphostin 25 caused an irreversible and complete block of the enhancement by GTP(gamma)S of the Ca2+-induced contraction without affecting this Ca2+ contraction. The inactive genistein analogue daidzein did not modify the effect of GTP(gamma)S. The Ca2+ sensitizing effects of phenylephrine and histamine were also blocked by these tyrosine kinase inhibitors. 5. These results suggest that rho p21 predominantly mediates GTP(gamma)S-induced Ca2+ sensitization of beta-escin-skinned smooth muscle of rabbit mesenteric artery, while the Ca2+ sensitizing actions of heterotrimeric G protein-coupled receptor agonists do not involve this small G protein. However, it seems that tyrosine phosphorylation, but not PKC activation, plays an important role in both of the rho p21 protein- and heterotrimeric G protein-mediated Ca2+ sensitization mechanisms.
Assuntos
Toxinas Botulínicas , Cálcio/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Artérias Mesentéricas/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Tirosina/metabolismo , ADP Ribose Transferases/metabolismo , ADP Ribose Transferases/farmacologia , Difosfato de Adenosina/metabolismo , Animais , Carcinógenos/farmacologia , Dimerização , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/química , Genisteína/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Trifosfato/farmacologia , Histamina/farmacologia , Técnicas In Vitro , Masculino , Artérias Mesentéricas/fisiologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/fisiologia , Naftalenos/farmacologia , Fenilefrina/farmacologia , Dibutirato de 12,13-Forbol/farmacologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Coelhos , Ribose/metabolismo , Estaurosporina/farmacologia , Tirfostinas/farmacologia , Vasoconstritores/farmacologia , Proteínas rho de Ligação ao GTPRESUMO
ADP-ribosyltransferase from Clostridium botulinum type C strain was found to induce an increase of inositol phosphates (IPs) formation in murine thymocytes membranes. Incubation of electropermeabilized murine thymocytes with the enzyme also caused an increase of IPs formation in the cells. This increase of IPs formation in the enzyme-treated membranes and electropermeabilized cells was dependent on the amount of both NAD and the enzyme, suggesting that the stimulation of phosphoinositide-specific phospholipase C (PLC) was related to ADP-ribosylation of membrane proteins by the enzyme. On the other hand, in calf and murine thymocytes two proteins with the same molecular weight of 21,000 were found to be ADP-ribosylated by the botulinum ADP-ribosyltransferase. A minor ADP-ribosylation substrate was shown by two-dimensional polyacrylamide gel electrophoresis to be G21k, a low-molecular-weight GTP-binding protein (G protein) suggested previously by us to be involved in PLC regulation [Wang, P. et al. (1987) J. Biochem. 102, 1275-1287; (1988) 103, 137-142; and (1989) 105, 461-466], and the other major ADP-ribosylation substrate was identified as a rho A protein. Under the experimental conditions of the IPs formation study, ADP-ribosylation of both G21k and rho A proteins by botulinum ADP-ribosyltransferase in membranes and permeabilized cells was observed. These results suggest that botulinum ADP-ribosyltransferase-induced PLC stimulation in thymocytes is closely correlated with ADP-ribosylation of the low-molecular-weight G proteins.
Assuntos
Clostridium botulinum/enzimologia , Proteínas de Ligação ao GTP/química , Fosfatidilinositóis/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Linfócitos T/metabolismo , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Dados de Sequência Molecular , Peso Molecular , Fosfatidilinositol Diacilglicerol-Liase , Diester Fosfórico Hidrolases/metabolismo , Especificidade por Substrato , Linfócitos T/enzimologiaRESUMO
Listeria monocytogenes replicates in a phagocytic cell following escape into the host cytoplasm. Listeriolysin O, secreted by L. monocytogenes, which belongs to the thiol-activated hemolysin family, is known to play an important role in the escape of the bacterium into the host cytoplasm. In this study, we demonstrated that expression of listeriolysin O by infecting L. monocytogenes was lightly induced in J774.1 macrophage-like cells pretreated with lipopolysaccharide, although the growth of the bacteria was suppressed. The number of viable L. monocytogenes decreased until 4 h post-infection and then increased between 4 and 8 h post-infection in untreated J774.1 host cells, but it decreased until 8 h post-infection in lipopolysaccharide-treated host cells. However, expression of listeriolysin O by L. monocytogenes was not induced in the untreated host cells, while it increased 0 and 4 h post-infection in the lipopolysaccharide-treated host cells. Expression of listeriolysin O mRNA in the lipopolysaccharide-like cells. These results suggest that macrophage activation induced with lipopolysaccharide could lead to the expression of the listeriolysin O gene and the synthesis of listeriolysin O protein under suppression of the intracellular growth of L. monocytogenes.
Assuntos
Toxinas Bacterianas , Proteínas de Choque Térmico/biossíntese , Proteínas Hemolisinas/biossíntese , Lipopolissacarídeos/farmacologia , Listeria monocytogenes/metabolismo , Macrófagos/microbiologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Compartimento Celular , Citoplasma/microbiologia , Expressão Gênica , Vetores Genéticos , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Fagossomos/microbiologia , RNA Bacteriano/análise , RNA Mensageiro/análise , Proteínas RecombinantesRESUMO
Botulinum type D neurotoxin inhibited Ca(2+)-evoked norepinephrine secretion in digitonin permeabilized PC12 cells. Inhibition by the toxin required prior incubation with dithiothreitol (DTT). The inhibition was dependent on both concentration and incubation times of the toxin, and was affected by Ca2+ concentration. With less than 0.7 microM Ca2+ almost complete inhibition was observed; however, above 0.7 microM, Ca2+ stimulated additional norepinephrine release in a dose-dependent manner.
Assuntos
Toxinas Botulínicas/farmacologia , Digitonina/farmacologia , Norepinefrina/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Cálcio/metabolismo , Cálcio/fisiologia , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Células PC12 , Ésteres de Forbol/farmacologia , RatosRESUMO
The molecular properties of Clostridium botulinum type D South African (D-SA) were compared with those of neurotoxins from type D strain 1873 (D-1873) and type C strains Stockholm and 6813. D-SA toxin, purified 610-fold from the culture supernatant in an overall yield of 30%, consisted of an intact peptide chain with a molecular weight of 140,000. Limited proteolysis of the toxin by trypsin formed a dichain structure consisting of a light chain (Mr, 50,000) and a heavy chain (Mr, 90,000) linked by a disulfide bond(s) and enhanced the lethal activity about fourfold. Antibodies against the D-SA toxin light chain reacted with D-1873 toxin but not with C1 toxins. On the other hand, antibodies against the heavy chain of D-SA toxin cross-reacted with type C strain Stockholm, D-1873, and type C strain 6813 toxins in that order. Amino-terminal sequences of heavy and light chains of D-SA and D-1873 toxins were similar but not identical. These results indicate that within the type D strains, neurotoxins differ in molecular structure and antigenicity.
Assuntos
Toxinas Botulínicas/isolamento & purificação , Clostridium botulinum/imunologia , Neurotoxinas/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/isolamento & purificação , Antígenos de Bactérias/imunologia , Toxinas Botulínicas/imunologia , Reações Cruzadas , Ponto Isoelétrico , Dados de Sequência Molecular , Neurotoxinas/imunologiaRESUMO
The region encoding proline-rich units of actA genes was amplified from 24 strains of Listeria monocytogenes using polymerase chain reaction (PCR). PCR products of 13 strains showed the expected size of 623 bp, whereas those of 11 strains showed a short size of 518 bp. The shortening of these PCR products resulted from the deletion of one proline-rich unit. These results indicate that ActA proteins are divided into at least two different types which are unrelated to bacterial serotypes.
Assuntos
Genes Bacterianos/genética , Listeria monocytogenes/genética , Adulto , Idoso , Sequência de Bases , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA/métodosRESUMO
Lactoferrin (LFR) plays an important role in the anti-microbial defense through iron binding, lipopolysaccharide binding and immunomodulation. In this study, we demonstrate that bovine LFR specifically inhibits the hemolytic activity of listeriolysin O (LLO) produced by Listeria monocytogenes. The hemolytic activity of LLO was completely inhibited in the presence of bovine LFR that was highly purified on two cation-exchange columns, whereas that of streptolysin O or perfringolysin O was not inhibited at all. A rabbit anti-LFR antibody canceled this inhibitory activity of bovine LFR. Although human transferrin exhibits 62% amino acid identity with bovine LFR, human apo-transferrin could not inhibit LLO-induced hemolysis. An increase in the concentration of FeCl3 or the Fe3+-saturation of bovine LFR, however, slightly reduced its inhibition of the hemolysis. The inhibitory activity of bovine LFR was dependent on pH, since it was observed under neutral and alkali conditions, but not under acidic conditions. These results suggest that the inhibition of LLO-induced hemolysis by bovine LFR is influenced by pH and iron ions, both of which may lead to conformational changes of LFR.
Assuntos
Toxinas Bacterianas , Proteínas de Choque Térmico/farmacologia , Proteínas Hemolisinas/farmacologia , Hemólise/efeitos dos fármacos , Lactoferrina/farmacologia , Animais , Proteínas de Bactérias , Bovinos , Endopeptidases/química , Eritrócitos/efeitos dos fármacos , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/biossíntese , Proteínas Hemolisinas/genética , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ferro/sangue , Ferro/farmacologia , Lactoferrina/isolamento & purificação , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Coelhos , Ovinos , Estreptolisinas/farmacologia , Tiorredoxinas/farmacologiaRESUMO
Neurotoxin from Clostridium botulinum type D strain South African (neurotoxin D) has shown ADP-ribosylation activity as well as toxic activity (Matsuoka, I., Sakuma, H., Syuto, B., Moriishi, K., Kubo, S., and Kurihara, K. (1989) J. Biol. Chem. 264, 706-712). Separation of these activities from each other was attempted by means of gel filtration, hydroxylapatite column chromatography, or immunoaffinity chromatography. Approximately 90% of toxic activity was recovered in each chromatography. Although ADP-ribosylation activity was incompletely separated from neurotoxin D by gel filtration, it was separated by hydroxylapatite column chromatography. In immunoaffinity chromatography with a column of Sepharose 4B coupling antibodies against botulinum ADP-ribosyltransferase, no ADP-ribosylation activity was detected by autoradiography in the unabsorbed toxic fraction. These results indicate that neurotoxin D does not have ADP-ribosylation activity.
Assuntos
Toxinas Botulínicas/isolamento & purificação , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/toxicidade , Cromatografia , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Durapatita , Hidroxiapatitas , Peso Molecular , Poli(ADP-Ribose) Polimerases/isolamento & purificação , Poli(ADP-Ribose) Polimerases/metabolismo , Especificidade por SubstratoRESUMO
We examined production of ADP-ribosyltransferase C3 in 11 strains of Clostridium botulinum type C and D and their nontoxigenic derivatives. Antisera to C3 proteins of type C organisms divided C3 proteins roughly into at least two groups, bearing no relation to their bacterial types. The C3 gene of type D strain South African was isolated from a toxigenic phage library, and the complete sequence of the C3 gene was determined. The C3 protein of type D strain South African had 98% homology to the C3 protein of type C strain 003-9 and 66% homology to that of type D strain 1873. These results indicate that there are two types of C3 protein in type D organisms, as there are in type C organisms.
Assuntos
ADP Ribose Transferases/genética , Toxinas Botulínicas , Clostridium botulinum/enzimologia , Clostridium botulinum/genética , ADP Ribose Transferases/classificação , ADP Ribose Transferases/imunologia , Sequência de Aminoácidos , Anticorpos Antibacterianos , Sequência de Bases , Clonagem Molecular , Clostridium botulinum/imunologia , DNA Bacteriano/genética , Genes Bacterianos , Lisogenia , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
By cation-exchange column chromatography followed by gel filtration or hydroxylapatite column chromatography, ADP-ribosyltransferases (exoenzyme C3) were isolated from culture supernatants of Clostridium botulinum type C strains Stockholm (CST) and 6813 (C6813) and from type D strains South African (DSA) and 1873 (D1873), and their molecular properties were compared. The purified C3 enzymes were homogeneous in polyacrylamide gel electrophoresis. The C3 enzymes existed as single-chain polypeptides with molecular masses of 25.0 to 25.5 kDa and transferred ADP-riboses to the same substrates in rat brain membrane extract. The C3 enzymes could be roughly classified into two groups with respect to amino acid composition, amino-terminal sequence, and antigenicity. One group contains the C3 enzymes of strains C6813 and DSA, and the other contains those of strains CST and D1873. The specific activity of the C3 enzyme of strain C6813 was about 15 times higher than that of the C3 enzyme of strain CST. These results indicate that the classification of the C3 molecules differs from that of the neurotoxin molecules.
Assuntos
ADP Ribose Transferases/química , Toxinas Botulínicas , Clostridium botulinum/enzimologia , ADP Ribose Transferases/isolamento & purificação , Sequência de Aminoácidos , Dados de Sequência Molecular , Peso Molecular , Especificidade por SubstratoRESUMO
Clostridium botulinum D (strain South Africa) produces ADP-ribosyltransferase which modifies eukaryotic 24-26-kDa proteins. ADP-ribosyltransferase activity was associated with a neurotoxin of 150 kDa (Dsa toxin) as confirmed by the elution profile of Dsa toxin from high performance anion-exchange column. The 24-kDa substrate of Dsa toxin-catalyzed ADP-ribosylation was detected in several tissues examined including rat brain, heart, and liver; bovine adrenal medulla; sea urchin eggs; electric organs of electric fish; and cell lines of neural (N18, N1E115, NS20Y, NG108, PC12, and C6) and non-neural (3T3) origins, suggesting its ubiquitous localization in eukaryotic cells. On the other hand, the 26-kDa substrate was detected only in membrane fractions of neural tissues and neuronal cells, suggesting its specific localization in membrane of nerve terminals. ADP-ribosylation of both the 24-kDa substrate in PC12 membrane and the 24-26-kDa substrates in rat brain membrane was potentiated by either divalent cations or guanine nucleotides, whereas adenine nucleotides did not affect the ADP-ribosylation reaction. Trypsin digestion of the 24-kDa substrate in PC12 membrane and the 24-26-kDa substrates in rat brain membrane extract produced different tryptic fragments indicative of the structural difference between the 24- and 26-kDa substrates. Both the 24- and 26-kDa substrates were less sensitive to trypsin digestion before being ADP-ribosylated by Dsa toxin than after, suggesting the conformational alterations of the 24-26-kDa proteins induced by ADP-ribosylation. These results suggest that Dsa toxin modifies two distinct low molecular mass GTP-binding proteins by ADP-ribosylation to alter their putative function(s).
Assuntos
ADP Ribose Transferases/metabolismo , Toxinas Botulínicas/metabolismo , Encéfalo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Nucleotídeos de Adenina/metabolismo , Animais , Toxinas Botulínicas/isolamento & purificação , Linhagem Celular , Órgão Elétrico/metabolismo , Electrophorus , Nucleotídeos de Guanina/metabolismo , Cinética , Peso Molecular , Especificidade de Órgãos , Ratos , TorpedoRESUMO
The genetic structure of neurotoxin genes in a Clostridium botulinum strain producing both type A and B neurotoxins (type AB) was investigated. Analyses by polymerase chain reaction (PCR) using type-specific primers corresponding to the coding regions for N-terminals of light-chains and C-terminals of heavy-chains of type A and type B neurotoxins, and Southern hybridization of total DNA showed that the type AB strain I.P.7212 carries at least one copy each of type A and B neurotoxin genes. Partial nucleotide sequences obtained by direct sequencing of the PCR products indicate that the type A and B genes carried by this strain are not classical type A and non-proteolytic type B genes, but are similar to the type A gene present in a strain which had caused infant botulism in Kyoto and the type B gene present in a proteolytic type B strain.
Assuntos
Toxinas Botulínicas/genética , Clostridium botulinum/genética , Neurotoxinas/genética , Sequência de Bases , Southern Blotting , Clostridium botulinum/química , DNA Bacteriano , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido NucleicoRESUMO
The Bcl-2 family of proteins regulates apoptosis, the cell death program triggered by activation of certain proteases (caspases). An attractive model for how Bcl-2 and its closest relatives prevent caspase activation is that they bind to and inactivate an adaptor protein required for procaspase processing. That model has been supported by reports that mammalian prosurvival Bcl-2 relatives bind the adaptor Apaf-1, which activates procaspase-9. However, the in vivo association studies reported here with both overexpressed and endogenous Apaf-1 challenge this notion. Apaf-1 could be immunoprecipitated together with procaspase-9, and the Apaf-1 caspase-recruitment domain was necessary and sufficient for their interaction. Apaf-1 did not bind, however, to any of the six known mammalian prosurvival family members (Bcl-2, Bcl-x(L), Bcl-w, A1, Mcl-1, or Boo), or their viral homologs adenovirus E1B 19K and Epstein-Barr virus BHRF-1. Endogenous Apaf-1 also failed to coimmunoprecipitate with endogenous Bcl-2 or Bcl-x(L), or with two proapoptotic relatives (Bax and Bim). Moreover, apoptotic stimuli did not induce Apaf-1 to bind to these family members. Thus, the prosurvival Bcl-2 homologs do not appear to act by sequestering Apaf-1 and probably instead constrain its activity indirectly.