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The pathogenesis of diabetes mellitus is a complicated process involving much gene regulation. The molecular mechanism of mulberry (Morus alba L.) leaf in the treatment of diabetes is not fully understood. In this study, we used the Illumina HiSeq™ 2,500 platform to explore the liver transcriptome of normal mice, STZ-induced diabetic mice, and mulberry leaf-treated diabetic mice, and we obtained 52,542,956, 52,626,414, and 52,780,196 clean reads, respectively. We identified differentially expressed genes (DEGs) during the pathogenesis of diabetes in mice. The functional properties of DEGs were characterized by comparison with the GO and KEGG databases, and the results show that DEGs are mainly involved in the metabolic pathway. qRT-PCR was used to analyse 27 differential genes involved in liver expression in different groups of diabetic mice. Among the DEGs, the expression of Scube1, Spns3, Ly6a, Igf2 , and other genes between the control (C) and diabetic control (DC) groups was significantly upregulated; the expression of Grb10, Mup2 , and Fasn was significantly downregulated; the expression of the Sqle, Lss , and Irs2 genes between the C group and diabetic group treated with mulberry (DD) was significantly upregulated; the expression of Fabp2, Ly6a , and Grb10 was significantly downregulated; and the expression of Sqle and Lss was significantly upregulated in the DC and DD groups, but Tap1, Igf2 , and Spns3 were significantly downregulated. The results of Western blot validation showed that dynamic changes in proteins, such as IGF2, Ly6a, Grb10, and UBD, occurred to regulate the incidence of diabetes by influencing the insulin receptor substrate (IRS) signaling pathway. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensovirus ( BmBDV) VD1-ORF4 consists of 3,318 nucleotides, which codes for a predicted protein with molecular weight of about 127 kDa. However, the authentic proteins encoded by VD1-ORF4 in silkworm midguts infected with BmBDV and their interacting proteins are still unclear. In this study, Western blot analysis revealed that a 127-kDa protein was confirmed to be translated from the VD1-ORF4 transcript using polyclonal antibodies and monoclonal antibodies against VD1-ORF4 deduced amino acid. Moreover, four smaller proteins with molecular weight of about 70, 60, 53, and 42 kDa were also examined in the infected midguts. Transient expression assay indicated that the expression amount of VD1-ORF4 fused with egfp was at least 30-fold lower than that of egfp gene, and immunofluorescence staining result indicated that these proteins encoded by VD1-ORF4 were located in both the cytoplasm and nucleus. Co-immunoprecipitation result showed that Aminopeptidase and Heat shock protein 90 can be captured by these proteins encoded by VD1-ORF4. In conclusion, multiple proteins were produced from the transcripts of VD1-ORF4 gene by an uncertain expression strategy, which may play important roles in viral replication and assembly. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensovirus ( BmBDV) is a new designated species of the new genus Bidensovirus in the new family Bidnaviridae, which contains two single-stranded linear DNAs (VD1 and VD2) and causes the chronic densonucleosis disease of silkworm. Previous researches revealed that VD1-ORF3 encodes the major structural proteins VPs. In this work, through western blot, we found that VPs expressed from 48 h post-inoculation and kept increasing until 120 h post-inoculation in midgut of Bombyx mori. In order to further investigate the translation of vp gene, the ORFs ( vp1 and vp2) of the VP started just up-stream of the first two candidate initiation codons were expressed in Sf9 cells by a baculovirus expression system. The expression products were purified by gradient density centrifugation and analyzed by Western blot and electron microscopy. The results showed that the expressions of vp1 yielded three proteins (VP1, VP1′, and VP2), which are the same with the viral VPs expression in midgut of Bombyx mori, and vp2 generated two VPs with the molecular weights of about 51 kDa (VP2) and 37 kDa. The observation by electron microscopy indicated that these VPs can auto-assemble into virus-like particles that could not be distinguished from virus particles. These findings will provide materials for studying the structure of BmBDV and be helpful in the studies on BmBDV-based disease in silkworms. [ABSTRACT FROM AUTHOR]

Orf101 ( Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, Bm101 was characterized. Transcripts of Bm101 were detected from 24 through 96 h post infection (h p.i.) by RT-PCR. The corresponding protein was also detected from 24 to 96 h p.i. in BmNPV-infected BmN cells by Western blot analysis using a polyclonal antibody against Bm101. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm101 encodes a 28-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. In addition, confocal analysis showed that the protein was localized in the cytosol and cytoplasmic membrane in virus-infected cells. In conclusion, the available data suggest that Bm101 is a functional ORF of BmNPV and encodes a protein expressed in the late stage of the infection cycle that is associated with the BV envelope. [ABSTRACT FROM AUTHOR]

Open reading frame 17 ( Bm17) of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this report, we describe the characterization of Bm17. Reversed transcriptive-PCR (RT-PCR) and Western blot analysis demonstrated that Bm17 was expressed as a late gen. Immunofluorescence analysis by confocal microscopy showed that BM17 protein was localized on cytoplasm and nucleus of infected cells. These results show that BM17 was a late protein localized in cytoplasm and nucleus. [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA) is the major protein component of human plasma. To date, HSA for clinical uses is mostly produced by fractionation of human whole blood, which is accompanied by a lot of limitations. To obtain long-term bioactive albumin, we used hsa as a foreign gene and constructed a recombinant plasmid pJS700-HSA which carries a recombinant gene cotC- hsa under the control of cotC promoter. Plasmid pJS700-HSA was transformed into Bacillus subtilis by double cross-over and an amylase inactivated mutant was produced. After induction of spore formation, western blot and fluorescence immunoassay were used to monitor HSA surface expression on spores. We estimated that HSA displayed on the spore accounted for 0.135 % of the total spore proteins and about 0.023 fg HSA were exposed on the surface of each spore. Oral administration to mice with spores displaying HSA implied that the recombinant spores may have potential ability to increase the serum albumin level in vivo due to the resistant characters of spores. [ABSTRACT FROM AUTHOR]

Bombyx mori parvo-like virus ( BmPLV) has two complementary single-stranded DNA genome (VD1 and VD2) and owns a self-encoding DNA polymerase motif, but its replication mechanism is unclear . In our previous research, a protein encoded by VD1-ORF1 was indentified in the midgut of BmPLV China Zhenjiang isolate-( BmPLV-Z) infected silkworm larvae via two-dimensional gel electrophoresis (2-DE). This protein was named as non-structural protein 2 (NS2), which showed no similarity to that of parvoviruses. To date, little is known about it. In this study, sequence alignment results showed that NS2 shared homology with some chromosomal replication initiator protein dnaA and DNA-binding response regulators. The ns2 was cloned and expressed in E. coli, and then a polyclonal antibody of the NS2 protein was prepared successfully. The data from real-time quantitative PCR displayed that the transcription of VD1-ORF1 from BmPLV-Z-infected midguts started from 28-h post inoculation (h p.i.) in low amounts, but in high amounts at late stages of infection. Immunofluorescence showed that NS2 ultimately concentrated on the nuclear membrane in BmN cells at late stages, indicating that NS2 might be associated with integral membrane protein. [ABSTRACT FROM AUTHOR]

Objective: ORF134 of Bombyx mori nucleopolyhedrovirus (BmNPV) is a homolog of Autographa californica multiple NPV ORF5, but its function is unknown so far. The aim of this study is to characterize BmNPV ORF134 (Bm134). Methods: Transcription, protein expression and subcellular localization of Bm134 in BmN cells and silkworm midguts were analyzed using real-time polymerase chain reaction (RT-PCR), Western blot and immunofluorescence, respectively. Results: Both the transcription and protein expression of Bm134 were not detected until 24 h post-infection (p.i.) by RT-PCR and Western blot, indicating that Bm134 is a late gene. Western blot revealed that Bm134 encodes an expected 12.4-kDa structural protein that is associated with occlusion-derived virus (ODV), not with budded virus. Immunofluorescence analysis showed that the Bm134 was first detected in the cytoplasm 24 h p.i. and then transported to the nucleus during later infection. Transcripts of Bm134 and the corresponding protein were only detected 48-72 h p.i. in BmNPV-infected larvae of 306, a highly BmNPV-susceptible silkworm strain, but not in the NB strain that is resistant to BmNPV infection. Conclusion: Taken together, our data suggest that Bm134 is a late gene of BmNPV and may function as an ODV structural protein. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Objective:Bombyx mori parvo-like virus is a type of virus containing two single-stranded linear DNA molecules (VD1, VD2). In the present work, the structural proteins of B. mori parvo-like virus (China Zhenjiang isolate) (BmDNV-Z) were identified. Methods: The virions were purified by cesium chloride density gradient centrifugation from the feces of B. mori larvae infected with BmDNV-Z and analyzed by SDS-PAGE, Western blotting and MALDI-TOF/mass spectometry. Results: There were seven polypeptides of the virions, named P1 to P7, which reacted with the anti-virion serum. In addition, two polypeptides, P5 and P6, were recognized by the antibody against a viral protein encoded by VD1-ORF3. Conclusion: The results showed that the viral polypeptides were mainly composed of 7 proteins named P1 to P7. Western blotting with anti-virion serum indicated that structural proteins P5, P6 and P7 had molecular weights of around 50, 55 and 130 kDa, respectively. They were the major immunogens of the virion. Among them, P5 and P6 were coded by VD1-ORF3 and P7 was encoded by VD2-ORF1. Taken together, three viral structural proteins encoded by VD1-ORF3 and VD2-ORF1 were identified in BmDNV-Z for the first time. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Orf91 (Bm91) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that encodes a predicted 105-amino-acid protein, but its function remains unknown. In the current study, 5′-RACE revealed that the transcription initiation site of Bm91 was – 12 nucleotides upstream of the start codon ATG, transcription of Bm91 was detected from 12 to 96h postinfection (p.i.) and Bm91 protein was detected from 24 to 96h p.i. in BmNPV-infected BmN cells. Furthermore, Western blot analysis revealed that Bm91 was in occlusion-derived virus (ODV) but not in budded virus (BV). To investigate the role of Bm91 in baculovirus life cycle, a Bm91-knockout virus was constructed by bacmid recombination in E. coli. Fluorescence and light microscopy showed that the production of BV and occlusion bodies (OBs) in Bm91-deficient-virus-infected BmN cells were similar to those in wild-type-virus-infected ones. Bioassay results showed that genetic deletion of Bm91 did not significantly affect BmNPV infectivity, but extended the median lethal time (LT50). Taken together, these results indicate that Bm91 is not essential for viral propagation in vitro, but absence of the gene may affect the virulence of ODVs in silkworm larvae. [Copyright &y& Elsevier]

Abstract: A gene encoding Bombyx mori arginine kinase (BmAK) has been indentified differentially expressed in the midguts of Bombyx mori strain NB which is resistant to nucleopolyhedrovirus (BmNPV), strain 306 which is susceptible to NPV and a near isogenic line BC8 with similar genetic background to 306 but resistant to NPV by two-dimensional gel electrophoresis (2-DE). In this study, we characterized the expression profiles of BmAK using RT-PCR and real-time quantitative PCR. The expression level of BmAK fluctuated in various developing stage and various tissue. Remarkably, the expression level of BmAK increased more than 10-fold 24 hours post inoculation (h p.i.) of NPV in strain NB and BC8, while such increment was abraded in strain 306 although the basal expression level of BmAK in strain 306 was higher than that of strain NB and BC8. Western blotting analysis using polyclonal antibody against BmAK verified such observation, and immunofluoresence analysis indicated for the first time that BmAK was mainly located to the cytoplasm or some structures in cytoplasm. These findings suggest that arginine kinase is involved in the antiviral process of Bombyx mori larvae against NPV infection. [Copyright &y& Elsevier]