RESUMO
Orf virus (ORFV) infects sheep and goat tissues, resulting in severe proliferative lesions. To analyze cellular protein expression in ORFV-infected goat skin fibroblast (GSF) cells, we used two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ). The proteomics approach was used along with quantitative reverse transcription polymerase chain reaction (RT-qPCR) to detect differentially expressed proteins in ORFV-infected GSF cells and mock-infected GSF cells. A total of 282 differentially expressed proteins were identified. It was found that 222 host proteins were upregulated and 60 were downregulated following viral infection. We confirmed that these proteins were differentially expressed and found that heat shock 70-kDa protein 1B (HSPA1B) was differentially expressed and localized in the cytoplasm. It was also noted that HSPA1B caused inhibition of viral proliferation, in the middle and late stages of viral infection. The differentially expressed proteins were associated with the biological processes of viral binding, cell structure, signal transduction, cell adhesion, and cell proliferation.
Assuntos
Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP70/fisiologia , Vírus do Orf/fisiologia , Proteoma/genética , Replicação Viral , Animais , Células Cultivadas , Cromatografia Líquida , Fibroblastos/virologia , Cabras , Interações Hospedeiro-Patógeno , Vírus do Orf/genética , Proteômica , Espectrometria de Massas em TandemRESUMO
Four new protopanaxatriol-type triterpenes (1-2) and glucosides (3-4), were isolated from the rot roots of Panax notoginseng (Burk.) Chen, along with four known ones (5-8). Their structures were elucidated on the basis of extensive spectroscopic analysis (HRESIMS, NMR, UV, IR, and OR) and acidic hydrolysis. The possible transformation pathway of these compounds were also speculated from ginsenoside Rg1. Compound 1, with a unique α,ß-unsaturated ketene in its side chain, showed significant inhibitory effects against NO production on Murine macrophage cells (IC50 = 4.12 ± 0.20 µM) and comparable cytotoxicities against five human cancer cell lines (myeloid leukemia HL-60, lung cancer A-549 cells, hepatocellular carcinoma SMMC7721, breast cancer MCF-7, and colon cancer SW480) to positive control, cisplatin (DDP).
RESUMO
p53 is a sequence-specific DNA-binding transcription factor and key regulator of cell cycle arrest and apoptosis. p53 is mutated in most human cancers and these mutations generally impair its ability to activate transcription. When expressed in Saccharomyces cerevisiae, p53 acts as a strong transcriptional activator allowing yeast to be used as a model system to study the effects of p53 mutations on activity. However, little is known about the exact mechanisms by which p53 functions in yeast. Using 76 mutant yeast strains, we have evaluated the effect of deleting components of the ADA, COMPASS, INO80, ISW1, Mediator, RSC, SAGA, SAS, SLIK, SWI/SNF, and SWR1 transcriptional regulatory complexes on p53-dependent transcription. In addition, we examined the role of histone H2B ubiquitylation by Rad6/Bre1 on p53 activation. Overall, our analysis indicates that there are several remarkable similarities between p53-dependent transcription in yeast and mammalian cells, suggesting that yeast can serve as a valid model system for at least some aspects of p53 function.
Assuntos
Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/fisiologia , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Metilação , Ligação Proteica , Interferência de RNA/fisiologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitinas/metabolismoRESUMO
Composite scaffolds of nano-hydroxyapatite (nHAp) and silk fibroin (SF) have been reported to promote bone regeneration mainly through signaling pathways associated with cell-biomaterial interaction. However, it is unclear whether soluble factors also play a role in osteoinduction with nHAp-SF. In this study, we confirmed the biocompatibility and superior osteoinductivity of nHAp-SF scaffolds versus SF scaffolds both in vitro and on a calvarial defect model in vivo. This was followed by further analysis with microarray assay. The cDNA microarray results identified 247 differentially expressed genes in bone marrow mesenchymal stem cells (BMSCs) cultured on SF-nHAp scaffolds versus SF scaffolds. The greatest disparity in gene expression levels were observed with Il1α and Ilr2. Real-time PCR assay validated the results. The addition of IL-1α into cultures of BMSCs with SF significantly increased both Bmp2 and Ilr2 expression. However, with BMSCs alone, the Il1r2 expression increased substantially, whereas Bmp2 expression exhibited a decrease rather than increase. These data suggested that nHAp may exert osteoinductive effects on BMSCs via the secretion of IL-1α in an autocrine/paracrine fashion, and IL-1α activity could be regulated through the synthesis of IL1R2 by BMSCs upon interaction with nHAp. These results complemented our understanding of the underlying mechanisms of biomaterial osteoinductivity.
Assuntos
Comunicação Autócrina/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Durapatita/farmacologia , Fibroínas/farmacologia , Interleucina-1alfa/metabolismo , Células-Tronco Mesenquimais/citologia , Nanopartículas/química , Comunicação Parácrina/efeitos dos fármacos , Alicerces Teciduais/química , Animais , Materiais Biocompatíveis/farmacologia , Células da Medula Óssea/citologia , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Análise por Conglomerados , Modelos Animais de Doenças , Feminino , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/ultraestrutura , Nanopartículas/ultraestrutura , Osteogênese/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores Tipo II de Interleucina-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Crânio/efeitos dos fármacos , Crânio/patologiaRESUMO
Elucidating the regulatory mechanisms of osteogenesis of human mesenchymal stem cell (hMSC) is important for the development of cell therapies for bone loss and regeneration. Here we showed that hsa-miR-199a-5p modulated osteogenic differentiation of hMSCs at both early and late stages through HIF1a pathway. hsa-miR-199a expression was up-regulated during osteogenesis for both of two mature forms, miR-199a-5p and -3p. Over-expression of miR-199a-5p but not -3p enhanced differentiation of hMSCs in vitro, whereas inhibition of miR-199a-5p reduced the expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and mineralization. Furthermore, over-expression of miR-199a enhanced ectopic bone formation in vivo. Chitosan nanoparticles were used for delivery of stable modified hsa-miR-199a-5p (agomir) both in vitro and in vivo, as a proof-of-concept for stable agomir delivery on bone regeneration. The hsa-mir199a-5p agomir were mixed with Chitosan nanoparticles to form nanoparticle/hsa-mir199a-5p agomir plasmid (nanoparticle/agomir) complexes, and nanoparticle/agomir complexes could improve the in vivo regeneration of bone. Further mechanism studies revealed that hypoxia enhanced osteogenesis at early stage and inhibited osteogenesis maturation at late stage through HIF1a-Twist1 pathway. At early stage of differentiation, hypoxia induced HIF1a-Twist1 pathway to enhance osteogenesis by up-regulating miR-199a-5p, while at late stage of differentiation, miR-199a-5p enhanced osteogenesis maturation by inhibiting HIF1α-Twist1 pathway.