In the present study, the effects of different degumming methods on the structural characteristics and properties of regenerated silk fibroin (SF) were examined. The crystallinity index of the ...degummed silk increased with the degumming ratio. The crystallinity index at any given degumming ratio differed depending on the degumming method. The soda method and the soap/soda method using sodium carbonate resulted in a higher crystallinity index than the other methods The degumming method strongly affects the molecular weight (MW) and solution viscosity of the regenerated SF. The MW and viscosity of the regenerated SF, according to the degumming method, was in the order of urea method>HTHP method≈acid method>soap/soda method≈soda method. The turbidity of a silk formic acid solution decreased as a result of increasing the degumming ratio and was a minimum at a degumming ratio of around 26%. However, it was not affected by the degumming method. The mechanical properties of a regenerated SF film were strongly affected by the degumming method and the trend in the strength and elongation with the various degumming methods was the same as that of the MW and viscosity of the regenerated SF.
Cecropins belong to the antibacterial peptides family and are induced after injection of bacteria or their cellwall components. By silkworm cDNA microarray analysis, a novel type of Cecropin family ...gene was identified as a cDNA up-regulated in early embryo, 1 day after oviposition. The cDNA isolated was 394 bp with 198 ORF translating 65 amino acids, encoding BmCecropin-E (BmCec-E). Using Southern hybridization and genome search analysis, the number of BmCec-E gene was estimated to be at least two per haploid, which consisted of two exons, as in other Cecropin family members. BmCec-E mRNA was expressed transiently 1 day after egg-laying (AEL, germ-band formation stage), and was specifically expressed in the degenerating intestine during the pre-pupal and pupal stages, unlike other Cecropin family genes. Immune challenge analysis showed that BmCec-E gene expression was more strongly induced by Escherichia coli (gram-negative) than by Micrococus luteus (gram-positive), and not by virus injection. By bacterial challenge, expression of BmCec-E mRNA was induced 12 h after injection, and was maintained for 24 h. Expression of BmCec-E after immune challenge was observed strongly in excretory organs, such as hindgut and malphigian, slightly in fat body, skin, and midgut.
Effect of envenomation of ectoparasitoid
Bracon hebetor was determined on the heart rate and the expression of
shsp,
hsc70 and
hsp90 of the lepidopteran host
Plodia interpunctella. Envenomated host ...larvae were promptly immobilized but heart rate was not changed until 4 days after envenomation. Northern hybridization showed that each
hsp gene was differentially influenced by envenomation: continued high induction of
shsp, gradual strong induction of
hsc70, but no induction of
hsp90. Our results suggest that upregulation of both
shsp and
hsc70 may produce potent factors that have important roles in the mechanism of host–parasitoid relationship.
We prepared a cDNA library for a microarray from eggs of the silkworm, Bombyx mori, at the germ-band formation (24 hours after fertilization) stage. Using a microarray constructed with 2,445 ESTs, we ...screened gene expression profiles during germ-band formation at six specific time points in the early embryonic stages (from the unfertilized egg to the formation of abdominal leg appendages), and determined 241 of these cDNAs to represent genes that were expressed differentially during the germ-band formation stage. These differentially expressed genes grouped into two clusters. In the early and late clusters, 203 and 38 genes were upregulated, respectively. In the upregulated clusters, we isolated several genes that were associated with development and cell communication, including egalitarian, RAD23b, innexin 2, and senescence-associated protein. Northern blot hybridization revealed that the expression patterns of 14 genes had changed in each of the stages. In this study, we assessed changes in the levels of gene expression in relation to the germ-band formation stages in whole Bombyx embryos.
Translationally controlled tumor protein (Tctp/p23) is known to be synthesized preferentially in cells during the early growth phase of tumors, but is also expressed in normal cells. To elucidate its ...molecular basis of the expression and physiological significance, a cDNA encoding for the
Bombyx mori Tctp (BmTctp) was deduced by editing the partial cDNA sequences registered in a
Bombyx EST database. RT-PCR analyses indicated that the
BmTCTP mRNA was transcribed in all larval organs examined and was present constantly during the cell cycle of BmN4 cells. A genomic clone of 4255 nucloetide residues produced by inverse PCR contained the 5′-flanking region, two introns and three exons of the
BmTCTP gene. Sequence analysis of the 5′-flanking region indicated that a putative promoter region contains several canonical transcription elements such as GATA box, CCAAT motif, MEF2, E4BP4.01 and AP-1, but lacks a TATA box element. Luciferase reporter assay of the deletion constructs of the 5′-flanking region revealed that the −676 to +66 region enhanced the promoter activity the most markedly. In addition to this, there were at least two enhancer-like elements and several repressor elements.
Gap junctions are clusters of intercellular channels that are associated with embryonic development and neural signaling. Innexins, invertebrate gap junction proteins, have been identified in
...Drosophila and
Caenorhabditis. Here, we report the isolation and characterization of two novel members of the insect innexin family,
Bm inx2 and
Bm inx4, from embryos of the silkworm,
Bombyx mori, during the germ-band formation stage.
Bm inx2 is a single copy gene with one exon, while
Bm inx4 is a single copy gene with four exons and three introns. The predicted proteins show structural similarities with other innexin family members, including four transmembrane (TM) domains, two extracellular loops (ELs), one cytoplasmic loop (CL), and typical conserved amino acids.
Bm inx2 is phylogenetically orthologous to the other insect
inx2 genes, but
Bm inx4 is not orthologous to any known innexin including
Dm inx4. Interestingly, Northern blotting and in situ hybridization showed that
Bm inx2 was variously expressed across all developmental stages and in various tissues, with high expression seen in the nervous system at the time of embryogenesis. In contrast,
Bm inx4 was transiently expressed at the germ-band formation stage of embryogenesis, and was specifically expressed in the ovary and testis during the larval and pupal stages. The isolation and characterization of these novel genes should form the basis for further study of the functional events that occur during development and neuronal communication in
B. mori.
In the course of screening anti-dementia agents from natural products, two β-secretase (BACE1) inhibitors were isolated from the ethyl acetate soluble fraction of Sanguisorbae Radix by the ...activity-guided purification using silica gel, Sephadex LH-20, and RP-HPLC. They were identified as 1,2,3-trigalloyl-4,6-hexahydroxydiphenoyl-β-D-glucopyranoside (Tellimagrandin II,1) and 1,2,3,4,6-pentagalloyl-β-D-glucopyranoside (2) and were shown to non-competitively inhibit β-secretase (BACE1) with the IC₅₀ values of 3.10×10⁻⁶ M and 3.76×10⁻⁶ M, respectively. TheKi values of1 and2 were 6.84×10⁻⁶ M and 5.13×10⁻⁶ M. They were less inhibitory to α-secretase (TACE) and other serine proteases such as chymotrypsin, trypsin, and elastase, suggesting that they were relatively specific inhibitors of BACE1.
We report the isolation of activating transcription factor of chaperone (ATFC), a novel cDNA from
Bombyx mori BM5 cells that encodes a putative transducer of endoplasmic reticulum (ER) stress. The ...236 amino acids of ATFC include both a basic region and a leucine zipper at the C-terminus, in contrast to Hac1p of yeast which features these structures at its N-terminus. ATFC expression was strongly up-regulated by ER stress. ATFC could specifically bind to the unfolded protein response element. BM5 cells transfected with ATFC cDNA displayed enhanced ER chaperone expression in response to ER stress. These results indicate that ATFC encodes a putative transducer of ER stress.
We isolated a calreticulin cDNA from the silkworm, Bombyx mori. The cDNA encodes 398 amino acid residues of B. mori calreticulin, with an endoplasmic reticulum retentional HDEL motif at its ...C-terminus and a predicted molecular mass of 45,801 Da. The B. mori calreticulin shows high protein homology with calreticulin from G. mellonella (88%), A. aegypti (71%), D. melanogaster (69%) and H. sapiens (63%). The highest level of mRNA expression of B. mori calreticulin was exhibited in the fat body of this insect. Although expression of B. mori calreticulin was affected by disturbances in intracellular calcium levels, other ER stress conditions such as inhibition of intracellular protein transport, reduction of disulfide formation, glycosylation inhibition, heat shock and oxidative stress did not disrupt induction of B. mori calreticulin.
A complex type marner-like element (MLE) was isolated by PCR with genomic DNA of Bombyx mandarina. The clones of this MLE, homologous to the 'Cecropia-ITR-MLE' in BmTNML locus that we have isolated ...previously from B. mori, were roughly classified into four groups according to the state of inserted elements: (1) Containing a retrotransposon (L1Bm) and another element, (2) containing L1Bm alone, (3) containing another element alone and (4) without insertions. This inserted element was named BmamaT1, as it was homologous to the B. mori MLE called Bmmar6, which was previously reported to be similar to a maT-family member, Bmmar1. Many of the current Cecropia-ITR-MLE clones had, as the BmamaT1 insertion site, the sequence of TA/TATA, which may be a transposition target site. Moreover, the BmamaT1 elements that inserted into the Cecropia-ITR-MLE region were highly homogeneous, making one group when a phylogenetic tree was made together with BmamaT1 members isolated directly from the B. mandarina genome. Many of the inserted type BmamaT1 elements had complete ORF which possessed the so-called DDD catalytic triad. All these findings were taken to indicate that a large fraction of the BmamaT1 elements are still highly active.
