Proteomic analysis was performed on the fat body of the fifth instar female silkworm (Bombyx mori, p50) larvae because it is thought that numerous biological substances are synthesized in the fat body. The Drosophila genome and silkworm expression sequence tag (EST) data were applied to the analysis of the fat body proteins and served as the database for protein identification. Protein spots were cut manually from two-dimensional polyacrylamide gel electrophoresis gels, and these spots were treated with 4-vinylpyridine for alkylation. Each spot was analyzed by capillary HPLC coupled with ion-trap mass spectrometry (MS) after proteolysis using modified trypsin. A total of 177 proteins analyzed by MS were identified using the Drosophila genome and silkworm EST data, and these proteins were mapped on 2D-gel. The fat metabolism-related proteins identified in this study were diacylglycerol binding protein, triacylglycerol lipase, and putative hydrolases. Fewer proteins than expected were identified by fat body proteomic analysis. However, nine glycolysis-related proteins were identified in the extracts of the fat bodies. Many cytoskeleton proteins and defense-related proteins were observed in the protein profile. However, some of these proteins were thought to have been derived from the muscles on the epidermis. Calreticulins and chaperonins, including heat-shock proteins, were observed in the fat body extracts.

View full abstract

In transient expression assays, hycu-hr6, the largest homologous region (hr) among the six hrs identified in the genome of Hyphantria cunea nucleopolyhedrovirus (HycuNPV) has been shown to stimulate the activity of an early promoter of the HycuNPV gp64 gene (hycu-gp64) which is located immediately downstream of hycu-hr6. In this study, we generated a recombinant HycuNPV defective in hycu-hr6 (vHycuΔhr6) and characterized its biological properties in permissive SpIm cells. Analyses by Northern blotting and immunoblotting showed that the transcription of hycu-gp64 in vHycuΔhr6-infected SpIm cells was significantly lower than that in HycuNPV-infected SpIm cells until 12 h postinfection, and the observed difference in hycu-gp64 transcription was reflected in the production of Hycu-GP64 protein in the infected cells. In later stages of viral infection, however, no substantial difference was observed between vHycuΔhr6 and HycuNPV in both hycu-gp64 transcription and Hycu-GP64 protein production. Viral DNA replication also showed no substantial difference between vHycuΔhr6 and HycuNPV throughout the experiments. Furthermore, there were little, if any, differences in the ultimate yields of budded virions (BVs) and polyhedrin between vHycuΔhr6 and HycuNPV. These results demonstrate that vHycuΔhr6 is able to complete its infection cycle in SpIm cells and yields BVs and polyhedrin at levels comparable to those of HycuNPV, indicating that hycu-hr6 is a dispensable viral element for HycuNPV replication in SpIm cells.

View full abstract

Using an acidic polyacrylamide gel electrophoresis followed by activity staining, three basic chymotrypsin inhibitors have been found in larval hemolymph of the silkworm, Bombyx mori. These components, named CIs-b1, b2 and b3 in order of migration towards the cathode, were subjected to linkage analysis. Results indicated that the genes coding for CIs-b1 and b2 were found to be novel multiple alleles of the Ict-H locus linked with the Y gene on the second linkage group.

View full abstract

Through that diapause hormone enhances activity of membrane-bound trehalase in developing ovaries, Bombyx oocyte is shown to actively incorporate the blood trehalose and accumulate higher level of glycogen. Because the trehalase-1 gene encoding a soluble type is activated by diapause hormone in ovaries, it is believed that the soluble trehalase becomes to be localized on oolemma via the post-translation modification. Recently, we isolated the trehalase-2 gene encoding a novel membrane-penetrating type. Therefore, in this study it was examined whether this gene was activated by diapause hormone. The trehalase-2 gene was shown to be also activated in developing ovaries by the hormone, although the enhancement was at a lower rate than observed for the trehalase-1 gene. The structure of the trehalase-2 gene was determined and compared with those of trehalase-1 and mammalian trehalase. Several common nucleotide sequences were found in the 5’-upstream regions of the trehalase-1 and -2 genes. Thirteen intron sites of the trehalase-2 gene were much more similar to mouse or human trehalase rather than trehalase-1 that contains only one intron. In B. mori, the ancestral gene similar to trehalase-2 and mammalian trehalase is believed to have duplicated and then diverged into trehalase-1 and trehalase-2.

View full abstract

Calreticulin (CRT) is a multifunctional endoplasmic reticulum (ER) lumen protein, which is also involved in innate immunity. To study the involvement of CRT in the insect immune response, silkworm larvae of Bombyx mori were treated with lipopolysaccharide (LPS). LPS treatment induced an increase in CRT mRNA in the fat body only, and not in hemocytes, the midgut, silk gland, or Malpighian tubule. Time course analysis indicated that significant CRT mRNA expression by the fat body was induced 6h after LPS injection, and returned to the normal level within 24h. The NIAS-Bm-aff3 cell line, which was established from the fat body of B. mori, showed a similar expression pattern on LPS exposure. Since the fat body is capable of a humoral response in insect innate immunity, our results suggests that CRT is induced by the activation of the humoral immune system in the fat body of Bombyx mori.

View full abstract

Sericin, which is about 25% of the total cocoon shell of the silkworm Bombyx mori, has a skin moisturizing effect, and an inhibitory effect against lipid peroxidation and tyrosinase activity. According to these properties, it has come into use in the skin care industry. To effectively produce intact sericin protein, a new silkworm race “Sericin Hope” was developed by cross breeding an Nd mutant (naked pupa) and a high cocoon yielding strain KCS83. The new strain spins sericin cocoons at a frequency of 99%. The cocoon weight is more than 4times heavier than that of the Nd mutant. The Sericin Hope cocoon, named “Virgin Sericin”, can be gelled in water with less hydrolyzing by autoclaving at 110°C for 10min. The Virgin Sericin gel can be easily emulsified, firmly creamed or made into foam with 5% oil.

View full abstract

To investigate the effect of a promoter sequence for detecting enhancer activity in the silkworm Bombyx mori, we constructed six different GAL4 vectors under the control of different minimum promoters. In addition, a homozygous UASGFP strain was constructed and used to test expression of the GAL4 gene. Green fluorescent protein (GFP) expression was compared in the transgenic strains with the different GAL4 gene inserts. The most prominent difference in the expression pattern in the different strains was observed when the Bombyx cytoplasmic actin gene (BmA3) promoter was used. We concluded that using the GAL4 gene with the BmA3 promoter would be the most useful way for detecting the positional effect of genes inserted in the silkworm.

View full abstract