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Effects of heavy-ion radiosurgery on the hemopoietic function of the silkworm Bombyx mori.

To study the effects of heavy-ion radiosurgery on the hemopoietic function of a silkworm, hemopoietic organs of larvae were locally irradiated with carbon-ion beams, and the changes in the hemocyte density and in the hemocyte function were investigated. When the larvae were irradiated by 50 Gy to 300 Gy carbon ions on the 3rd day of the 4th instar, the hemocyte densities did not change for a while, though they gradually increased at a later stage, but were finally still significantly lower than those of unirradiated controls. The hemocyte densities of the larvae irradiated at different developmental stages showed suppressed increments, and carbon-ion irradiation given to larvae at early stages compared to the later stages had a significant suppressive effect on the hemocyte densities. On unilateral irradiated larvae a hemocyte intermediate increment between those of bilateral irradiated larvae and unirradiated controls was observed. The percentage of dead hemocytes was obviously higher for irradiated larvae than unirradiated controls during the later 5th instar. Thus, it is evident that carbon-ion radiosurgery on hemopoietic organs of silkworm induced not only a quantitative change, but also a qualitative change in the hemocytes.

Animals↗

Purification and refolding of recombinant human IGF II from silkworms infected with recombinant Bombyx mori nuclear polyhedrosis virus.

We have reported that insulin-like growth factor II (IGF II) was produced as a fusion protein in Bombyx mori (silkworm) larval bodies infected with recombinant B. mori nuclear polyhedrosis virus [J. Gen. Virol., 68, 2599-2606 (1987)]. In this study, the purification of IGF II from the infected silkworms is reported. The fusion protein was extracted with 6.0 M guanidine-HCl from the infected larval bodies homogenized in water. The use of organic solvents to remove the impurities, such as lipid derived from the larval bodies, was a very effective method of purification. IGF II was released from the partially purified fusion protein by treatment with CNBr, purified by HPLC, and refolded by air-oxidization. Refolded IGF II had an identical primary structure including disulfide bonds and showed identical thymidine uptake stimulation activity with human IGF II. Furthermore, protein disulfide-isomerase was shown to be able to refold scrambled IGF II rapidly.

Amino Acid Sequence↗

Cloning and expression of the Momordica charantia trypsin inhibitor II gene in silkworm by using a baculovirus vector.

MCTI-II (Momordica charantia trypsin inhibitor II) isolated from bitter gourd (Momordica charantia LINN.) seeds is one of the serine protease inhibitors of the squash family. We cloned cDNA that encodes MCTI-II and constructed an expression system for MCTI-II by using a baculovirus vector. The recombinant baculovirus was inoculated to early fifth-instar larvae of the silkworm (strain: Shunrei x Shougetsu). Four days after infection, the hemolymph of silkworm larvae was collected and the recombinant protein was purified. Two kinds of expressed MCTI-II protein were obtained. An amino acid sequence analysis of the two proteins indicates that both were similar to the authentic inhibitor, except for the addition of a tripeptide derived from the vector at the N-terminus. One of the two inhibitors (MCTI-II A) resulted in a single PTH-amino acid in each Edman degradation cycle, while the other (MCTI-II B) resulted in two PTH-amino acids, suggesting the occurrence of cleavage of the reactive site. The inhibitory activities of MCTI-II expressed toward trypsin are examined in terms of the Ki value, these being 6.4 x 10(-10)M for MCTI-II A and 5.2 x 10(-10) M for MCTI-II B.

Amino Acid Sequence↗

Promoting protein, a silkworm hemolymph protein promoting in vitro replication of nucleopolyhedrovirus, binds to beta-glucans.

A protein which bound to 125I-labeled peptidoglycan (PGN) was isolated from hemolymph of silkworm larvae. The N-terminal amino acid sequence and the molecular weight of the protein were in accord with those described for Promoting Protein (PP) from the silkworm. The binding of the protein to [125I]PGN was competitively inhibited by various beta-glucans. The binding kinetics of PGN and chitin to the protein were analyzed in a biosensor.

Animals↗

Stress induction of Bm1 RNA in silkworm larvae: SINEs, an unusual class of stress genes.

This study surveys the induction of RNA polymerase III (Pol III)-directed expression of short interspersed element (SINE) transcripts by various stresses in an animal model, silkworm larvae. Sublethal heat shock and exposure to several toxic compounds increase the level of Bm1 RNA, the silkworm SINE transcript, while also transiently increasing expression of a well-characterized stress-induced transcript, Hsp70 messenger RNA (mRNA). In certain cases, the Bm1 RNA response coincides with that of Hsp70 mRNA, but more often Bm1 RNA responds later in recovery. Baculovirus infection and exposure to certain toxic compounds increase Bm1 RNA but not Hsp70 mRNA, showing that SINE induction is not necessarily coupled to transcription of this particular heat shock gene. SINEs behave as an additional class of stress-inducible genes in living animals but are unusual as stress genes because of their high copy number, genomic dispersion, and Pol III-directed transcription.

Animals↗

Identification of ommin in the integument of the silkworm, Bombyx mori.

An ommin was isolated from the integument of the quail mutant of the silkworm, Bombyx mori, using column chromatography on SP-Sephadex and cellulose powder. As a standard, ommin was isolated from diapause eggs of the normal silkworm. The red pigment from the larval integument of quail mutants was identical to the standard compound with respect to absorption spectrum, infrared spectrum and RF values in thin-layer chromatography (TLC). After acid hydrolysis of the pigment, 3-hydroxykynurenine was detected by TLC. This is the first report of an ommin in a lepidopteran larval integument.

Animals↗

Construction of a single nucleotide polymorphism linkage map for the silkworm, Bombyx mori, based on bacterial artificial chromosome end sequences.

We have developed a linkage map for the silkworm Bombyx mori based on single nucleotide polymorphisms (SNPs) between strains p50T and C108T initially found on regions corresponding to the end sequences of bacterial artificial chromosome (BAC) clones. Using 190 segregants from a backcross of a p50T female x an F1 (p50T x C108T) male, we analyzed segregation patterns of 534 SNPs between p50T and C108T, detected among 3840 PCR amplicons, each associated with a p50T BAC end sequence. This enabled us to construct a linkage map composed of 534 SNP markers spanning 1305 cM in total length distributed over the expected 28 linkage groups. Of the 534 BACs whose ends harbored the SNPs used to construct the linkage map, 89 were associated with 107 different ESTs. Since each of the SNP markers is directly linked to a specific genomic BAC clone and to whole-genome sequence data, and some of them are also linked to EST data, the SNP linkage map will be a powerful tool for investigating silkworm genome properties, mutation mapping, and map-based cloning of genes of industrial and agricultural interest.

Animals↗

Computational prediction of microRNA genes in silkworm genome.

MicroRNAs (miRNAs) constitute a novel, extensive class of small RNAs (approximately 21 nucleotides), and play important gene-regulation roles during growth and development in various organisms. Here we conducted a homology search to identify homologs of previously validated miRNAs from silkworm genome. We identified 24 potential miRNA genes, and gave each of them a name according to the common criteria. Interestingly, we found that a great number of newly identified miRNAs were conserved in silkworm and Drosophila, and family alignment revealed that miRNA families might possess single nucleotide polymorphisms. miRNA gene clusters and possible functions of complement miRNA pairs are discussed.

Animals↗

Studies on the effect of pesticides on the reproduction of the silkworm, Bombyx mori L. I. Effects of chemicals administered during the larval stage on egg-laying and hatching.

The administration of sublethal pesticide doses to silkworm (Bombyx mori L.) larvae resulted in marked reproductive abnormalities: a decrease in the total number of eggs laid; an increase in the number of non-fertilized eggs; the death of embryos in the early developmental stage; the inability of embryos, developed to the stage just before hatching, to hatch; and the death of newly hatched silkworms.

Animals↗

Effect of rearing at non-permissive temperature on silkworm larvae infected with temperature-sensitive lef-8 mutant of bombyx mori nucleopolyhedrovirus.

A temperature-sensitive (ts) mutant of bombyx mori nucleopolyhedrovirus (BmNPV), ts-S1, contains a mutation in a putative RNA polymerase gene, which is involved in late viral gene expression. When 4th-instar silkworm larvae were infected with 1.0 x 10(5) TCID50 of ts-S1 per larva and reared at 33 degrees C, the titre of budded virus (BV) and number of occlusion bodies (OBs) in the haemolymph of the infected larvae were very low in the early stage but markedly increased in the late stage of infection. In contrast, a rapid increase in both BV titre and OB number was detected in the haemolymph of infected larvae reared at 25 degrees C. LD50 values of ts-S1 and wild type BmNPV (wtBmNPV) for 4th-instar larvae were 2.41 and 0.96 TCID50 per larva at 25 degrees C, and > 1.0 x 10(6) and 1.70 TCID50 per larva at 33 degrees C, respectively. These results indicate that the virulence of ts-S1 for the larvae reared at 33 degrees C was markedly reduced. To examine further the reduction of ts-S1 virulence at the non-permissive temperature of 33 degrees C, silkworm larvae were infected with ts-S1 at the multiplicity of 1.0 x 10(2) - 1.0 x 10(6) TCID50 per larva, reared for various time at 33 degrees C and then shifted to 25 degrees C. Longer rearing periods at 33 degrees C resulted in better survival rates indicating that the reduction of virulence of ts-S1 was proportional to cumulative rearing time at 33 degrees C. When large virus inocula were used, a growth alteration of larvae was preferentially induced. However, when small virus inocula were used, the appearance of abortive infection due to the non-permissive temperature became more evident.

Animals↗

Production of chimeric antibody in cells and larvae of silkworm.

By using the recombinant baculovirus expression system, the chimeric antibody against human small cell lung carcinoma was expressed in the cells and larvae of silkworm Bomyx mori N (BmN). The chimeric antibodies produced in BmN cells and larvae which were infected with recombinant viruses rNPVL2, rNPVH17 and dual-recombinant virus rNPVLH19 were detected by Western blot analysis. The co-expression products of heavy- and light-chain genes in silkworm larvae revealed stronger immune binding activity than the single product of heavy or light chain gene expression.

Animals↗

The heuristic function of 'error' in the scientific methodology of Louis Pasteur: the case of the silkworm diseases.

With the aid of the Cahiers de laboratoire, the Correspondence and, of course, the Oeuvre de Pasteur, this work reconstructs the extraordinary scientific undertakings of the great French scientist in his study of silkworm diseases. The focus of this study consists in the attempt to explain the initial perplexing behaviour of Pasteur, even in the presence of correct interpretations regarding the causes of these diseases (cfr. the results obtained by Béchamp); for a good three years he insisted on maintaining that the aetiology of silkworm diseases could not be attributed to pathogenetic germs from outside. And this was in spite of the fact that previously (through fermentation and spontaneous generation) he had been able to demonstrate the importance of microorganisms in biological processes. Finally it is intended to highlight the extraordinary methodological depth of that initial 'error', which was capable of paving the way for the future conquests of Pasteur in the field of aetiology and the prevention of infectious diseases.

Animal Diseases↗

Characterization of cypovirus isolates from tropical and temperate Indian saturniidae silkworms.

Cytoplasmic polyhedrosis virus (cypovirus, CPV) isolates from larvae of saturniidae silkworms Antheraea mylitta, Antheraea proylei, and Antheraea assamensis were obtained and characterized. Scanning electron microscopic (SEM) studies revealed the presence of hexahedral (rhombic dodecahedron) polyhedral bodies of various size (1-4 microns) in individual hosts while transmission electron microscopic (TEM) studies of isolated virions showed their uniform (icosahedral) shape and size (approximately 55 nm). Genomes of these isolates were obtained from purified polyhedral bodies and analyzed by agarose and polyacrylamide gel electrophoresis. Eleven bands ranging from about 350 bp to 3.9 kb (total of 25.35 kb) were detected. These bands were completely degraded after treatment of the genome preparations with RNase A at low salt but not at high salt concentration. The banding pattern of the genomes remained unaffected by treatment of the respective preparations with DNase I, indicating that the genomes were composed of 11 double-stranded (ds) RNA segments. The RNA banding patterns of the CPV isolates from the saturniidae silkworms were similar to each other. These characteristics were similar to that of Antheraea mylitta cypovirus 4 (AmCPV-4) but different from that of Bombyx mori cypovirus 1 (BmCPV-1). In comparison to AmCPV-4 the isolates contained an additional, eleventh RNA segment and the size of the RNA segments was also different. Dot blot hybridization showed hybridization of the cloned 9th and 10th segment cDNAs from the A. mylitta CPV (AmCPV) isolate with RNAs from the A. proylei and A. assamensis CPV (ApCPV, AaCPV) isolates but not with RNA from BmCPV-1. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) of polyhedral proteins from the AmCPV isolate showed the presence of 3 major and 5 minor bands of M(r) from 29.4 K to 163 K, but BmCPV-1 polyhedral bodies showed different protein banding pattern. Two heterologous cell lines infected with the AmCPV isolate showed very slow virus growth with the accumulation of polyhedral bodies in the cytoplasm of infected cells indicating a restricted host range of this CPV isolate.

Animals↗

Effect of temperature and relative humidity on spinning behaviour of silkworm (Bombyx mori.L).

The rate of spinning of silkworm larva (Bombyx mori.L) was slow at 22 degrees C and fast at 38 degrees C. The time taken for completion of cocoon was longest at 98 +/- 2% and least at 40 +/- 2% RH. However, good quality cocoons were spun at 22 degrees C and 65 +/- 5% RH, hence it would be advantageous to maintain this temperature and relative humidity at the time of cocoon spinning in the case of CB race of silkworm.

Animals↗

[A study of the complex of proteolytic enzymes and their protein inhibitors in embryogenesis of the silkworm].

We studied changes in the activities of serin, thiol, and aspartyl proteinases and their protein inhibitors during embryogenesis of the silkworm. The dynamics of activities of the protein inhibitors and specific proteinases were interrelated, thus providing for coordination and fine regulation of functioning of the proteolytic complex of enzymes during embryogenesis. Possible functions of peptidohydrolases and their protein inhibitors in the silkworm are discussed.

Animals↗

[Impact of high temperature on testicular growth and development of valuable silkworm Antheraea yamamai(Lepidoptera: Saturiidae)].

The results indicated that high temperature had a significant impact on the growth and development of testis in Japanese oak silkworm Antheraea yamamai. The testicular size of the 3rd and 4th instar larvae increased within the range from 20 to 29 degrees C, and slightly decreased at 32 degrees C. While in the 5th instar, it decreased within the range from 20 to 26 degrees C, with the biggest size at 20 degrees C. The testis was almost hard to grow and develop because the larvae could not survive at 29 to 32 degrees C. When the mature larvae were treated at 32 degrees C at the beginning of coconing or at the 1st or 6th day of pupating, the growth of testicular size and spermatogenesis were significantly arrested, and the sperm number and the content of soluble protein in the testis markedly decreased. When the male moth of the 1st day was treated at 32 degrees C, the activity of the sperms in the testis obviously decreased, in contrast to the treatment at 20 degrees C. It is concluded that for the egg raising of the silkworm, its mature larvae, puape in cocoons and male moths should not be exposed to 32 degrees C from cocooning to adult stage. The proper temperature for rearing 3rd, 4th and 5th instar larvae was discussed.

Animals↗

[Expression of human interleukin-11 in cell culture and larvae of silkworm].

A recombinant transfer vector, pBacIL-11, containing hIL-11 cDNA of 546 nucleotides lacking leader sequence was constructed and co-transfected into BmN cells with linearized BmBacPAK(modified BmNPV) DNA for construction of a recombinant baculovirus carrying the hIL-11 gene. Southern hybridization analysis suggested that the recombinant baculovirus DNA contained hIL-11 cDNA fragment. RNA dot blotting demonstrated that the hIL-11 gene was transcribed. The recombinant baculovirus has a strong infectivity to BmN cell line and to silkworm larvae and pupae. Specific hIL-11 bands were detected from all the samples of cell extract, culture supernatant, haemolymph of larvae and pupae by SDS-PAGE analysis. Biological activity of the expressed product was determined with IL-11 dependent B9-11 cell line and by MTT colorimetric assay, which indicated that biologically active rhIL-11 protein was overexpressed in BmN cell line and in silkworm larvae and pupae.

Animals↗

The Autonomous Replication Function (ARS) of the Scaffold-associated Region (SAR) of Silkworm Attacus ricini rDNA in Yeast.

The one kilo-base scaffold-associated region (SAR) of silkworm Attacus ricini rRNA gene (rDNA) has been identified previously([1]). To investigate the critical sequence and the relative activity of ARS (autonomously replicating sequence), a set of restriction from covered the whole rRNA gene unit were subcloned into the nonreplicative pSKY vector. Among the seven plasmids constructed, the plasmid pSEY, having SAR, gave obvious positive replication activity in yeast as determined by the transformation efficiency. Further dissection of SAR demonstrated that plasmid pAAY, having a 0.26 kb small fragment of SAR was 40-50 fold more active then the whole SAR, while pSAY, having the remaining part of SAR, showed no activity. There were fifteen ARS consensus sequences (ACSs) within SAR being identified through sequenced alignment. It was found that only three ACSs in pAAY, located at the 3' end of SAR displayed a positive ARS activity and the remaining sequence having the other twelve ACSs functioned as a repressor. The in vitro binding assay showed that SAR from the silkworm rRNA gene bound to the yeast nuclear scaffold. These results suggest that SAR is evolutionarily conserved, and there is a close correlation between SAR and ARS activity. Detailed analysis of the positive and negative regulatory elements in SAR can be carried out based on these results.

Journal Article↗