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Fibroin silk proteins from the nonmulberry silkworm Philosamia ricini are biochemically and immunochemically distinct from those of the mulberry silkworm Bombyx mori.

Silk proteins were isolated from the cocoons of the nonmulberry silkworm, Philosamia ricini. Three polypeptides of 97, 66, and 45 kDa were identified. The 66-kDa molecule represented sericin, whereas the 97-kDa and the 45-kDa polypeptides linked together through a disulfide bond constituted the fibroin protein. Antibodies raised against the 97-kDa P. ricini fibroin heavy chain reacted specifically with this molecule and did not recognize fibroin heavy chain from another nonmulberry silkworm, Antheraea assama or from the mulberry silkworm, Bombyx mori, suggesting the presence of P. ricini species-specific determinants in this heavy chain. Antibodies generated against fibroin light chain of P. ricini also showed similar reactivity pattern. Immunoblot analysis with proteins isolated from the silk glands of P. ricini at different stages of larval development showed that the expression of fibroin heavy chain was developmentally and spatially regulated. The protein was most abundant in the 5th instar larva, and could be detected in the middle and the posterior but not the anterior silk glands. The amino acid composition of the 97-kDa fibroin protein showed abundance of glutamic acid and did not contain (Gly-Ala)(n) motifs, a characteristic feature of B. mori fibroin heavy chain. Our study reveals significant differences between the nonmulberry silkworm P. ricini and the mulberry silkworm B. mori in the biochemical composition and immunochemical characteristics of fibroin heavy chain. These differences might be responsible for the differences seen in the quality of silk produced by these two silkworms.

Amino Acids↗

The effect of W chromosome origin on sex-chromosome pairing in ZZWW tetraploid females of the domesticated silkworm, Bombyx mori, and the congenic wild silkworm, Bombyx mandarina.

To analyze the degree of pairing of the Z and W chromosomes in ZZWW tetraploid female silkworms that have the W chromosomes of the domesticated silkworm, Bombyx mori, and those of the wild silkworm, Bobyx mandarina, we induced two types of ZZWW tetraploid female silkworms (Cr4n, Wr4n) through cold treatment of the eggs. The Wr4n female is congenic to the Cr4n female for W chromosomes; namely, the W chromosomes of the Wr4n female are derived from those of B. mandarina. Each of the sex ratios (female/male) in filial triploids from the Cr4n females was shown to be in the range of 3.9-5.3 (4.6 as an average of six cases). On the other hand, each of the sex ratios (female/male) in filial triploids from the Wr4n females was shown to be in the range of 6.2-9.0 (6.9 as an average of nine cases). The results of a t-test indicated that the difference in sex ratios in the two groups is highly significant (at the 0.1% level). These results suggest that, in the meiosis of the ZZWW tetraploid female, the frequency of pairing of the W chromosome of B. mandarina and the Z chromosome of B. mori is lower than that of the pairing of the W and Z chromosomes of B. mori. Furthermore, the t-test results are evidence that the W chromosomes have undergone significant evolutional change.

Animals↗

Antibody production in silkworm cells and silkworm larvae infected with a dual recombinant Bombyx mori nuclear polyhedrosis virus.

We have examined the efficiency of coexpression of two heterologous genes from a recombinant Bombyx mori nuclear polyhedrosis virus for the production of antibodies in silkworm larvae. The cDNAs encoding the light and the heavy chains of a murine immunoglobulin, directed against lipoprotein I of Pseudomonas aeruginosa, were brought under the control of two separate copies of the viral polyhedrin promotor. Infection of silkworm cells with the recombinant baculovirus yielded a maximum of 6.4 micrograms/ml IgG2A in the culture supernatant 72 hours post infection, while 800 micrograms/ml IgG2A was found in the hemolymph of infected fifth instar silkworm larvae seven days after infection with the same construct. The recombinant antibody exhibited a similar antigen specificity and avidity to that of the monoclonal antibody derived from ascites fluid.

Animals↗

Identification of novel random amplified polymorphic DNAs (RAPDs) on the W chromosome of the domesticated silkworm, Bombyx mori, and the wild silkworm, B. mandarina, and their retrotransposable element-related nucleotide sequences.

Genomic DNAs were compared between males and females of the domesticated silkworm, Bombyx mori, strains C108, C137, J137, p50, and WILD-W (constructed by crossing a wild silkworm, B. mandarina, female with a male of strain C108) by polymerase chain reaction (PCR) with 700 arbitrary 10-mer primers. Four female-specific RAPDs (W-Kabuki, W-Samurai, W-Kamikaze, and W-Yamato) were found. The sex chromosome formulas of B. mori and B. mandarina are ZW (XY) for the female and ZZ (XX) for the male. The four female-specific RAPDs are assumed to be derived from the W chromosome because the other chromosomes are shared by both sexes. A computer search for deduced amino acid sequences of these four RAPDs revealed that all of them showed homology to previously reported amino acid sequences encoded in known retrotransposable elements from various organisms.

Amino Acid Sequence↗

Studies on the posterior silk gland of the silkworm Bombix mori. IV. Ultracentrifugal analyses of native silk proteins, especially fibroin extracted from the middle silk gland of the mature silkworm.

Ultracentrifugal analyses of the native silk proteins extracted from the various parts of the middle silk gland of the mature silkworm have revealed that there exist four components with S degrees (20,w) values of 10S, 9-10S, 9S, and 4S in the extract. It is suggested that the fastest 10S component is the native fibroin synthesized in the posterior silk gland and transferred to the middle silk gland to be stored there, while the slower three components probably correspond to inner, middle, and outer sericins which were synthesized in the posterior, middle, and anterior portion of the middle silk gland, respectively. Native fibroin solution was prepared from the most posterior part of the middle silk gland. Ultracentrifugal analyses have shown that the solution contains considerable amounts of aggregates in addition to the main 10S component. Treatment with lithium bromide (LiBr), urea, or guanidine hydrochloride solution up to 6 M all have failed to dissociate the 10S component. From the sedimentation equilibrium analyses and partial specific volume of 0.71(6), the molecular weight of the 10S component of the native fibroin solution was found to be between 3.2 - 4.2 x 10(5), with a tendency to lie fairly close to 3.7 x 10(5).

Animals↗

Rapid degradation of the silkworm diapause hormone by trypsin and its suppression by VAP-map, a synthetic analog of the cuticular peptide of silkworm, Bm ACP-6.7 (VAP-peptide).

Very fast tryptic degradation of the silkworm diapause hormone was found and the degradation pathway was analyzed by moderating the reaction conditions. It proceeded via cleavage at Arg23 and finally at Arg15 of DH. As the C-terminal structure of DH was essential for exhibiting bioactivity, the first cleavage caused rapid inactivation of the hormone. This tryptic digestion was strongly suppressed by adding VAP-map, a synthetic analog of the cuticular peptide of silkmoths, Bm ACP-6.7 (VAP-peptide), which is a natural synergist of DH. VAP-map suppressed the enzymic reaction by interacting with the substrate, but not with the enzyme.

Amino Acid Sequence↗

Lipid and fatty acid composition of muga silkworm, Antheraea assama, host plants in relation to silkworm growth.

The lipid and fatty acid composition of the leaves (tender, medium and mature) of muga host plants, Machilus bombycina, Litsaea monopetala (primary food plants) and L. cubeba and L. salicifolia (family: Lauraceae) (secondary food plants) was investigated by standard procedures, gas chromatography after saponification and esterification. The total lipid content of M. bombycina and L. monopetala leaves was recorded to be higher (16 and 18 g%), respectively, than that of L. cubeba (10 g%) and L. salicifolia (12 g%). GC analysis identified the presence of eight fatty acids (C14 to C22) and the concentration varied from 0.0297 to 8.1572 g% dry leaf powder. Among the fatty acids, (C14 to C22), polyunsaturated fatty acids were recorded to be highest in concentration in mature leaves of the primary host plants. The concentrations of saturated and polyunsatuated fatty acids were found to be at a minimum level in all the types of leaves of secondary muga host plants.

Animals↗

M chromosome of the wild silkworm, Bombyx mandarina (n = 27), corresponds to two chromosomes in the domesticated silkworm, Bombyx mori (n = 28).

Chromosomes of Bombyx mori (n = 28) and of Bombyx mandarina (n = 27) were studied cytogenetically to resolve the origin of the large M chromosome in the Japaneses type of B. mandarina. In the F1 progeny from the reciprocal cross between B. mandarina and B. mori, the mitotic chromosome number was 2n = 55, and a chromosome configuration of 26 bivalents plus 1 trivalent was observed at metaphase I of germ cells. The trivalent chromosome consisted of the M chromosome from B. mandarina and two chromosomes from B. mori. When males of B. mori were mated to the F1 females, nuclei with two types of chromosome number (2n = 55 and 2n = 56) and two sets of chromosome pairs (26 bivalents plus 1 trivalent versus 28 bivalents) were observed in the metaphase I stage. Linkage analysis showed that the 14th chromosome of B. mori was involved in these two types of chromosome segregation. This result indicates that the M chromosome in B. mandarina arose from a fusion between a chromosome corresponding to the 14th linkage group and another, yet unidentified linkage group.

Animals↗

[Effect of low-dose gamma-irradiation of silkworm eggs on the development of the mulberry silkworm (Bombyx mori L.)].

Chronic gamma-irradiation of an egg at a dose-rate 100, 1000 and 4000 times as high as that of the natural radiation background significantly accelerates the development of Bombyx mori L. The caterpillar development may also be stimulated by a single exposure of the egg to 2 Gy radiation. The acceleration of the caterpillar growth promotes the increase in the cocoon weight and the raw-silk mass.

Animals↗

Production of silkworms with conjugated linoleic acid (CLA) incorporated into their lipids by dietary CLA.

Silkworms with conjugated linoleic acid (CLA) incorporated into their lipids (designated CLA silkworms) were produced to enhance the quality of silkworms having a synergistic effect with CLA functions by dietary synthetic CLA. Silkworm larvae were fed fresh mulberry leaves (control diet) until the third instar stage and were then subjected to various levels (0%, 0.1%, 1%, 5%, and 10%) of CLA-sprayed mulberry leaves (designated CLA diet) beginning on the first day of the fourth instar stage and continuing to the third day of the fifth instar stage. CLA contents in CLA silkworms increased proportionally with increasing CLA levels of CLA diets. CLA silkworms on a 1% CLA diet contained 2.2 g CLA/100 g lipid without body weight reduction, whereas CLA silkworms on a 10% CLA diet contained 14.8 g CLA/100 g lipid with a significant reduction of body weight, relative to the control silkworms. The CLA content in the lipids of CLA silkworms on a 10% CLA diet was significantly higher than that of CLA silkworms on a 5% CLA diet. A 0.1% CLA diet was not sufficient to accumulate CLA in the silkworms. Most of the CLA (approximately 99%) of silkworm lipids was present in triglyceride (TG) with a similar ratio of c9,t11 and t10,c12 CLA isomers. These results suggest that a 1% CLA diet was suitable for the production of CLA silkworms.

Animals↗

Causative allergens of allergic rhinitis in Japan with special reference to silkworm moth allergen.

To determine the importance of silkworm moth allergens, we tested 267 patients with allergic rhinitis with CAP-RAST, a new assay system for detecting specific IgE in sera. We used the following allergen sources: house-dust mite, Candida, Alternaria, cat dander, orchard grass, ragweed, mugwort, Japanese hops, Japanese cedar, Japanese cypress, cotton, silk, larval chironomid midge, adult chironomid midge, and silkworm moth. As expected, the highest positive rate of reaction, as determined by CAP-RAST, was 73.8% for Japanese cedar, followed by 53.2% for mite and 50.6% for Japanese cypress. Although the positive rate for silkworm moth was not as high as for cedar pollen, one-third of patients had specific IgE against silkworm moth. The positive rate of reaction to silkworm moth was much higher than that to chironomid. It was interesting to note that the patients reported had not been documented as having frequent contact with silkworm moth allergen. Correlations between silkworm moth and silk, and silkworm moth and chironomid midge were found to be significant (P < 0.001). Silkworm moth allergen showed the third highest reaction rate in patients with severe symptoms. These results suggest that silkworm moth allergen should be considered to be important in patients with allergic rhinitis in Japan.

Adolescent↗

Productive infection of Autographa californica nucleopolyhedrovirus in silkworm Bombyx mori strain Haoyue due to the absence of a host antiviral factor.

We have reported that several silkworm strains are permissive to intrahemocoelical infection of Autographa californica nucleopolyhedrovirus (AcNPV), contrary to the general belief that AcNPV cannot infect silkworm. In the present study, we address whether the intrahemocoelical infection of AcNPV to the silkworm was an exceptional phenomenon, and the possible genetic basis underlying it. Wilder range test of 31 strains of silkworm Bombyx mori for intrahemocoelical AcNPV infection led to the identification of 14 permissive strains and 17 nonpermissive strains, indicating that the intrahemocoelical infection of AcNPV to the silkworm was not a rare and isolated phenomenon. Productive infection was shown in permissive silkworms, by EGFP fluorescence in various tissues when expression of reporter gene controlled by a very late viral promoter polh. The viral titer in larval hemolymph of permissive silkworms increased and maintained at a higher level hundredfold more than the initial amount of virus, indicating viral replication. A series of genetic cross experiments suggested the existence of only one dominant host anti-AcNPV gene or a set of genetically linked genes, which prevent AcNPV infection in nonpermissive silkworm strain Qingsong and are absent in permissive silkworm strain Haoyue.

Animals↗

[Characterization and stage-specific change of proteins during the embryonic development of silkworm Bombyx mori].

The gene expression pattern was analysed employing protein two-dimensional electrophoresis and amino acid sequencing during the embryonic development of silkworm Bombyx mory. The 2D-PAGE pattern of the silkworm embryonic proteins had little change in the long developmental period from the Critical Stage until the Head Pigmentation Stage of silkworm embryo. The percentage of matched proteins that found between the spots in the 2D-PAGE pattern of the Critical Stage of silkworm embryo and the spots in the 2D-PAGE pattern of the Head Pigmentation Stage of silkworm embryo was 63%. Silkworm Egg-specific Protein and 30K Protein were in large amount. However, 2D-PAGE pattern of the silkworm embryonic proteins showed considerably varied in shorter developmental stage from the Head Pigmentation Stage to the Body Pigmentation Stage and until the Newly Hatched Larva. The percentage of matched proteins that found the spots in the 2D-PAGE pattern of each developmental stage of silkworm embryo became lesser. The protein spots that the isoelectric points lying on the acidic side greatly increased, the Silkworm Egg-specific Protein and 30K Protein were gradually disappeared and the gene that resembles BmLSP's, Enolase's and Superoxide dismutase's gene was activated and expressed with the embryonic development.

Amino Acid Sequence↗

Inhibition of human cell apoptosis by silkworm hemolymph.

Many studies on preventing apoptosis have been carried out from the viewpoint of anti-apoptotic cloned-gene expressions inside cells, whereas in this study, we investigated the inhibition of apoptosis by the addition of silkworm hemolymph, a natural compound, from outside of the cells. In a previous study, we reported the inhibition effect of silkworm hemolymph on the baculovirus-induced insect cell apoptosis. Using the vaccinia virus-HeLa cell system as a model system in this study, we found that silkworm hemolymph, the insect serum, inhibits apoptosis not only in the insect cell system but also in the human cell system. The vaccinia virus-induced HeLa cell apoptosis was analyzed using DNA electrophoresis, TUNEL, and flow cytometry, and the resulting data confirmed that silkworm hemolymph inhibits human cell apoptosis. The inhibition of apoptosis due to silkworm hemolymph was not caused by an inhibition of virus binding and internalization steps, nor did silkworm hemolymph interfere with the virus production. The inhibition of apoptosis by silkworm hemolymph decreased the cell detachment from an adhering surface. With these characteristics, silkworm hemolymph can be effectively used to minimize cell death in commercial animal cell culture.

Animals↗