EA4, an ATPase purified from Bombyx diapause eggs, exhibits one-time transitory burst activation two weeks after chilling at 5°C. The time of sudden increase in the EA4 activity is thought to be equivalent to that observed in vivo and is coincident with the chilling period indispensable for diapause termination. EA4 is a glycoprotein, and the carbohydrate moiety is involved in the EA4 activation through the interaction with peptides named PIN that corresponds to fragments of egg-specific protein. PINs inhibit the ATPase activity to delay the initiation of the activation and also inhibit deglycosylation of EA4 by PNGase F. The rate of inhibition increases as a function of increasing PIN amounts added. When 80nmol of PINs are added to 1nmol of EA4, the deglycosylation is almost completely inhibited as well as the ATPase activities. Those peptides of similar molecular weight to PINs, of hydrophobicity and others do not inhibit the deglycosylation. PINs may recognize the sugar chain of EA4 specifically and they also may bind the sugar directly or the site close to the sugar where PNGase F works, by which PINs regulate the initiation of EA4 activation.

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An ommin-binding protein (OMBP) in the acid-methanol extract of diapause eggs of the silkworm, Bombyx mori was found. OMBP was purified by a modification of the purification method for ommin, and the molecular mass of OMBP was 32kDa by SDS-PAGE. The purified protein was similar to a kind of 30-kDa protein that is known to be a major plasma protein in the larval hemolymph on the basis of immunochemical reactivity. The changes of OMBP during two days after oviposition was compared with diapause eggs and non-diapause eggs by the Western blot analysis using anti-OMBP antiserum, but there were no differences in the immuno positive signals. OMBP was recognized just before hatching, but disappeared after hatching. However, the immuno positive signal was found in the larvae after hatching using anti-30-kDa protein antiserum. According to the results, OMBP seems to be similar to a strand of 30-kDa protein found during the egg stages, but OMBP is different from 30-kDa proteins in the larvae after hatching. The relationship between 30-kDa proteins and OMBP in the eggs of the silkworm is discussed.

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Mariner-like elements (MLEs) were amplified by PCR against genomic DNAs from several Japanese lepidopteran species, using the inverted terminal repeats (IR) of the Hyalophora cecropia MLE as a primer. Clones thus obtained were of a size of about 1.3kb, and expected to contain the full-length MLE. A 1.3-kb band was also amplified against genomic DNA from the grasshopper, Traulia ornata, and from a coral allocated to the Fungia family. All of the 1.3-kb bands were cloned and analyzed for nucleotide sequence. Multi-alignment analyses of the results indicated that the clones were highly similar to each other and classified into the cecropia subfamily of MLEs. The coral MLE was found to have a complete ORF coding for transposase, a situation similar to that previously found in the Emperor moth, Attacus atlas, from the Ryukyu island. These findings, together with the fact that all of the insects and coral species that exhibited a positive signal for the full-length MLE were collected in relatively close regions around Japan, indicated that the horizontal transfer of MLEs had taken place even among phylogenetically remote organisms.

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Two novel crystal protein genes, designated as cry39A and 39orf2, were cloned from Bacillus thuringiensis (Bt) subsp. aizawai Bun1-14. When sequenced, these genes were found to contain open reading frames for proteins of 75 kDa and 63 kDa, respectively. The Cry39A protein, which is a typical Bt crystal protein, revealed five conserved blocks (block1 to block5). The 39Orf2 protein showed high homology to the carboxyl half of the 130 kDa Cry proteins such as Cry4A and had three conserved blocks (block6 to block8). The cry39A and 39orf2 genes were expressed in an acrystalliferous B. thuringiensis strain. Cry39A formed bipyramidal crystal inclusions only in the presence of 39Orf2 and was toxic to mosquito larvae as a wild type strain Bun1-14. No crystal inclusion was observed when 39Orf2 was truncated.

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Two Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) mutants defective in conserved pcna 1 and pcna 2 sequences and in substantial portion of the C-terminal region of pcna open reading frame were generated. Biological characterization revealed that these pcna-defective mutants were both viable in the cell culture and the kinetics of viral DNA replication, viral structural protein synthesis, budded virion yield and polyhedrin production in the pcna-defective AcMNPV-infected cells were comparable to those in the cells infected with wild-type (wt) AcMNPV. Localization experiments showed that the amount of cellular PCNA in the nuclear fraction was significantly higher in the cells infected with pcna-defective AcMNPVs than in the cells infected with wt AcMNPV from 8 to 16h postinfection. These results indicate that AcMNPV PCNA is not essential not only for viral DNA replication but also for the yield of progeny virions and suggest that cellular PCNA functions in the viral DNA replication in the cells infected with pcna-defective AcMNPV mutants. On the basis of our data from cellular PCNA localization experiments and the fact that pcna-missing NPVs are able to multiply to high titers in the cell culture, it is also suggested that virus-encoded PCNA is crucial for viral DNA replication only in the cells that provide an environment unfavorable for DNA replication.

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Hyphantria cunea nucleopolyhedrovirus (HycuNPV) homologue (hycu-gp64) of baculovirus gp64 gene was identified and characterized. The hycu-gp64 open reading frame encoded a polypeptide of 509 amino acids with a predicted molecular weight (MW) of 58, 378, that shared 72 to 82% amino acid sequence identities with GP64's from other group I NPVs. The Hycu-GP64 possessed ten putative N-glycosylation sites that represented the largest number among GP64's so far sequenced. Analyses of the hycu-gp64 promoter region revealed a number of elements responsible for transcriptional regulation, that included an early transcription start motif (CAGT), three late transcription start motifs (A/T/GTAAG), an IE1 binding motif (IBM: 5′-ACBYGTAA-3′)-like sequence, a TATA box, two GATA elements, and a heat shock factor motif. Northern blot analysis revealed the presence of two major transcripts (1.7 and 1.6kb) that were detectable from 2h postinfection (pi), whereas Western blot analysis showed a polypeptide with an approximate MW of 68, 000 that was detected from 4h pi onward. Transient expression assays suggested that basal activity of the hycu-gp64 early promoter depended on the presence of the TATA box, whereas full activity of the hycu-gp64 early promoter on a HycuNPV homologous repeated sequence, hycu-hr6, that was located immediately upstream of the hycu-gp64 promoter. Cotransfection experiments with a plasmid containing the HycuNPV ie1(hycu-ie1) showed that Hycu-IE1 stimulated the transcription from the hycu-gp64 early promoter, although gene expression from the full hycu-gp64 promoter was two times lower than that from the construct carrying the hycu-gp64 promoter with a deletion of an IBM-like and GATA sequences. The transient expression assays also demonstrated that hycu-gp64 contained a secretory signal sequence that was active in uninfected Splm cells.

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The extraction of sericin from the cocoon of Bombyx mori was re-investigated by incubation in 8M urea solutions with and without 2-mercaptoethanol concerning the yield and the integrity of the polypeptide components on the basis of Gamo's method reported in 1973. When the incubation temperature was raised from 40 to 80°C, the yield of sericin first decreased, and then increased to reach 100% at 80°C. The addition of 5% 2-mercaptoethanol also raised the yield of sericin. Gel filtration chromatography revealed that sericin solutions reduced by 2-mercaptoethanol consisted of at least two main components, and that high temperature and long heating time caused the degradation of sericin molecules. The most practical method to recover intact sericin as much as possible was determined; the incubation in 8M urea with 5% 2-mercaptoethanol of 80°C for 2-10min enables the extraction of more than 95% of the total cocoon sericin without degradation.

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Silk fibroin and cellulose were dissolved in cuprammonium solution using degummed silk of various wild silkmoths and spun into thread. Produced composite fibers exhibited lustrous color, typical of each component silk fibroin, and showed the mechanical properties necessary to withstand the practical use of it as clothing and as a functional polymeric material as well. There is a possibility to make the composite fibers more durable against stretching by improving the spinning procedure. The cocoons of S. c. ricini, C. trifenestrata and of Rhodinia fugax are unable to be spun to obtain a long thread. This kind of disadvantage can be overcame by producing the composite fiber.

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Mori silk and a wild silk were dyed with mononicotinic acid triazine reactive dyes, which were high temperature neutral-fixing dyes, and the following results were obtained; 1. The dye absorption and fixing ratio were best in a neutral bath of pH 6.5-7.5. 2. When a mori silk was dyed at the boiling temperature in a bath containing 60-80g/l of anhydrous sodium sulphate, the absorption and fixing ratio were both higher than 80%, which was about the same level of dyeability as with vinyl sulphone type reactive dyes (Ogawa, 1977). The results suggested that mononicotinic acid triazine reactive dyes could be used for dyeing mori silk. 3. The wild silk gave about half the dye absorption and fixing ratio as mori silk, suggesting that these reactive dyes were not practically suitable for dyeing wild silks. 4. The addition of a nonionic surfactant in the dye bath slightly improved the dye absorption by wild silk, because the surfactant promoted the penetration of the dye to the interior of the fibres. 5. The mononicotinic acid triazine reactive dyes did not give a complete sericin fixing effect, unlike dichlorotriazine-based reactive dyes. But mononicotinic acid triazine reactive dyes appeared to be suited for dyeing half-degummed mori silk.

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To further understand the function of Samui protein which is a member of the BAG-protein family, the temporal profiles of mRNAs for the cold-inducible genes, Samui and sorbitol dehydrogenase (SDH), were examined in Bombyx diapause eggs exposed to 5, 0 and -10°C after incubation at 25°C for 30 days after oviposition Incubation at 5°C activated the expression of both genes, incubation at 0°C activated only the Samui gene, and incubation at -10°C did not activate the expression of either gene. Although incubation at 0°C was less effective for the termination of diapause than at 5°C, from about half of the eggs that had been incubated at 0°C for 250 days, larvae hatched within 60 days after being transferred to 25°C. This changing profile in the hatching ability appears to be correlated with the amount of Samui mRNA. These results are consistent with a proposal from a previous study which stated that the Samui protein may play a role in transmitting the signal from low temperatures, such as 5 and 0°C, to a downstream cascade in a diapause egg. In addition, a possible strategy for the long-term preservation of Bombyx eggs was discussed.

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The baculovirus vector is a widely utilized, highly efficient protein production system, which, with the incorporation of larvae from the silkworm, Bombyx mori, can be used for commercial mass production. Using recombinant baculovirus encoding human serum albumin (HSA) and B. mori larvae, we studied the effects of larval developmental stage and timing of virus infection on recombinant HSA (rHSA) expression. Silkworm larvae were infected with virus on fourth instar day 1 (D1), day 2 (D2), day 3 (D3), or day 4 (D4). Although all cohorts died by postinfection day 4, rHSA expression levels and total soluble protein levels differed between cohorts. The rHSA expression levels decreased from 273μg/larva in cohort D1 to 246μg/larva in cohort D2, to 195μg/larva in cohort D3, and to 138μg/larva in cohort D4. Our results helped identify optimal conditions for rHSA production in silkworm larva using the baculovirus vector.

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A non-flavonoid component with antioxidative activity was isolated from cocoons of the silkworm, Bombyx mori. This antioxidant was purified by reversed-phase HPLC from the water-soluble extract rich in flavonoids. The LC/ESI-MS mass spectrometry of the antioxidant gave an estimated molecular weight of 167.7. Based on UV and IR spectrometry, it was identified as uric acid. The content of uric acid varied depending on silkworm strains, with yellow-green cocoons (Daizo) containing more uric acid than non-colored cocoons (Kinshu×Showa).

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