STAT1-mediated down-regulation of Bcl-2 expression is involved in IFN-γ/TNF-α-induced apoptosis in NIT-1 cells.

Tumor necrosis factor (TNF)-α and interferon (IFN)-γ are the major pro-inflammatory cytokines involved in beta-cell destruction. The fate of islet beta-cells in the cytokine-induced intrinsic mitochondrial apoptotic pathway is determined by the interaction between members of the Bcl-2 family. However, the mechanism through which beta-cell apoptosis is regulated remains unclear. In this study, we treated the murine beta-cell line NIT-1 with TNF-α and IFN-γ and then investigated the regulation of signal transducer and activator of transcription-1 (STAT-1) and expression of the members of the Bcl-2 family in this apoptotic pathway. Results showed that TNF-α and IFN-γ synergistically reduced NIT-1 cell viability. In addition, the decrease in cell growth was due to apoptosis as shown by apoptotic body formation, detected by confocal laser microscope, and a significant increase in Annexin-Vup(+) cell percentage, detected by flow cytometry. Combination treatment with TNF-α and IFN-γ caused a remarkable increase in the release of cytochrome c, and in the activation of caspase-9 and caspase-3, as well as, an obvious enhancement in STAT-1 phosphorylation; the treatment, however, resulted in the down-regulation in Bcl-2 expression. The enhancement in STAT-1 activity and a down-regulation in Bcl-2 expression was also observed in MIN6 cells, another murine beta-cell derived line, after cells exposure to the combination of TNF-α and IFN-γ treatment. Knockdown of STAT-1 gene expression by siRNA or inhibition of STAT-1 activation with fludarabine reversed Bcl-2 down-expression and led to a significant decrease in apoptosis in TNF-α- and IFN-γ-treated NIT-1 cells. Taken together, our results suggest that STAT1-mediated down-regulation of Bcl-2 is involved in NIT-1 cell apoptosis induced by combination treatment with TNF-α and IFN-γ.

Caspase-3 is involved in IFN-γ- and TNF-α-mediated MIN6 cells apoptosis via NF-κB/Bcl-2 pathway.

TNF-α and IFN-γ are the major pro-inflammatory cytokines in the ß-cell destruction. However, the underlying mechanism remains unclear. The present study used a murine insulinoma cell line MIN6 for further investigation of the effect of Caspase-3 on the cytokines-induced pancreatic ß-cell apoptosis and analyzed the mechanisms involved in the activation of Caspase-3. It was showed that the combination of IFN-γ and TNF-α significantly reduced the viability of MIN6 cells and the observed cells growth inhibition was due to cell apoptosis as judged by the morphological changes under a confocal laser scanning microscopy and FACS assay of Annexin-V/7-AAD double staining. Accompanying with NF-κB activation and Bcl-2 downregulation, both the cleaved Caspase-3 and PARP, a known substrate of Caspase-3 in vivo, were observed at 24 and 12 h, respectively, after cells exposure to IFN-γ and TNF-α treatment. Pretreatment of Caspase-3 inhibitors remarkably attenuated IFN-γ- and TNF-α-induced cells apoptosis. Inhibition of NF-κB activation led to the increase in Bcl-2 expression, a significant attenuation in Caspase-3 activity, and an obvious amelioration in cells viability in IFN-γ- and TNF-α-treated MIN6 cells. Taken together, our results indicate that Caspase-3 is critical for the induction of MIN6 cells apoptosis and it's activation is further confirmed to be related to the NF-κB-mediated Bcl-2 downregulation, which may be the underlying mechanism of IFN-γ- and TNF-α-mediated MIN6 cells apoptosis.

[Full sequence analysis and characterization of the Shenzhen Norovirus strain SZ2010422].

OBJECTIVE: To obtain information on viral molecular structural and evolutionary characteristics, we conducted the SZ2010422 full-length genomic analysis. METHODS: Primers were designed by New Orleans full sequence, SZ2010422 full genome was amplified by RT-PCR, the whole genome sequence and the capsid domain amino acid sites was analysised after cloned and sequenced. RESULTS: The genome of G II-4 Norovirus SZ2010422 strain was consist of 7559 bp, it revealed three ORFs composites of the whole genome, ORF1 (5100 bp), ORF2 (1623 bp), ORF3 (807 bp) respectively, ORF1 and ORF2 had 19 nucleotide overlap. By evolutionary comparative analysis found SZ2010422 genomic nucleotide sequences with reference strains of G II-4 New Orleans1805 strains the highest homology with a total length of homology was 99.3%, of ORF1 (99.5%), ORF2 (99.2%), ORF3 (98.6%). Phylogenetic analyses showed SZ2010422 belonging to G II-4 New Orleans variant. Date of 541 amino acid analyses showed: New Orleans variant strains of popular sites: aa310N or K, --> S aa341D --> of N, aa359T--> S, aa396H --> P, aa460H --> Y. CONCLUSION: Norovirus SZ2010422 belonged to the G II-4 New Orleans variant. In This study, SZ2010422 full sequence can be used not only as a full-length NoV variant sequence standard for future comparison studies, but also as useful material for the public health field by enabling the diagnosis, vaccine development, and prediction of new emerging variants. Noroviruses; Genes; Sequence analysis

[Detection and typing assay of norovirus in acute hospitalizations among children less than 5 years old from 2008 to 2009 in Lulong, Hebei province].

OBJECTIVE: To investigate the molecular epidemiologic characteristics and genotypes of norovirus in children less than 5 years of age in Lulong area from 2008 to 2009. METHODS: 325 stool specimens and epidemiological data from hospitalized children with diarrhea less than 5 years of age were collected. Rotavirus was detected by using the ELISA kit. Norovirus, adenovirus and astrovirus were detected by multiple reverse transcription-polymerase chain reaction (RT-PCR). Partial norovirus strains were sequenced and the tree was conducted by using the phylogenetic analyses. RESULTS: Norovirus was detected in 37 out of 325 (11.3%) specimens,ranked only second to rotavirus (48.6%), and higher than adenovirus (6.5%) and astrovirus (4.3%). Norovirus predominantly infected children less than 2 years of age and the season peak of norovirus occurred in November. Phylogenetic analysis showed that the predominant strain was the GII. 4/2006b variant. Interestingly, a novel unreported GII-4 variant was found in this study. CONCLUSION: Norovirus was one of the most important pathogens causing acute gastroenteritis from 2008 to 2009 in Lulong area. The GII. 4/2006b vairant was still the predominant strain. It is important to keep on monitoring the novel GII. 4 variant.

[Study on concentration of nuorovirus genegroup II from environmental water].

A new viral sampling concentration device was designed which was equipped with a new cationic filter membrane-Nanoceram suitable for field sampling. Norovirus Genegroup II was detected from environmental water with the aid of this device. The effects on virus recovery of prefiltration, various second-concentration methods, and different eluants were investigated through pre-experiment. The concentration optimized process, and the optimal concentration process were then determined. The results showed that the prefiltration had a profound effect on virus recovery, and two second-concentration method: PEG-NaC1 precipitation and celite adsorption, had almost the same concentration effects. The Na2 HPO4 solution of 0.15 mol/L was selected as the final eluant to elute the adsorbed Nuorovirus from the celite. The virus recovery of Nanoceram was determined to be 3.02%. Finally, successful detection of Norovirus GII in sewage from Yangqiao River, Fengtai District, Beijing was acheived. All these data had shown that the Naneceram filter concentration method could concentrate Norovirus from environmental water with a steady effects.