We prepared conjugates (COS-CY-SF) by the chemical modification of solubilized silk fibroin (SF) with polycationic chitooligosaccharides (COS) using cyanuric chloride (CY) as a coupling spacer, and examined initial attachment of fibroblast cells and hepatoma cells employed as a model for hepatocytes on the conjugate-coated surfaces. The attachment of fibroblast cells on the COS-CY-SF conjugate-coated surfaces was significantly lower than that on unmodified SF-coated surfaces. Fibroblast cells attached to the conjugate-coated surfaces showed only round-shaped morphology while fibroblast cells attached to SF-coated surfaces showed predominantly spread morphology. In the case of hepatoma cells, the attachment on the COS-CY-SF conjugate-coated surfaces was lower than that on SF-coated surfaces. Hepatoma cells attached to the conjugate-coated surfaces showed round shapes. These results indicated the inhibitory effect of polycationic COS moiety immobilized into SF on initial cell attachment. Based on the round and spread morphologies of the hepatoma cells on SF-coated surfaces, SF cannot be considered as a suitable substrate for hepatocyte attachment.

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In vitro gene expression of attacin, an antibacterial protein from the silkworm, Bombyx mori, was analyzed using nuclear extracts from the fat body (FB) and posterior silk gland (PSG). TATA box-dependent basal promoter activity was observed in vitro in the FB and PSG, even though the Attacin gene is known to be expressed in vivo in the FB but not in the PSG. Comparison of nucleosomal structure near the Attacin gene promoter regions between the two tissues indicated clear nucleosomal arrangements in the PSG but not in the FB, suggesting that regulation of tissue-specific Attacin gene expression occurs at the chromatin level. Electrophoresis mobility shift assay (EMSA) was conducted using different regulatory regions of the Attacin gene as probes. Results showed that nuclear proteins bind tissue-specifically to the sequence CATTT in addition to a NF-κB-like (κB) motif, suggesting the presence of an additional regulatory motif for Attacin gene expression. Nuclear protein binding to CATTT was not competitively prevented by the Cecropin B1 (CecB1) regulatory region containing two CATTA sequences, suggesting different nuclear protein(s) bind to the CATT(T/A) motif. Functional analysis of the CATTT motif of the Attacin gene showed that a mutation of this motif still retains 78.9% of the activity of the wild type control, suggesting that the CATT(T/A) motif plays a different role in Attacin and CecB1 even though they are similarly involved in immune reactions against bacterial infection.

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In the silkworm, Bombyx mori, diapause hormone (DH) produced in the female subesophageal ganglion (SG) and released into the hemolymph primarily determines embryonic diapause by targeting developing ovaries. DH consists of 24 amino acid residues with an amidated C-terminus and belongs to the FXPRLamide peptide family. In order to quantify DH levels, we previously developed a sandwich enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal and rabbit polyclonal antibodies to the N- and C-terminal regions of DH, respectively. This procedure demonstrated that DH levels in SG during the middle pupal stage decreased markedly in the diapause-egg producers when compared with the nondiapause-egg producers from a bivoltine race, Daizo, thus indicating active release of DH into the hemolymph. In this study, in order to further confirm this result, we measured and compared DH levels in SGs of diapause- and nondiapause-egg producers from Bombyx three races. When DH release into hemolymph was stimulated by administration of the Na⁺-K⁺ ATPase inhibitor ouabain, DH levels in SG were decreased. These results suggest that the reduced levels of DH in SG during the middle pupal stage are the result of active release of DH into the hemolymph.

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Susceptibility to Autographa californica NPV (AcNPV) of a cell line derived from Antheraea pernyi (AnPe) was drastically improved by changing the culture medium from Sf-900II serum free medium (Sf (−)) to serum-supplemented TC-100 medium (TC (+)). When AnPe cells maintained in Sf (−) and TC (+) were adapted to the mixed medium (Sf (−):TC (+)=1:1) or to serum-supplemented Sf (−), amounts of budded virus and polyhedrin produced in the AcNPV-infected cells as well as, in most cases, expression levels of 5 medium-responsive cellular transcripts, which had been identified by differential display and real-time PCR analyses, resulted in intermediate values between those observed in Sf (−) and TC (+). The results indicated that susceptibility to AcNPV and expression of 5 medium-responsive transcripts in AnPe cells were mainly altered by the balance of components contained in the fetal bovine serum and Sf (−). In addition, expression levels of several medium-responsive transcripts were transiently enhanced after the virus infection, suggesting the possibility that they encode factors promoting and suppressing the AcNPV replication.

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The production and excretion of silk proteins in the silk gland cells are very marked processes at the final larval instar of the silkworm, Bombyx mori. Here, proteins in the posterior silk gland tissues and lumen contents in the mutant called flimsy cocoon (flc), wherein fibroin is hardly secreted into the lumen, were separately analyzed by two-dimensional gel electrophoresis. It was shown that the putative small subunit fibroin was accumulated in the cells, and that this protein did not appear in the lumen. In the normal silkworm, this protein was scarce in the tissues but rich in the lumen. Radioactivity counting in the hot-acid insoluble fractions after intraperitoneal injection of [³H]glycine also exhibited a rapid incorporation of label into the posterior silk gland tissues both in the normal and mutant larvae, but differential label was found in the lumen, being very small in the mutant. These results support the idea that the secretion machinery in the flc silk gland cells is deficient.

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Non-viral 45.5K and 44K polypeptides have been shown to accumulate in virus-infected degenerate cells in the midgut epithelium of the silkworm, Bombyx mori. These polypeptides are detectable first temporally at around 3 days postinfection (pi) and then persistently at around 8 days pi onwards, when day 1 fifth-instar larvae of the silkworm are infected with B. mori densovirus type 2 (BmDNV-2). In this study, we found that the amount of 45.5K and 44K polypeptides accumulated in BmDNV-2-infected midgut epithelium was inversely correlated with that of BmDNV-2 structural polypeptides. TUNEL staining showed that the degeneration of BmDNV-2-infected cells enriched in 45.5K and 44K polypeptides was not due to apoptosis. These results suggest that 45.5K and 44K polypeptides are involved in the clearance of the cells damaged by virus multiplication from BmDNV-2-infected midgut epithelium by a mechanism different from apoptosis induction, resulting in a restricted accumulation of viruses in BmDNV-2-infected midgut epithelium.

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