RESUMO
New Zealand isolates of the entomopathogenic fungus Beauveria were examined for the presence of dsRNAs and virus-like particles. Seven out of nine isolates contained one or more high-molecular-weight dsRNAs and all seven contained isometric virus particles ranging in size from 30 to 50 nm. B. bassiana isolate ICMP#6887 contained a single dsRNA band of ~6 kb and isometric virus-like particles of ~50 nm in diameter. Sequencing revealed that the virus from ICMP#6887 had a genome of 5,327 nt with two overlapping ORFs coding for a putative coat protein (CP) and an RNA-dependent RNA-polymerase (RdRp). The sequence showed a highest CP identity of 58.3 % to Tolypocladium cylindrosporum virus 1 (TcV1) and a highest RdRp identity of 48.8 % to Sphaeropsis sapinea RNA virus 1 (SsRV1). Since both TcV1 and SsRV1 belong to the genus Victorivirus, the new virus from B. bassiana ICMP#6887 was tentatively assigned the name Beauveria bassiana victorivirus 1 (BbVV1-6887).
Assuntos
Beauveria/virologia , Genoma Viral , RNA Viral/genética , Análise de Sequência de DNA , Totiviridae/classificação , Totiviridae/genética , Animais , Análise por Conglomerados , Nova Zelândia , Fases de Leitura Aberta , Filogenia , Homologia de Sequência de Aminoácidos , Totiviridae/isolamento & purificação , Proteínas Virais/genéticaRESUMO
TGF-beta induces vascular endothelial growth factor (VEGF), a potent angiogenic factor, at the transcriptional and protein levels in mouse macrophages. VEGF secretion in response to TGF-beta1 is enhanced by hypoxia and by overexpression of Smad3/4 and hypoxia-inducible factor-1alpha/beta (HIF-1alpha/beta). To examine the transcriptional regulation of VEGF by TGF-beta1, we constructed mouse reporters driven by the VEGF promoter. Overexpression of HIF-1alpha/beta or Smad3/4 caused a slight increase of VEGF promoter activity in the presence of TGF-beta1, whereas cotransfection of HIF-1alpha/beta and Smad3/4 had a marked effect. Smad2 was without effect on this promoter activity, whereas Smad7 markedly reduced it. Analysis of mutant promoters revealed that the one putative HIF-1 and two Smad-binding elements were critical for TGF-beta1-induced VEGF promoter activity. The relevance of these elements was confirmed by chromatin immunoprecipitation assay. p300, which has histone acetyltransferase activity, augmented transcriptional activity in response to HIF-1alpha/beta and Smad3/4, and E1A, an inhibitor of p300, inhibited it. TGF-beta1 also increased the expression of fetal liver kinase-1 (Flk-1), a major VEGF receptor, and TGF-beta1 and VEGF stimulated pro-matrix metalloproteinase 9 (MMP-9) and active-MMP-9 expression, respectively. The results from the present study indicate that TGF-beta1 can activate mouse macrophages to express angiogenic mediators such as VEGF, MMP-9, and Flk-1.
Assuntos
Neovascularização Fisiológica/imunologia , Fator de Crescimento Transformador beta1/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Células Cultivadas , Proteína p300 Associada a E1A/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Perfilação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Proteína Smad4/metabolismo , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Double-stranded RNAs and virus particles were identified in Pleurotus ostreatus strain Shin-Nong in Korea. Isometric virus particles with a diameter of 33 nm were purified, which are similar to other Pleurotus viruses reported previously. This strain contains 5 dsRNAs, 8.0, 2.5, 2.4, 2.0, and 1.8 kb in size. The virus particles contain 2 dsRNAs, designated RNA-1 (2.5 kb), and RNA-2 (2.4 kb) which is a typical pattern of Partitiviridae. A non-encapsidated dsRNA of about 8.0 kb also was identified. Partial cDNA from RNA-1 was cloned, and sequence analysis revealed that this gene codes for RdRp. The comparison of the sequence from partial cDNA clone showed 35% amino acid homology with the C-terminal end of the RdRp gene of Helicobasidum mompa virus and Rosalinia necatrix virus. Specific primers designed from the partial sequences successfully amplified RT-PCR product from the infected mycelium and a single spore culture. We used these primers to determine the pattern of distribution of viruses in spores. Of the 96 different single spore cultures generated from Shin-Nong strain, a specific RT-PCR product was identified in 25 cultures, indicating that about 26% of basidiospores contain viruses.
Assuntos
Pleurotus/virologia , Vírus de RNA/isolamento & purificação , RNA de Cadeia Dupla/genética , Esporos Fúngicos/virologia , Sequência de Aminoácidos , Clonagem Molecular , Primers do DNA , DNA Complementar/genética , Dados de Sequência Molecular , Pleurotus/fisiologia , RNA/genética , Vírus de RNA/genética , RNA de Cadeia Dupla/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência , Esporos Fúngicos/genética , Proteínas Virais/genética , Vírion/isolamento & purificação , Vírion/ultraestruturaRESUMO
Cyclic AMP-responsive element binding protein (CREB) is known to be associated with angiogenesis. In the present study we investigated the possible role of CREB in the expression of vascular endothelial growth factor (VEGF) by mouse macrophages. Over-expression of CREB increased VEGF secretion by cells of the RAW264.7 mouse macrophage cell line. It also increased the promoter activity of a mouse reporter driven by the VEGF promoter, while a dominant negative CREB (DN-CREB) abrogated the activity, suggesting that CREB mediates VEGF transcription. Forskolin, an adenylyl cyclase activator, stimulated VEGF transcription, and the PKA inhibitor H89 abolished this effect. IFN-gamma, a potent cytokine, stimulated VEGF expression only in part through the PKA-CREB pathway. These results indicate that PKA phosphorylates CREB and so induces VEGF gene expression. An analysis of mutant promoters revealed that one of the putative CREB responsive elements (CREs), at 399 approximately 388 in the promoter, is critical for CREB-mediated VEGF promoter activity, and the significance of this CRE was confirmed by chromatin immunoprecipitation assays.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Macrófagos/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Imunoprecipitação da Cromatina , Colforsina/farmacologia , Proteína Quinase Tipo II Dependente de AMP Cíclico , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica/efeitos dos fármacos , Elementos de Resposta/efeitos dos fármacos , Elementos de Resposta/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
The nucleotide sequences of the genomic dsRNA mycovirus infecting Pleurotus ostreatus (P. ostreatus virus 1; PoV1) were determined and compared to the sequences of the other mycoviruses belonging to partitiviruses and totivirues. PoV1 dsRNA-1 and dsRNA-2 had genomes of 2296 and 2223 nucleotides, respectively. The purified virus preparations contained isometric particles of 28-30 nm in diameter, and also the same two dsRNAs were isolated from purified virus preparations. The sequences of PoV1 dsRNA-1 and dsRNA-2 had GC contents of 48.4 and 51.5%, respectively. dsRNA-1 had 78 and 97 nucleotides of 5'- and 3'-untranslated region (UTR) while dsRNA-2 had 114 and 198 nucleotides of 5'- and 3'-UTR, respectively. Computer analysis of putative open reading frame (ORF) shows that dsRNA-1 and dsRNA-2 contain a single ORF encoding proteins of 82.2 and 71.1 kDa that show high sequence identity with RNA-dependent RNA polymerase and capsid protein of partitiviruses, respectively. When compared to other dsRNA mycoviruses in a phylogenetic analysis they were found to form a distinct virus clade with partitiviruses, and were more distantly related to totiviruses.
Assuntos
Genoma Viral , Pleurotus/virologia , Vírus de RNA/genética , Vírus de RNA/isolamento & purificação , RNA Viral/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Proteínas do Capsídeo/genética , Sequência Conservada , Microscopia Eletrônica , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta/genética , Filogenia , Vírus de RNA/classificação , Vírus de RNA/ultraestrutura , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/isolamento & purificação , RNA Viral/isolamento & purificação , RNA Polimerase Dependente de RNA/genética , Homologia de SequênciaRESUMO
The WRKY proteins are a major family of plant transcription factors implicated in the regulation of plant defense mechanisms against pathogens. OsWRKY6 was isolated based on expression profiling data carried out with samples infected by Xanthomonas oryzae pv. oryzae (Xoo). OsWRKY6 encodes a DNA binding protein that contains one WRKY domain, a nuclear localization signal and C(2)H(2)-type zinc finger motif. OsWRKY6 is a member of the group II family of WRKY proteins. Based on the result of yeast one hybrid assay this OsWRKY6 protein binds to the typical W box ((T)TGACC/T). OsWRKY6 functions as a transcriptional activator in yeast. OsWRKY6 enhanced the expression of the reporter gene downstream of OsPR1 promoter, indicating that OsWRKY6 is a transcriptional activator in rice as well. Heterologous expression of OsWRKY6 enhanced disease resistance to pathogen. Defense-related genes were constitutively expressed in Arabidopsis transgenic lines overexpressing OsWRKY6. All together, OsWRKY6 functions as a positive transcriptional regulator of the plant defense response.
Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Oryza/genética , Doenças das Plantas/genética , Fatores de Transcrição/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Genes de Plantas , Imunidade Inata , Oryza/metabolismo , Oryza/microbiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , XanthomonasRESUMO
Transplantation of human cord blood-derived endothelial progenitor cells (EPCs) is reported to contribute to neovascularization in various ischemic diseases. However, the possible beneficial role and underlying mechanisms in diabetes-impaired wound healing have been less well characterized. In this study, EPC transplantation stimulated keratinocyte and fibroblast proliferation substantially as early as 3 days after injury, leading to significantly accelerated wound closure in streptozotocin-induced diabetic nude mice, compared to PBS control. RT-PCR analysis showed that EPCs secreted various wound healing-related growth factors. Among them, keratinocyte growth factor and platelet-derived growth factor were highly expressed in the EPCs and were present at substantial levels in the EPC-injected dermal tissue. Using EPC-conditioned medium (CM), we found that paracrine factors from EPCs directly exerted mitogenic and chemotactic effects on keratinocytes and fibroblasts. Moreover, injection of EPC-CM alone into the same diabetic wound mice promoted wound healing and increased neovascularization to a similar extent as achieved with EPC transplantation. These results indicate that the beneficial effect of EPC transplantation on diabetic wounds was mainly achieved by their direct paracrine action on keratinocytes, fibroblasts, and endothelial cells, rather than through their physical engraftment into host tissues (vasculogenesis). In addition, EPC-CM was shown to be therapeutically equivalent to EPCs, at least for the treatment of diabetic dermal wounds, suggesting that conditioned medium may serve as a novel therapeutic option that is free from allograft-associated immune rejection concern.
Assuntos
Meios de Cultivo Condicionados/química , Complicações do Diabetes/terapia , Células Endoteliais/fisiologia , Sangue Fetal/citologia , Transplante de Células-Tronco , Células-Tronco/fisiologia , Cicatrização , Animais , Movimento Celular , Proliferação de Células , Diabetes Mellitus Experimental , Células Endoteliais/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Fisiológica , Células-Tronco/citologiaRESUMO
Endothelial progenitor cells (EPCs), endothelial precursors that promote neovascularization in ischemic tissues, have shown the limited vascular regeneration efficacy due to their poor homing into injured sites and low survival, so that a variety of biosynthetic scaffolds have been employed as cell delivery vehicles to overcome the current cell transplantation methods. However, few paralleled studies that directly compare the efficacy of EPCs seeded within synthetic scaffolds to that of EPCs delivered by the conventional transplantation techniques used for EPC therapies have been performed. To address these issues, RGD-g-PLLA biosynthetic scaffold was developed for the targeted EPC delivery and was found to successfully support the in vitro growth and endothelial functions of EPCs. This scaffold also appeared to be good as in vivo targeted delivery carriers of EPCs as it promoted vascular regeneration in a murine dermal wound models. Furthermore, direct comparison with the intradermal EPC injection revealed that the targeted delivery of EPCs by using the RGD-g-PLLA scaffold was superior to their conventional local injection method in terms of the localization and survival/retention of the transplanted EPCs, and their vascular repairing potential. These results suggest that the development of an effective stem cell delivery system may help to maximize the tissue-repairing efficacy with a limited number of stem cells, thereby resolving the limited clinical success of current stem cell therapies that have utilized simple cell injections or infusions.
Assuntos
Derme , Células Endoteliais/transplante , Ácido Láctico/metabolismo , Neovascularização Fisiológica , Oligopeptídeos/metabolismo , Polímeros/metabolismo , Transplante de Células-Tronco , Alicerces Teciduais , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Adesão Celular/fisiologia , Derme/citologia , Derme/patologia , Células Endoteliais/citologia , Ácido Láctico/química , Masculino , Teste de Materiais , Camundongos , Camundongos Nus , Nitritos/metabolismo , Oligopeptídeos/química , Poliésteres , Polímeros/química , Regeneração/fisiologia , CicatrizaçãoRESUMO
Orysa sativa pathogenesis-related protein 10a (OsPR10a) was induced by pathogens, salicylic acid (SA), jasmonic acid (JA), ethephon, abscisic acid (ABA), and NaCl. We tried to analyze the OsPR10a promoter to investigate the transcriptional regulation of OsPR10a by SA. We demonstrated the inducibility of OsPR10a promoter by SA using transgenic Arabidopsis carrying OsPR10a:GFP as well as by transient expression assays in rice. To further identify the promoter region responsible for its induction by SA, four different deletions of the OsPR10a promoter were made, and their activities were measured by transient assays. The construct containing 687-bp OsPR10a promoter from its start codon exhibited a six-fold increase of induction compared to the control in response to SA. Mutation in the W-box like element 1 (WLE 1) between 687 and 637-bp from TGACA to TGAAA completely abolished induction of the OsPR10a promoter by SA, indicating that the WLE 1 between -687 and -637 of OsPR10a promoter is important in SA-mediated OsPR10a expression. We show for the first time that the W-box like element plays a role in SA mediated PR gene expression.
Assuntos
Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Ácido Salicílico/farmacologia , Sequência de Bases , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oryza/genética , Elementos de Resposta , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Activation-induced cytidine deaminase (AID) is an inducible gene that plays a critical role in Ig class switch recombination and somatic hypermutation in B cells. We explored the mechanisms by which IL-4 induces AID expression in mouse B cells. IL-4 increased AID expression and over-expression of Stat6 further augmented IL-4-induced promoter activity. The involvement of Stat6 in the promoter activity was confirmed using ChIP assays and site-directed mutagenesis. Treatment with H89, a PKA inhibitor, markedly decreased IL-4-induced AID expression, and over-expression of CREB enhanced it. These results indicate that Stat6 and PKA/CREB are involved in IL-4-induced AID expression. The relevance of these signal transducing molecules was verified using the TGFbeta1-induced IgA isotype switching model. Our results indicate that IL-4, through Stat6 and PKA/CREB, induces AID expression leading to Ig isotype switching event.
Assuntos
Citidina Desaminase/biossíntese , Imunoglobulina A/genética , Switching de Imunoglobulina/genética , Interleucina-4/farmacologia , Recombinação Genética/genética , Animais , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citidina Desaminase/genética , Indução Enzimática/efeitos dos fármacos , Switching de Imunoglobulina/efeitos dos fármacos , Camundongos , Modelos Imunológicos , Regiões Promotoras Genéticas/genética , Recombinação Genética/efeitos dos fármacos , Fator de Transcrição STAT6/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) is a phosphoprotein possessing various functions. We have previously reported that the HCV NS5A protein interacts with tumor necrosis factor (TNF) receptor-associated factor (TRAF) domain of TRAF2 (Park, K.-J., Choi, S.-H., Lee, S. Y., Hwang, S. B., and Lai, M. M. C. (2002) J. Biol. Chem. 277, 13122-13128). Both TNF-alpha- and TRAF2-mediated nuclear factor-kappaB (NF-kappaB) activations were inhibited by NS5A-TRAF2 interaction. Because TRAF2 is required for the activation of both NF-kappaB and c-Jun N-terminal kinase (JNK), we investigated HCV NS5A protein for its potential capacity to modulate TRAF2-mediated JNK activity. Using in vitro kinase assay, we have found that NS5A protein synergistically activated both TNF-alpha- and TRAF2-mediated JNK in human embryonic kidney 293T cells. Furthermore, synergism of NS5A-mediated JNK activation was inhibited by dominant-negative form of MEK kinase 1. Our in vivo binding data show that NS5A does not inhibit interaction between TNF receptor-associated death domain and TRAF2 protein, indicating that NS5A and TRAF2 may form a ternary complex with TNF receptor-associated death domain. These results indicate that HCV NS5A protein modulates TNF signaling of the host cells and may play a role in HCV pathogenesis.