In order to determine the properties of trehalase in the developing eggs of Bombyx mori, soluble-type trehalase was purified from the supernatant of egg homogenate by acid treatment, column chromatographies of Sephacryl S-300, DEAE-cellulofine and Con A-Sepharose, and preparative polyacrylamide gel electrophoresis. Two peaks of trehalase activity were detected by gel filtration column chromatography, and designated as P I (73kDa) and P II (140kDa). P I and P II were eluted as a single peak from ion exchange column chromatography at about 0.2M and 0.25M of NaCl, respectively. Subunit molecular masses of P I and P II were estimated as approximately 64kDa on SDS-PAGE and Western blot analysis. P I had a K_m of 1.56mM for trehalose and an optimal pH around 6.5 with an activation energy of 11.03kCal/mol. P II had a K_m of 0.44mM for trehalose and an optimal pH around 5.5 with an apparently lower activation energy of 5.92kCal/mol. These results suggest that two types of soluble trehalase are present in the embryonic larvae of the silkworm, Bombyx mori.

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Micropylar structures of chorion in the bd mutant group of Bombyx mori were observed under a scanning electron microscope, with special reference to the external features of the micropylar rosette, orifice and internal channel tubes. These structures were normal in the control eggs, i.e., heterozygous for bd, bd^sw and bd^f alleles, but were deformed in the bd and bd^sw homozygous eggs, both of which were sterile. In particular, the channel tubes were not present in these eggs. On the other hand, the micropylar architecture of the bd^f homozygous eggs, which were fertile, was normal like control eggs. Based on these results, we suggest that the sterility of the bd and bd^sw females is caused by the abnormality of the micropylar apparatus, which may be crucial for fertilization processes.

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Small heat shock proteins (sHsps), which are constitutively expressed in the absence of stress during development in many organisms, have been correlated with not only enhanced thermoresistance but also the developmental, tissue-, and cell-specific induction and expression. This study demonstrates the constitutive sHsps 21 and 23 of an entomoresouce, the leaf beetle, Gastrophysa atrocyanea, display a double chaperone function in vitro. The RNAi suppression of two sHsp genes in adults resulted in decreased thermoresistance. A strong correlation was observed between the in vitro chaperone function and in vivo thermotolerance analysis, and the results support a possible double chaperone function critical for the survival of the leaf beetles against higher temperatures.

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The silkworm, Bombyx mori, is known to be more resistant to ionizing radiation (IR) compared with mammals, but detailed processes underlying this resistance have remained largely unexplored. In this study, we have confirmed that silkworm larvae survived and showed no effect in the apparent reproduction ability after the irradiation of high doses of γ-ray. We have then observed the effects of γ-irradiation to cells of the silkworm cell line BmN4, which showed marked cell-cycle arrest at the G₂/M phase, but not at the G₁ or S phases. These cells did not undergo apoptosis after γ-ray irradiation in contrast to the mammalian cells wherein G₁ arrests after IR causes apoptosis. After UV-C radiation to cells of BmN4 and Sf21 (a Spodoptera frugiperuda cell line), S-phase checkpoint activation was provoked. But there was also no observation of cell death in BmN4 cells, in contrast to Sf21 cells. Based on these results, we proposed that the extraordinary tolerance of induced DNA injury or the elimination of cell death programs after irradiation is a possible cause of the irradiation resistance in the silkworm cells.

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A recombinant Bombyx mori nucleopolyhedrovirus (BmNPV), vBmΔpolh/lacZ/hrf1 (vBm/hrf1), that possessed the hrf-1 (host range factor 1) gene derived from Lymantria dispar multicapsid NPV (LdMNPV) was generated and examined for its biological properties in nonpermissive L. dispar Ld652Y cells. Although budded virus (BV) production was undetectable in vBm/hrf1-infected Ld652Y cells under the conditions used, a significant amount of BVs were yielded in Ld652Y cells transfected with vBm/hrf1 DNA, while transfection of DNA from a control recombinant BmNPV, vBmΔpolh/lacZ (vBm), that did not possess the hrf-1 gene, produced no detectable BVs in Ld652Y cells. BV yields increased when vBm/hrf1 DNA-transfected Ld652Y cells were cotransfected with a plasmid expressing HRF-1. Immunoblot analysis showed that viral structural proteins were produced in vBm/hrf1-infected Ld652Y cells, while viral structural proteins were not detected in vBm-infected Ld652Y cells. Pre-transfection of Ld652Y cells with a plasmid expressing HRF-1 increased viral structural proteins produced in vBm/hrf1-infected Ld652Y cells. No difference was observed between vBm/hrf and vBm in viral DNA replication in infected Ld652Y cells, indicating no direct effects of hrf-1 on viral DNA replication. These results indicate that HRF-1 expressed from the hrf-1 gene embedded within the vBm/hrf1 genome functions to preclude global protein synthesis shutdown and aids the vBm/hrf1 replication in Ld652Y cells, providing additional evidence for the hypothesis that HRF-1 is an essential viral factor commonly required for NPVs to replicate productively in Ld652Y cells.

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Enzymatic properties of fibroinase of silk gland from day one B. mori pupa were investigated to obtain additional comparable data with those of the fibroinases of silk gland from the fourth molt period (Watanabe et al., 2004a) and from the spinning period (Sutthikhum et al., 2004a, b). Fibroinase of silk gland from day one pupa hydrolyzed liquid fibroin efficiently. Subunit and native molecular masses were estimated as 34.2 and 19.8kDa. Optimum pH was at pH3.71 and at pH5.5 with liquid fibroin and benzyloxycarbonyl (Z)-Phe-Arg-4-methyl-coumaryl-7-amide (MCA) being used as substrates. Proteinase inhibitor concentrations (nM) required to inhibit activity by 50% were:leupeptin (1.14), E-64 (1.22), Z-Phe-Phe-CHN₂ (3.46), antipain (8.26), chymostatin (10.1), TLCK (12.5), Z-Phe-Ala-CHN₂ (29.3), TPCK (577.5), iodoacetic acid (1096) and pepstatin (37077) with no inhibition by APMSF. Fibroinase stored at 30°C for 1h at pH's 4.0 and 5.0 was stable retaining half the initial activity but was unstable at pH's 6.0 and above. These properties clearly indicate that fibroinase of silk gland from day one pupa is a cathepsin L-like cysteine proteinase. Properties of fibroinase of silk gland from day one pupa determined with liquid fibroin as a substrate were similar to those of the fibroinase of silk gland from the fourth molt period. However the properties determined with Z-Phe-Arg-MCA as a substrate, fibroinase of silk gland from day one pupa was similar to the fibroinase of silk gland from spinning period. These differences should result from the differences in physical properties of the substrate which affected the responses of fibroinase of silk gland from respective developmental period. Based on several evidences, fibroinase of silk gland that is similar in enzymatic properties to the fibroinase of silk gland from day one pupa is highly likely responsible for enzymatic digestion of fibroin and sericin remaining in the silk gland of B. mori pupa prior to apoptosis of pupal silk gland.

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