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At least 217 records · Page 12Linked to original sources
PubMed2006

Production of a cellulase in silkworm larvae using a recombinant Bombyx mori nucleopolyhedrovirus lacking the virus-encoded chitinase and cathepsin genes.

The expression efficiency of an insect-derived cellulase was assayed in silkworm larvae infected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) mutants lacking the virus-encoded chitinase (chiA) and/or cathepsin (v-cath) genes. Expression was increased by approx. 10% in mutants lacking chiA or v-cath and 17% in a mutant lacking both chiA and v-cath compared with that of the unmodified recombinant BmNPV. The recombinant BmNPV lacking both chiA and v-cath can therefore be used for a large-scale production of foreign proteins in silkworms.

Animals↗
PubMed1975

Cyclic nucleotide phosphodiesterase in silkworm. Separation and characterization of multiple forms of the enzyme.

The existence of two forms of cyclic AMP phosphodiesterase (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) was demonstrated in silkworm larvae by kinetic analysis and DEAE-cellulose column chromatography. The two forms of the enzyme (phosphodiesterase II and III) differ apparently in their characteristics from the previously reported cyclic nucleotide phosphodiesterase (phosphodiesterase I) of silkworm. The higher K-m form (phosphodiesterase II) has a molecular weight of approx. 50 000 and optimum pH of 7.8, and requires Mn-2-+ for maximum activity. The lower K-m form (phosphodiesterase III) has a molecular weight of approx. 97 000 and optimum pH of 7.2, and requires Mg-2-+ for maximum activity. Phosphodiesterase II and probably phosphodiesterase III are specific enzymes for the hydrolysis of cyclic AMP.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1983

Activation of pro-phenoloxidase by bacterial cell walls or beta-1,3-glucans in plasma of the silkworm, Bombyx mori.

Silkworm hemolymph plasma contains pro-phenoloxidase and the activating system for the pro-enzyme. The latter was triggered by elicitors such as Gram-negative or Gram-positive bacterial cell wall, glucans with beta-1,3-glycosidic linkages and denatured lipophorin, which is one of silkworm plasma proteins, but not by lipopolysaccharide, dextram sulfate, kaolin or inulin. Calcium was required for the elicitors to activate the system. However, putative pro-PO activating enzyme, which activity is induced in plasma by the action of the elicitors, could activate pro-PO in the absence of the ion, suggesting that at least two reaction steps are involved in the activation reaction of pro-PO in plasma. The activating enzyme was completely inhibited in the presence of p-NPGB, an inhibitor for serine protease.

Animals↗
PubMed1986

Glucosidation of estradiol-17 beta in the cultured ovaries of the silkworm, Bombyx mori.

Metabolic pathways of estradiol-17 beta and other vertebrate steroid hormones of cultured silkworm pupal ovaries were examined using 14C-labeled steroids. The isolated ovaries showed significant uptake and metabolic activity of the 14C-labeled estradiol-17 beta added to the medium. Analysis of the metabolized compounds by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed extensive metabolic conversion of [14C]estradiol-17 beta and estrone; i.e., estrone was reduced to estradiol-17 beta and estradiol-17 beta was metabolized to conjugates, including estradiol-3-beta-D-glucoside and estradiol-17-alpha-D-glucoside. [14C]Testosterone was not transformed appreciably by the ovaries. Metabolic activity and physiological significance of the vertebrate steroid hormones in the silkworm ovaries are discussed.

Animals↗
PubMed1988

Expression of the E2 open reading frame of papillomaviruses BPV1 and HPV6b in silkworm by a baculovirus vector.

To produce and characterize the nature of the E2 protein of papillomaviruses, we have developed a system to express the DNA sequence containing an open reading frame (ORF) as a fusion protein in cultured insect cells and silkworms. The DNA fragments of the E2 ORF predicted from the DNA sequence of bovine papillomavirus type 1 and human papillomavirus type 6b were linked to the N-terminal part of polyhedrin gene of the Baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) vector. Hybrid proteins composed of polyhedrin protein (52 amino acids) and the E2 proteins of BPV1 (410 amino acids) or HPV6b (346 amino acids) were efficiently produced in B. mori cells and silkworm larvae infected with recombinant viruses. The amount of E2 fusion proteins produced by recombinant viruses was comparable to that of polyhedrin produced by wild type BmNPV. The hybrid proteins were immunologically reactive to antiserum against polyhedrin.

Animals↗
PubMed1996

Induction of anti-malarial transmission blocking immunity with a recombinant ookinete surface antigen of Plasmodium berghei produced in silkworm larvae using the baculovirus expression vector system.

We have studied Pbs21, a major ookinete surface protein of Plasmodium berghei, for the development of a model transmission blocking immunogen. In the mouse, recombinant Pbs21 expressed in the Escherichia coli expression system (EcrPbs21) is not as effective in inducing transmission blocking antibodies as native Pbs21 (nPbs21), possibly because of differences in post-translational processing between EcrPbs21 and nPbs21. In an attempt to improve the efficacy of the recombinant molecule, we describe here the use of a baculovirus expression vector system in the silkworm Bombyx mori. Following an injection of recombinant baculovirus containing Pbs21 cDNA, B. mori larvae produced recombinant Pbs21 (BmrPbs21) with a molecular weight indistinguishable from nPbs21. Fifty micrograms of BmrPbs21 could be purified from the hemolymph of each infected larva using affinity chromatography. Immunization of Balb/c mice with BmrPbs21 induced high anti-BmrPbs21 and anti--ookinete antibodies but low anti-EcrPbs21 antibody. In contrast, EcrPbs21 induced high anti--EcrPbs21 antibody but low anti-BmrPbs21 and anti-ookinete antibodies. This suggests that most B-cell epitopes on nPbs21 are conformational and that many of the linear epitopes in EcrPbs21 are not normally exposed in nPbs21. Oocyst formation in Anopheles stephensi mosquitoes, which fed on mice immunized with purified BmrPbs21 and infected with P. berghei, was blocked by 85.5-97.1%. These results suggest that the baculovirus-silkworm system produces useful quantities of recombinant Pbs21 which in limited studies is structurally and immunogenically indistinguishable from the native molecule.

Animals↗
PubMed1995

Trehazolin, a slow, tight-binding inhibitor of silkworm trehalase.

Mechanisms of enzyme inhibition by trehazolin, a new inhibitor of trehalase (Ando et al. (1991) J. Antibiot. 44, 1165), were investigated using purified soluble silkworm trehalase and other glycosidases. Trehazolin inhibited trehalase with an IC50 value of 27 nM, whereas some other exo-alpha-glucosidases were inhibited only weakly, with IC50 values ranging from 7 to 370 microM. Other glycosidases tested were not inhibited by 500 microM trehazolin. The inhibition of trehalase by trehazolin was competitive with respect to trehalose. A notable feature of the inhibition was a slow progression of the association and dissociation of the enzyme-inhibitor complex. Preincubation of the enzyme and the inhibitor at 37 degrees C potentiated the inhibition by 10-times in a time-dependent manner up to 6 h. Dialysis of the inactivated enzyme recovered the enzymatic activity very slowly, and the rate constant for the dissociation at 37 degrees C was (7.3).10(-2) h-1. Trehalamine, a deglucosylated form of trehazolin, inhibited both silkworm trehalase and exo-alpha-glucosidases only weakly. The inhibition of trehalase by trehalamine was reversible. Rat isomaltase inhibition by trehazolin and sucrase inhibition by trehalamine were also reversible. Taken together, trehazolin is a specific slow, tight-binding inhibitor of trehalase, and the glucose moiety of the inhibitor is essential to the tight binding.

Animals↗
PubMed1982

Properties of protease inhibitors from the haemolymph of silkworms, Bombyx mori, Antheraea pernyi and Philosamia cynthia ricini.

1. Effects of inhibitors in the haemolymph from three silkworms on proteases from the alimentary canal of respective insects were studied as well as those on bovine trypsin and alpha-chymotrypsin. 2. In Bombyx haemolymph, three inhibitor fractions were separated by gel filtration on Sephadex G-75. These fractions differed in the specificity of inhibition for different proteases and in thermal stability. 3. In Antheraea haemolymph, a relatively different elution profile was observed compared to that of Bombyx. Philosamia haemolymph showed the intermediate type of elution pattern. 4. Inhibitors from the haemolymph of Antheraea and Philosamia were heat stable. 5. Distinct electrophoretic patterns of inhibitors were observed in three silkworms.

Animals↗
PubMed1982

Comparison of three alkaline proteases from digestive fluid of the silkworm, Bombyx mori L.

1. Digestive fluid proteases of the silkworm, 6B1-3, were separated, partially purified and their properties were compared. 2. These proteases were different in the substrate specificity, effect of inhibitors, Km and influence of Mn2+. 3. Hydrolyzing ability for natural substrates was comparatively high in 6B1, whereas the hydrolysis of synthetic substrates of trypsin by 6B1 was lower than that by 6B2 or 3. 4. The protease activity was sensitive to DFP and PMSF. The soybean trypsin inhibitor differentially affected three proteases. Silkworm haemolymph strongly inhibited the protease activity of 6B2 and 3, but scarcely affected 6B1.

Animals↗
PubMedDate not supplied

Antibacterial activity inducible in the haemolymph of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 during the fifth larval instar and pharate adult development.

1. Antibacterial activity inducible in the haemolymph of the silkworm, Bombyx mori, by immunization, i.e. by injection of formalin-treated Escherichia coli (E. coli) K-12 during the fifth larval instar and pharate adult development that was reared aseptically on an artificial diet was determined by inhibition zone assay using the same bacterium as a test organism. 2. A peak of antibacterial activity was observed in each development stage; approximately 8 mm in diameter of a clear zone at days 3 or 4 in the fifth larval instar and approximately 5 mm at day 1 in the pharate adults. 3. Acid polyacrylamide gel electrophoresis of immunized haemolymph followed by overlay assay showed that an activity band was associated with two peptide bands that were similar to the cecropin-like peptides A and B that were reported in the silkworm (Morishima et al., 1988, Agri. Biol. Chem. 52, 929-934). Any other activity bands were not observed. No activity band was detectable from the haemolymph of non-immunized insects. 4. Fractionation of antibacterial peptides in immunized haemolymph on a CM-cellulose column resulted in separation of two groups of activity, both in the fifth instar larvae and in the pharate adults with a slight difference in elution conditions. 5. Duration of high antibacterial activity induced by a single immunization was approximately 12 hr in the fifth instar day 3 larvae and 48 hr in the day 2 pharate adults.

Animals↗
PubMedDate not supplied

Induction of antibacterial activity in the haemolymph of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 in the anterior and posterior body part of the ligated larvae.

1. Induction of antibacterial activity was investigated in the ligated fifth instar larvae of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 into the haemocoel in the anterior and in the posterior body part, followed by activity determination by inhibition zone assay of the haemolymph at 12 and 24 hr after immunization. 2. At 12 hr after immunization, high antibacterial activity, approximately 6.8-7.5 mm in the anterior body part and 4.5-6.4 mm in the posterior body part in diameter of a clear zone (2.0 mm for no activity) was detectable in day 3 larvae. This result was in good contrast to expression of lectin gene in the ligated flesh fly, Sarcophaga peregrena (Shiraishi and Natori, 1988, FEBS Lett. 232, 163-166), in which only the anterior part of insect responded to stimulus of injury. 3. Antibacterial activity at 24 hr after immunization in days 3 or 4 ligated larvae was lower than that at 12 hr; 4.0-4.5 mm of activity was observed in the anterior body part, and no activity was observed in day 3 ligated larvae in the posterior body part. 4. Acid polyacrylamide gel electrophoresis of the haemolymph of immunized insects followed by overlay assay showed that the size of antibacterial activity bands were similar between the haemolymph from 12 hr and from 24 hr, and between the anterior and the posterior body part. This result was contradictory to the observation of activity by inhibition zone assay. The activity bands were associated with peptides that were similar to cecropin-like peptides A and B in the silkworm.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Partial release of aminopeptidase N from larval midgut cell membranes of the silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C.

1. The membrane anchor of aminopeptidase N associated with larval midgut cell membranes of the silkworm, Bombyx mori, was investigated by using phosphatidylinositol-specific phospholipase C (PIPLC) and proteases. 2. Aminopeptidase N, which was virtually all localized in the brush border membrane, was solubilized by PIPLC but not by papain or trypsin. 3. Detergent-solubilized amphiphilic aminopeptidase N was converted into a hydrophilic form by PIPLC but not by papain. 4. Either of these effects of PIPLC on aminopeptidase N was maximally 40%. 5. These results suggest that in larval midgut cells of the silkworm, B. mori, at least 40% aminopeptidase N is anchored in the brush border membrane via glycosyl-phosphatidylinositol.

Aminopeptidases↗
PubMed1995

Sequences of two cDNAs encoding silkworm homologues of Drosophila melanogaster squid gene.

The squid (sqd) gene of Drosophila melanogaster encodes a few isoforms of a heterogeneous nuclear (hn) RNA-binding protein. We isolated two types of cDNAs coding for homologues of the Sqd protein from the silkworm Bombyx mori. The two predicted amino acid (aa) sequences are identical up to aa 280 and then diverge. The silkworm and fruit fly proteins share 80% homology in the RNA-binding motif region. These cDNAs detect 2.0-, 1.8- and 1-kb mRNAs in the middle and posterior silk glands.

Alternative Splicing↗
PubMed2005

A novel Rel protein and shortened isoform that differentially regulate antibacterial peptide genes in the silkworm Bombyx mori.

Two cDNAs encoding novel Rel proteins were cloned from the silkworm, Bombyx mori. These cDNA clones (BmRelA and BmRelB) showed identical nucleotide sequences except for the 5'-region. BmRelB cDNA derived probably from an alternatively spliced mRNA lacked 241 bp nucleotides at the 5'-region of the BmRelA cDNA, resulting in a loss of the first 52 amino acids. Expression of antibacterial peptide genes was strongly inhibited upon infection with Micrococcus luteus in transgenic silkworms in which BmRel gene expression was knocked down, suggesting that these two Rel proteins are involved in activation of antibacterial peptide genes. Co-transfection experiments indicated that BmRelB activated the Attacin gene strongly and other genes to a lesser extent, whereas BmRelA activated Lebocin 4 gene strongly and Attacin and Lebocin 3 genes very weakly. The Rel homology domain of BmRelA and BmRelB was shown to bind specifically to kappaB sites of antibacterial peptide genes. Proline-rich domains of the BmRels were necessary for activation of antibacterial peptide genes. These results illustrate that a minor structural change in Rel proteins can provoke a dramatic differential activation of antibacterial peptide genes, suggesting a novel regulatory mechanism for insect antibacterial peptide gene expression.

Amino Acid Sequence↗
PubMed2003

Analysis of extrachromosomal homologous recombination in cultured silkworm cells.

Double-strand breaks (DSBs) are potentially lethal lesions causing the loss of chromosomal information. Eukaryotic cells have evolved the error-free repair systems of DSBs by homologous recombination (HR) through gene conversion with or without crossing over. In this study, we have developed a rapid assay system for extrachromosomal HR events in the cultured silkworm BmN4 cells. When HR occurs within the disrupted luciferase gene, an enzymatically active luciferase is restored and expressed. Our results strongly suggest that error-prone single strand annealing (SSA) accounts for the majority of extrachromosomal recombination processes in the cells. However, upon the substrates which cannot be repaired through SSA, DSBs were efficiently repaired though gene conversion. The rapid and sensitive HR assay system developed in the present study is expected to be a powerful tool for the identification and analysis of HR-related genes in the silkworm.

Animals↗
PubMed2006

In vivo analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori using recombinant baculovirus as vector.

In order to investigate the functional signal peptide of silkworm fibroin heavy chain (FibH) and the effect of N- and C-terminal parts of FibH on the secretion of FibH in vivo, N- and C-terminal segments of fibh gene were fused with enhanced green fluorescent protein (EGFP) gene. The fused gene was then introduced into silkworm larvae and expressed in silk gland using recombinant AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) as vector. The fluorescence of EGFP was observed with fluorescence microscope. FibH-EGFP fusion proteins extracted from silk gland were analyzed by Western blot. Results showed that the two alpha helices within N-terminal 163 amino acid residues and the C-terminal 61 amino acid residues were not necessary for cleavage of signal peptide and secretion of the fusion protein into silk gland. Then the C-terminal 61 amino acid residues were substituted with a His-tag in the fusion protein to facilitate the purification. N-terminal sequencing of the purified protein showed that the signal cleavage site is between position 21 and 22 amino acid residues.

Amino Acid Sequence↗
PubMed2006

The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor.

We generated germline transgenic silkworms bearing a fibroin light chain (FL) promoter-driven FL gene whose 3'-end was flanked with human basic fibroblast growth factor (bFGF) gene, FL/bFGF gene. The cocoons from transgenic worms were trypsinized to remove sericin layers, and treated with solution containing CaCl(2), ethanol, and water at a molar ratio of 1:2:8 (CaCl(2)/ethanol/water) to solubilize fibroin layers. Western blot analysis showed that the recombinant protein, r(FL/bFGF), was solubilized with CaCl(2)/ethanol/water, but not with trypsin, indicating that r(FL/bFGF) was in fibroin layers. Thus, it was concluded that the worms spun cocoons whose fibroin layers were composed of the inherent gene-derived natural fibroin (nF) and r(FL/bFGF). The mixture of nF and r(FL/bFGF) was dubbed r(FL/bFGF)nF. The solubilized r(FL/bFGF)nF was refolded using the glutathione redox system. Human umbilical vein endothelial cells (HUVECs) grew in the refolded r(FL/bFGF)nF-containing culture media, showing that bFGF in r(FL/bFGF) was biologically active. r(FL/bFGF)nF immobilized on a culture dish also supported the growth of HUVECs in bFGF-free media, suggesting the usefulness of r(FL/bFGF)nF as a new biomaterial for tissue engineering. The currently developed transgenic silkworms will be suitable for mass production of fibroins bearing a variety of biological activities.

Animals↗
PubMed2004

Structure of pentasaccharide of glycopeptide from TIME-EA4, N-glycoprotein in silkworm diapause eggs.

The TIME-EA4, from silkworm diapause eggs of pure strain C108, Bombyx mori, has glycosylated chain as tetrasaccharide (Man(2)GlcNAc(2)) attaching to the Asn(22) of T3 peptide from tryptic digests. On the other hand, from Showa silkworm strain we additionally observed a pentasaccharide (Man(3)GlcNAc(2)) on T3 at the same linkage site. The linkage pattern of the 5-sugar chain was studied through Smith degradation combined with LC-MS and MS/MS analyses. These advanced methods led us to conclude that the pentasaccharide was branching as Man 1-->3(Man 1-->6)Man 1-->4GlcNAc 1-->4GlcNAc.

Animals↗