RESUMO
Dibutyltin (DBT) is the degradation products of TBT, which is generally considered higher toxicity than TBT in the immune system. In order to learn more about the mechanisms of immune-toxic of DBT, we exposed zebrafish (Danio rerio) to 0, 1, 10 and 100 ng/L DBT for 8 weeks. At the end of the experiment, we determined the immune parameters and immune-related genes. The results showed that with an increase in TBT dose, lysozyme activities and IgM, C3, C4 content in intestine, skin and spleen were all significantly inhibited by the DBT exposure. Fish exposed to 10 ng/L and 100 ng/L showed significantly lower lysozyme activities and IgM, C3, C4 content than those of the control group. Zebrafish exposed to 10 ng/L and 100 ng/L DBT, the mRNA transcript levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), interferon γ2 (INFγ2), nuclear factor-κB p65 (NF-kB p65), inhibitor protein-κBα (IκBα), IκB kinases ß (IKKß), Janus family of protein tyrosine kinases (JAKs) and the signal transducers and activators of transcription proteins (STATs) all increased with the DBT levels in the intestine and spleen. Those parameters showed significantly higher values in 10 ng/L and 100 ng/L than those of fish in the control group. However, no significant difference was found in IκB kinases α (IKKα) and IκB kinase γ (IKKγ) mRNA levels in the intestine and spleen. These data imply that DBT might be via suppression on IKKß/IkBa/NF-kBp65 and JAK/STAT signaling pathways to regulate the immunity of zebrafish.
Assuntos
Compostos Orgânicos de Estanho/toxicidade , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/metabolismo , Animais , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/imunologia , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Pele/efeitos dos fármacos , Pele/imunologia , Pele/metabolismo , Baço/efeitos dos fármacos , Baço/imunologia , Baço/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Peixe-Zebra/imunologiaRESUMO
Foot-and-mouth disease virus is a picornavirus and its RNA genome encodes a large polyprotein. The N-terminal part of this polyprotein is the leader protein, a cysteine protease, termed Lpro. The virus causes the rapid inhibition of host cell cap-dependent protein synthesis within infected cells. This results from the Lpro-dependent cleavage of the cellular translation initiation factor eIF4G. Lpro also releases itself from the virus capsid precursor by cleaving the L/P1 junction. Using site-directed mutagenesis of the Lpro coding sequence, we have investigated the role of 51 separate amino acid residues in the functions of this protein. These selected residues either are highly conserved or are charged and exposed on the protein surface. Using transient expression assays, within BHK-21 cells, it was found that residues around the active site (W52, L53 and A149) of Lpro and others located elsewhere (K38, K39, R44, H138 and W159) are involved in the induction of eIF4G cleavage but not in the processing of the L/P1 junction. Modified viruses, encoding such amino acid substitutions within Lpro, can replicate in BHK-21 cells but did not grow well in primary bovine thyroid cells. This study characterizes mutant viruses that are deficient in blocking host cell responses to infection (e.g. interferon induction) and can assist in the rational design of antiviral agents targeting this process and in the production of attenuated viruses.
Assuntos
Endopeptidases/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Vírus da Febre Aftosa/enzimologia , Vírus da Febre Aftosa/fisiologia , Proteínas Mutantes/metabolismo , Proteólise , Animais , Bovinos , Células Cultivadas , Cricetinae , Análise Mutacional de DNA , Endopeptidases/genética , Vírus da Febre Aftosa/genética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genéticaRESUMO
The aim of the present study was to investigate effects of dietary Lactobacillus delbrueckii (L. delbrueckii) on immune response, disease resistance against Aeromonas hydrophila (A. hydrophila), antioxidant capability and growth performance of Cyprinus carpio Huanghe var. 450 fish (mean weight of 1.05 ± 0.03 g) were randomly distributed into five groups that fed diets containing different levels of L. delbrueckii (0, 1 × 105, 1 × 106, 1 × 107 and 1 × 108 CFU g-1) for 8 weeks. The results showed that intestinal immune parameters such as lysozyme, acid phosphatase, and myeloperoxidase activities, immunoglobulin M content, and the survival rate were improved in fish fed with 1 × 106 and 1 × 107 CFU g-1L. delbrueckii. In addition, 1 × 107 CFU g-1L. delbrueckii supplementation down-regulated mRNA levels of TNF-α, IL-8, IL-1ß and NF-κBp65, and up-regulated IL-10 and TGF-ß mRNA levels in the intestine. The survival rate was significantly (P < 0.05) higher (68.33%) in fish fed 1 × 106 CFU g-1L. delbrueckii than the control diet-fed group (40%) after challenge by A. hydrophila. Fish fed with diet containing 1 × 106 CFU g-1L. delbrueckii showed higher antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and total antioxidant capacity (T-AOC) and lower MDA concentrations than those of the control group (P < 0.05). The relative gene expression (SOD, CAT, GPX) showed the same trend with their activities. In addition, the growth performance was significantly improved in fish fed with the diet containing 1 × 106 and 1 × 107 CFU g-1L. delbrueckii (P < 0.05). These results demonstrated that dietary optimal levels of L. delbrueckii enhanced immunity, disease resistance against A. hydrophila antioxidant capability and growth performance in Cyprinus carpio Huanghe var.
Assuntos
Carpas , Suplementos Nutricionais , Resistência à Doença , Doenças dos Peixes/prevenção & controle , Infecções por Bactérias Gram-Negativas/veterinária , Imunidade Inata , Lactobacillus delbrueckii , Aeromonas hydrophila/fisiologia , Ração Animal/análise , Animais , Antioxidantes/metabolismo , Carpas/crescimento & desenvolvimento , Dieta/veterinária , Suplementos Nutricionais/análise , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/prevenção & controle , Lactobacillus delbrueckii/química , Lactobacillus delbrueckii/imunologia , Distribuição AleatóriaRESUMO
Fourteen strains representing 11 Caragana-nodulating Mesorhizobium genomic species were identified as representing Mesorhizobium amorphae, Mesorhizobium huakuii, Mesorhizobium septentrionale and groups related to Mesorhizobium plurifarium, Mesorhizobium temperatum, Mesorhizobium tianshanense and Mesorhizobium mediterraneum by sequencing of the 16S rRNA gene, 16S-23S internal transcribed spacer, partial housekeeping recA gene, and previously performed sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins and BOX-PCR fingerprinting. Despite their different taxonomic affiliation, highly similar symbiotic genes (>93% similarity for nodC and >91.8% similarity for nifH) were found among the Caragana strains and the three type strains for M. tianshanense, M. temperatum and M. septentrionale. Cross nodulation tests revealed that each of these 14 Caragana mesorhizobia and the three type strains mentioned above could effectively infect each of their original host plants, Caragana microphylla, Glycyrrhiza (host for M. tianshanense type strain) and Astragalus adsurgens (host for M. temperatum and M. septentrionale type strains). These results provide evidence that different Mesorhizobium species can nodulate with Caragana, and they have similar symbiotic genes (probably acquired by a phenomenon of lateral gene transfer) and common host ranges.
Assuntos
Alphaproteobacteria/classificação , Alphaproteobacteria/genética , Caragana/microbiologia , Genes Bacterianos , Simbiose , Alphaproteobacteria/isolamento & purificação , Alphaproteobacteria/fisiologia , Proteínas de Bactérias/genética , Impressões Digitais de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/genética , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , N-Acetilglucosaminiltransferases/genética , Oxirredutases/genética , Proteoma/análise , RNA Ribossômico 16S/genética , Recombinases Rec A/genética , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The foot-and-mouth disease virus capsid precursor, P1-2A, is cleaved by the 3C protease (3Cpro) to VP0, VP3, VP1 and 2A. The P1-2A precursor (wt or mutant) was expressed alone or with 3Cpro and processing of P1-2A was determined. The VP2 K217R and VP3 I2P substitutions (near the VP0/VP3 junction) strongly reduced the processing at this junction by 3Cpro while the substitution VP2 K217E blocked cleavage. At the VP3/VP1 junction, the substitutions VP3 Q2221P and VP1 T1P each severely inhibited processing at this site. Blocking cleavage at either junction did not prevent processing elsewhere in P1-2A. These modifications were also introduced into full-length FMDV RNA; only wt and the VP2 K217R mutant were viable. Uncleaved VP0-VP3 and the processed products were observed within cells infected with the mutant virus. The VP0-VP3 was not incorporated into empty capsids or virus particles. The three junctions within P1-2A are processed by 3Cpro independently.
Assuntos
Proteínas do Capsídeo/metabolismo , Cisteína Endopeptidases/metabolismo , Vírus da Febre Aftosa/enzimologia , Vírus da Febre Aftosa/fisiologia , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Virais/metabolismo , Proteases Virais 3C , Animais , ProteóliseRESUMO
Hepacivirus has been identified in cattle in Africa, Europe, and South America. In this survey of bovine hepacivirus (BovHepV) in 131 serum samples from Chinese cattle herds using RT-PCR, five of 131 sera were BovHepV positive, with the infection rate of 3.82%. Phylogenetic analysis based on the partial NS3 coding sequence showed that the BovHepV of the five positive samples clustered with other BovHepV but formed a separate branch. The results indicated that these new BovHepV represent emerging and novel strains. Further investigations are necessary to determine the epidemiology and viral pathogenesis of these BovHepV strains, as well as the potential threat to ruminant and livestock workers in China.
Assuntos
Hepacivirus , Hepatite C , Filogenia , Animais , Bovinos , China , Hepacivirus/isolamento & purificação , Hepacivirus/patogenicidade , Hepatite C/diagnóstico , Hepatite C/epidemiologia , HumanosRESUMO
Soil bacteria known as rhizobia are able to establish an endosymbiosis with legumes that takes place in neoformed nodules in which intracellularly hosted bacteria fix nitrogen. Intracellular accommodation that facilitates nutrient exchange between the two partners and protects bacteria from plant defense reactions has been a major evolutionary step towards mutualism. Yet the forces that drove the selection of the late event of intracellular infection during rhizobium evolution are unknown. To address this question, we took advantage of the previous conversion of the plant pathogen Ralstonia solanacearum into a legume-nodulating bacterium that infected nodules only extracellularly. We experimentally evolved this draft rhizobium into intracellular endosymbionts using serial cycles of legume-bacterium cocultures. The three derived lineages rapidly gained intracellular infection capacity, revealing that the legume is a highly selective environment for the evolution of this trait. From genome resequencing, we identified in each lineage a mutation responsible for the extracellular-intracellular transition. All three mutations target virulence regulators, strongly suggesting that several virulence-associated functions interfere with intracellular infection. We provide evidence that the adaptive mutations were selected for their positive effect on nodulation. Moreover, we showed that inactivation of the type three secretion system of R. solanacearum that initially allowed the ancestral draft rhizobium to nodulate, was also required to permit intracellular infection, suggesting a similar checkpoint for bacterial invasion at the early nodulation/root infection and late nodule cell entry levels. We discuss our findings with respect to the spread and maintenance of intracellular infection in rhizobial lineages during evolutionary times.
Assuntos
Evolução Biológica , Fabaceae/microbiologia , Rhizobium/genética , Simbiose/genética , Sistemas de Secreção Bacterianos/genética , Mutação , Raízes de Plantas/microbiologia , Rhizobium/patogenicidade , Fatores de Virulência/genéticaRESUMO
Twenty-two genospecies belonging mainly to Mesorhizobium, and occasionally to Rhizobium and Bradyrhizobium, were defined among the 174 rhizobia strains isolated from Caragana species. Highly similar nodC genes were found in the sole Bradyrhizobium strain and among all the detected Mesorhizobium strains. A clear correlation between rhizobial genospecies and the eco-regions where they were isolated was found using homogeneity analysis. All these results demonstrated that Caragana species had stringently selected the rhizobia symbiotic genotype, but not the genomic background; lateral transfer of symbiotic genes from Mesorhizobium to Bradyrhizobium and among the Mesorhizobium species has happened in the Caragana rhizobia; and biogeography of Caragana rhizobia exists. Furthermore, a combined cluster analysis, based upon the patterns obtained from amplified 16S rRNA gene and 16S-23S intergenic spacer restriction analyses, BOX PCR and SDS-PAGE of proteins, was reported to be an efficient method to define the genospecies.
Assuntos
Caragana/microbiologia , Variação Genética , Rhizobiaceae/classificação , Rhizobiaceae/isolamento & purificação , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , China , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Eletroforese em Gel de Poliacrilamida , Transferência Genética Horizontal , Geografia , Dados de Sequência Molecular , N-Acetilglucosaminiltransferases/genética , Filogenia , Proteoma/análise , RNA Ribossômico 16S/genética , Rhizobiaceae/química , Rhizobiaceae/genética , Análise de Sequência de DNARESUMO
Bacterial strain CCBAU 25048(T) was isolated from root nodules of Kummerowia stipulacea grown in Shandong province of China. Cells of the strain were Gram-negative, strictly aerobic, non-spore-forming, motile short rods. Phylogeny of 16S rRNA gene sequences revealed that the strain belonged to the genus Shinella, a member of family Rhizobiaceae. Its closest phylogenetic relatives were Shinella granuli Ch06(T) and Shinella zoogloeoides IAM 12669(T), respectively showing 98.3 and 98.9 % 16S rRNA gene sequence similarity. Strain CCBAU 25048(T) had DNA-DNA relatedness of 43.5 and 34.8 %, respectively, with S. zoogloeoides JCM 20728(T) and S. granuli JCM 13254(T). In addition, in TP-RAPD analysis, different patterns were obtained for these three strains and some rhizobial strains. The nifH, nodC and nodD sequences of CCBAU 25048(T) were identical or very similar to those of bean-nodulating Rhizobium tropici strains. Several phenotypic characteristics, including the use of citrate and d-ribose as carbon sources and growth at pH 11.0, as well as the fatty acid composition, could differentiate CCBAU 25048(T) from the two defined Shinella species. Therefore, a novel species Shinella kummerowiae sp. nov. is proposed, with strain CCBAU 25048(T) (=JCM 14778(T) =LMG 24136(T)) as the type strain.
Assuntos
Fabaceae/microbiologia , Raízes de Plantas/microbiologia , Rhizobiaceae/classificação , Rhizobiaceae/isolamento & purificação , Simbiose , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , China , DNA Bacteriano/análise , DNA Ribossômico/análise , Ácidos Graxos/análise , Genes de RNAr , Dados de Sequência Molecular , Fixação de Nitrogênio , Hibridização de Ácido Nucleico , Fenótipo , Filogenia , RNA Ribossômico 16S/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Rhizobiaceae/genética , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Five rhizobial strains representing the largest group in the genus Mesorhizobium associated with Caragana spp. in China were characterized taxonomically. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these microsymbionts belonged to the genus Mesorhizobium, with Mesorhizobium tianshanense USDA 3592(T), Mesorhizobium temperatum SDW018(T) and Mesorhizobium mediterraneum UPM-Ca36(T) as the closest neighbours (>/=99.5 % 16S rRNA gene sequence similarity). Genotypic fingerprinting by whole-cell protein electrophoresis, DNA-DNA hybridization, comparative housekeeping sequence analysis of the atpD, glnII and recA genes, fatty acid profiles and a series of phenotypic and physiological tests allowed the novel group to be differentiated from all previously recognized species of the genus Mesorhizobium. This group therefore represents a novel species, for which the name Mesorhizobium caraganae sp. nov. is proposed with the type strain CCBAU 11299(T) (=LMG 24397(T)=HAMBI 2990(T)). Cross-inoculation tests showed that strain CCBAU 11299(T) could form effective nodules on Caragana microphylla, Caragana intermedia, Glycyrrhiza uralensis, Astragalus adsurgens and Phaseolus vulgaris.