In order to identify fibroinase in the silk gland of B. mori, fibroinase was purified to homogeneity from the silk gland in the fourth instar B. mori larva at the fourth molt period, stage D₂. Optimum pH was 4.7 as determined with Z-Phe-Arg-MCA which was a good substrate for fibroinase. Concentrations of proteinase inhibitors required to inhibit 50% of activity and expressed in nM were: leupeptin (0.833), E-64 (4.57), Z-Phe-Phe-CHN₂ (6.97), TLCK (10.1), antipain (11.5), chymostatin (13.7), Z-Phe-Ala-CHN₂ (43.3), TPCK (467) and iodoacetic acid (3176); no inhibition by pepstatin and APMSF. Fibroinase stored at pHs 4.0 and 5.0 was fairly stable and unstable at the neutral and alkaline pHs. Purified fibroinase hydrolyzed liquid fibroin, and primary cleavage site was a peptide bond between Gly and Ala. Cleavage sites of oxidized β-insulin chain were those cleaved by cathepsin L. Subunit molecular mass was 34.9kDa and native molecular mass 31.6kDa, and fibroinase was considered to consist of a monomer with molecular mass of approx. 35kDa. N-terminal amino acid sequence was Leu-Pro-Glu-Gln-Val-Asp-Trp-Arg-Lys-His. These results indicate that fibroinase in the silk gland from the fourth instar B. mori larva at the fourth molt period, stage D₂, is cathepsin L-like cysteine proteinase.

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Fibroinase activity in the B. mori silk gland was known to occur at the fourth molt period in the fourth instar larva (Sumida et al., 1993b) and at day 1 in the pupa (Sumida et al., 1993a). In order to know if fibroinase activity is present in the silk gland in other developmental periods, i.e., feeding and spinning periods, we developed two methods; a homogenization method of silk gland containing large amount of fibroin and sericin in its lumen contents and a quantitative assay method of fibroinase using liquid fibroin as a substrate. The activity was detectable in the silk gland in the feeding and spinning periods; it was low in the early fifth instar larva, rose rapidly in the middle of the instar and reached a plateau just before the onset of spinning and this level was maintained during spinning. Silk gland fibroinase was partially purified from the spinning larva. The final preparation hydrolyzed fibroin efficiently. Subunit molecular mass was 32.5kDa and N-terminal amino acid sequence was LPEQVDWRKHGA. Similar properties were observed in the B. mori fibroinase of silk gland purified from the fourth instar larva and the day 1 pupa (unpublished data). Optimum pH was at 5.85 when the liquid fibroin was used as a substrate, the value of which was higher than those of the fibroinase from the fourth instar larva and the day 1 pupa. The concentrations of proteinase inhibitors required to inhibit fibroinase activity completely using liquid fibroin as a substrate and expressed in μM were: E-64, 0.92; leupeptin, 1.6; TLCK, 2.2; chymostatin, 2.6; antipain, 24.0; TPCK, 94.0; pepstatin, 100.0 and iodoacetic acid, 680.0 with no inhibition by APMSF. These results indicate that fibroinase of silk gland present in the spinning B. mori larva is a cathepsin L-like cysteine proteinase.

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To investigate the active movements of organs and tissues in a silkworm body, a system was developed to measure the electric potential of the body surface of a silkworm, which was derived from muscle contractions. The measurement system can record the behavior of the silkworm by a video camera. We measured the electric potential of the body surface of a silkworm was in different state and behavior, particularly in resting and moving, and analyzed the constituent interests of the electric potential signal using the spectral analysis method, and the relationship between the electric potential and the intersegmental distance using the cross-correlation analysis method. We obtained different spectral density patterns of electric potential signals and extracted distinctive peaks in frequency range. These results showed that the electric potential obtained by this system reflects the active movement of organs and tissues of the silkworm. With this measurement system, it is thus possible to investigate the active movement and existence states of the internal organs and tissues of the silkworm that is in normal growth condition, without any dissection and restriction of behaviors.

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When a cylindrical transparent filament such as a silk fibroin one is irradiated by a laser beam, a scattering intensity pattern appears on a plane perpendicular to the filament. Its diameter and refractive index are determinable by measuring this intensity pattern. On the basis of this principle, I have developed an automated measurement system for the pattern. Using the system, I have examined to determine the parameters for the sample filaments of the silk fibroin peeled from cocoons. As a result, the theoretical intensity patterns were corresponded well to the experimentally measured ones. Furthermore, it revealed that the refractive indices and diameters of the filaments were reasonably determined.

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In order to simplify the model of simulating the fixed size raw silk reeling process, we deduced the approximate distributions of the size of cocoon filament in feeding ends and dropping ends and gave the average slope of the size of raw silk. Furthermore, we validated the simplified model by the existing methods of simulation reeling process and discussed its application to the reeling process control.

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The correlation between antifungal metabolites produced by Streptomyces sp. 39L40C and its disease suppressive effect was studied. Mutants of strain 39L40C showing non-antagonism to a mulberry twig blight pathogen, Fusarium lataritium f. sp. mori (FL), were induced by ultraviolet irradiation. Forty-three strains were isolated as candidates. Among them, two strains (100s-19, 15m-1) did not show antifungal activity with not only plate assay but also the culture filtrate assay. Disease suppression by these mutants and the wild strain 39L40C was examined with inoculation tests using spring pruning branches of mulberry. As a result, strain 100s-19 and strain 15m-1 showed suppression to mulberry twig blight in the case of 2.0×10⁴, 2.0×10⁵cells/ml FL concentration. However, both strains were not able to suppress twig blight at an FL concentration of 2.0×10⁶ cells/ml. These results showed that the disease suppressive effect of strain 39L40C was attributed to the antifungal metabolites.

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A mutation named Set, which has been characterized by abnormal larval traits, shows embryonic lethality as a pleiotropic effect. Light microscopic observations of the dying eggs without hatching of this mutation indicated that the epithelial cells of the midgut, especially the microvilli, were abnormal. Other tissues and organs of Set embryos were confirmed to develop fully, but massive yolk remained in the lumen of the midgut, indicating that yolk proteins were not utilized. We infer that the Set mutation brings about functional impairment of the midgut digestive/absorption system.

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