Biomedical subjects

Yutaka Banno

Publications and source records attributed to Yutaka Banno.

At least 19 recordsLinked to original sources

PubMed2006

Genes encoding small heat shock proteins of the silkworm, Bombyx mori.

Small heat shock protein (sHSP) is a family of ubiquitous polypeptides involved in a variety of physiological phenomena. From the silkworm, Bombyx mori, we isolated and sequenced the following cDNAs encoding sHSPs: shsp19.9, shsp20.1, shsp20.4, shsp20.8, shsp21.4, and shsp23.7. shsp21.4 was nearly twice as large in size as other shsps. The deduced amino acid sequence of sHSP21.4 was similar to that of Drosophila melanogaster CG14207-PA. Other sHSPs were highly similar to each other and, in a phylogenetic tree, formed a cluster including Plodia interpunctella alphaCP25. It was speculated that shsp21.4 has at least one intron in genome while other shsps do not. The transcripts of all shsps were subtle, but were constitutively detected in various tissues. Heat shock triggered a substantial increase in the transcripts other than shsp21.4. The B. mori sHSPs are perhaps classified into at least two groups: sHSP21.4 and others.

Animals↗

PubMed2006

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↗

PubMed2006

Proteome analysis of silk gland proteins from the silkworm, Bombyx mori.

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.

Animals↗

PubMed2006

Silkworm midgut proteins interacting with a hemolymph protease inhibitor, CI-8.

CI-8 is the chymotrypsin inhibitor in hemolymph from the silkworm, Bombyx mori. It occurs in the midgut at the spinning stage of larva, but little information on the mechanism of its uptake in the midgut is available. We found that two polypeptides interacting with CI-8 are in the midgut membrane, and we purified them using a biotinylated CI-8, viz., p29 and p60, having molecular sizes of 29 kDa and 60 kDa respectively. The structures of p29 and p60 were examined by N-terminal amino acid sequencing and peptide mass mapping, including tryptic digestion. p29 was highly similar to the matured 19G1-30K lipoprotein from hemolymph, but p60 was novel. Purified p29 was recognized by anti-19G1-30K antibody, and was confirmed to be similar to 19G1-30K. The antibody also neutralized the CI-8 binding ability of p29 in the midgut membrane. p29 and p60 are perhaps proteinaceous factors involved in the uptake of CI-8 into the midgut through the membrane.

Amino Acid Sequence↗

PubMed2005

Molecular and biochemical characterization of manganese-containing superoxide dismutase from the silkworm, Bombyx mori.

Superoxide dismutase (SOD) is responsible for the removal of superoxide anion from living organisms. In this study, cDNA encoding the manganese-containing SOD (MnSOD) from the silkworm, Bombyx mori, was isolated by reverse transcriptase-polymerase chain reaction and sequenced. The deduced amino acid sequence of the MnSOD revealed 62% identity to that of the Drosophila melanogaster; both were close to each other in a phylogenetic tree. The MnSOD was overproduced in Escherichia coli and purified. The internal structure of the recombinant MnSOD was confirmed by peptide mass fingerprinting method. The recombinant MnSOD facilitating the reduction reaction of superoxide anion retained 75% of its original activity after incubation at pH 4-11 for 24 h at 4 degrees C. Its activity was never affected by incubation at pH 7 for 30 min below 50 degrees C.

Amino Acid Sequence↗

PubMed2005

Cloning, expression and characterization of theta-class glutathione S-transferase from the silkworm, Bombyx mori.

This study focused on glutathione S-transferase (GST), one of the detoxification enzymes, from the silkworm, Bombyx mori (GSTT1). A cDNA encoding a putative GST was amplified by reverse transcriptase-polymerase chain reaction and sequenced. The deduced amino acid sequence revealed 59%, 57% and 56% identities to theta-class GSTs of Musca domestica, Anopheles gambiae and Drosophila melanogaster, respectively. GSTT1 was also estimated to be close to those GSTs in a phylogenetic tree. Recombinant GST (rGSTT1) was functionally overexpressed in Escherichia coli in a soluble form, purified to homogeneity, and characterized. The pH-optimum of rGSTT1 was broad from pH 4 to 9 and rGSTT1 retained more than 75% of its original activity after incubation at pH 5-11. Incubation for 30 min at temperatures below 50 degrees C also affected the activity insignificantly. The Michaelis constant for 1-chloro-2,4-dinitrobenzene was 0.48 mM.

Amino Acid Sequence↗

PubMed2005

Catalase from the silkworm, Bombyx mori: gene sequence, distribution, and overexpression.

Living organisms require mechanisms regulating reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion. Catalase is one of the regulatory enzymes and facilitates the degradation of hydrogen peroxide to oxygen and water. Biochemical information on an insect catalase is, however, insufficient. Using mRNA from fat body of the silkworm, Bombyx mori, a cDNA encoding a putative catalase was amplified by reverse transcriptase-polymerase chain reaction and sequenced. The deduced amino acid sequence comprised 507 residues with more than seventy residues forming a scaffold for a heme cofactor conserved. The sequence showed 71% and 66% identities to those of the Drosophila melanogaster and Apis mellifera catalases, respectively; the catalase from B. mori was estimated to be phylogenetically close to that from A. mellifera. The transcripts of the gene and the catalase activity were distributed in diverse tissues of B. mori, suggesting its ubiquitous nature. Using the gene, a recombinant catalase (rCAT) was functionally overexpressed in a soluble form using Escherichia coli, purified to homogeneity, and characterized. The pH-optimum of rCAT was broad around pH 8.0. More than 80% of the original rCAT activity was retained after incubation in the following conditions: at pH 8-11 and 4 degrees C for 24 h; at pH 7 and temperatures below 50 degrees C for 30 min. The Michaelis constant for hydrogen peroxide was evaluated to be 28 mM at pH 6.5 and 30 degrees C. rCAT was suggested to be a member of the typical catalase family.

Adipose Tissue↗

PubMed2005

Integration of molecular and classical linkage groups of the silkworm, Bombyx mori (n = 28).

Previously published linkage groups (LGs) composed of molecular markers were assigned to classical LGs in the silkworm, Bombyx mori (n = 28). Four markers from the classical linkage map, og, w-1, Lp, and Pfl, were assigned to the molecular linkage maps using sequence tagged sites. In addition, linkage analysis was carried out using BF1 progeny between wild-type and mutant stocks carrying morphological phenotypic markers. As a result, the counterparts for 26 of 28 molecular LGs were identified with their counterparts of the classical LGs. Two visible markers, Sel and Xan, representing different classical LGs, were found to be linked.

Animals↗

PubMed2005

Glucan-binding activity of silkworm 30-kDa apolipoprotein and its involvement in defense against fungal infection.

The silkworm Bombyx mori 30-kDa lipoproteins (6G1 and 19G1), major components of the hemolymph, were shown to bind to glucans. 6G1 apolipoprotein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and assayed for its binding activity. The purified recombinant 6G1 apolipoprotein specifically bound to beta-glucan, but not to chitin, mannan, peptidoglycan, or oligosaccharide chains on glycoproteins. The beta-glucan binding of the recombinant 6G1 was inhibited by laminaribiose and laminarin, a soluble glucan, but not by lipopolysaccharide or insect blood sugar, trehalose at physiological concentration. Furthermore, the recombinant 6G1 was shown to participate in the activation of prophenoloxidase cascade and to interfere with hyphal growth of the entomopathogenic fungus Paecilomyces tenuipes, injected into pupae of B. mori. These results suggest that 6G1 lipoprotein plays a role in the protection of B. mori against invading fungi.

Amino Acid Sequence↗

PubMed2005

Proteomic studies of isoforms of the P25 component of Bombyx mori fibroin.

It is recognized that P25 is one of three polypeptide components of the fibroin synthesized in the larval silk gland (SG) of silkworm, having two glycosylated isoforms. In the present study, however, eight P25 isoforms were separated by proteomics, including two-dimensional gel electrophoresis of whole SG proteins, and were identified by the peptide mass fingerprinting method. Four of the eight isoforms were identified as Bombyx mandarina P25s, although the SG of Bombyx mori has never been considered to contain the P25 from B. mandarina. It is suggested that this diversity of P25 isoforms depends on phosphorylation modification in addition to glycosylation.

Amino Acid Sequence↗

PubMed2005

Superoxide dismutase from the silkworm, Bombyx mori: sequence, distribution, and overexpression.

Superoxide dismutase (SOD) is an enzyme facilitating the removal of superoxide anions from living organisms. This study focused on SOD from the silkworm, Bombyx mori (bmSOD). cDNA encoding bmSOD was amplified by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of bmSOD indicated that the residues forming the Cu/Zn binding site are conserved and that the sequence is in 60% identity to that of the Drosophila melanogaster. B. mori SOD was also close to the D. melanogaster SOD in a phylogenetic tree. The bmSOD mRNA and the enzyme activity were widely distributed in diverse tissues. bmSOD functionally overexpressed in Escherichia coli in a soluble form was purified, and its stability was examined. bmSOD at 4 degrees C retained almost all of its original activity after incubation at pH 4-11 for 24 h. Incubation (pH 7) for 30 min at temperatures below 40 degrees C also affected activity insignificantly.

Amino Acid Sequence↗

PubMed2004

A carotenoid-binding protein (CBP) plays a crucial role in cocoon pigmentation of silkworm (Bombyx mori) larvae.

We examined the role of carotenoid-binding protein (CBP) in yellow cocoon pigmentation. First, using yellow or white cocoon races, we investigated the linkage between the yellow pigmentation and CBP expression. CBP was expressed only in the silk gland of the yellow cocoon races, which utilize carotenoids for cocoon pigmentation. Furthermore, CBP expression in the silk glands of day 1-7 fifth instar larvae matched the period of carotenoid uptake into the silk gland. Finally, we gave double-stranded CBP RNA to Bombyx mori (B. mori) larvae to induce RNA interference. The significantly reduced expression of CBP in the silk gland of fifth instar larva was confirmed on day 4 and a decrease in yellow pigmentation was observed in the cocoon. We showed that CBP plays a key role in the yellow cocoon pigmentation caused by carotenoids.

Animals↗

PubMed2004

Characterization of the carotenoid-binding protein of the Y-gene dominant mutants of Bombyx mori.

Carotenoids play important and diverse roles in insects. Recently, we purified and partially characterized a carotenoid-binding protein (CBP) from the wild type of Bombyx mori. In this report, we utilized immunoblotting, ELISA and immunocytochemistry to further characterize and localize the expression of CBP in the larval midgut and silk gland obtained from the wild type and four naturally occurring mutants linked to carotenoids transport. CBP was expressed throughout the 5th stadium, with highest expressions on days 4-5 in the silk gland and days 3-5 in the midgut. Immunoblotting analyses demonstrated the presence of CBP along the middle part of the midgut. Microscopic immunocytochemistry demonstrated that the 33 kDa CBP was uniformly expressed along the brush border of columnar cells in the epithelium of the midgut typifying its function in aiding absorption of dietary carotenoids. Similarly, CBP was highly expressed along the distal membrane of the middle part of the silk gland demonstrating its function in uptake of carotenoids from lipophorin. When the middle silk glands and midguts of the four mutants were incubated with rabbit anti-CBP antibody, only proteins of the Y-gene dominant mutants cross reacted with the antibody further accentuating the hypothesis that the CBP is a Y-gene dependent protein.

Animals↗

PubMed2004

The basis for colorless hemolymph and cocoons in the Y-gene recessive Bombyx mori mutants: a defect in the cellular uptake of carotenoids.

Bombyx mori is an excellent model for the study of carotenoid-binding proteins (CBP). In previous papers, we identified and molecularly characterized a CBP from the Y-gene dominant mutants. In the present study, we attempted to correlate and establish lipid metabolism and distribution in these mutants. When [3H]-triolein was fed to the mutants, typical patterns of uptake of labeled fatty acids from midgut to hemolymph and subsequent delivery to fat body and silk glands were obtained in all mutants. Further analysis of lipid and carotenoid profiles revealed that the yellow coloration in the hemolymph associated with lipophorin is not attributed to a difference in lipophorin concentrations among the mutants, nor to its lipid composition, but rather to its carotenoid content. Lipophorin of the Y+I mutant exhibited the highest concentration of total carotenoids of 55.8 microg/mg lipophorin compared to 3.1 microg/mg in the +Y+I mutant, 1.2 microg/mg in the YI mutant and 0.5 microg/mg in the +YI mutant. Characteristic retention time in HPLC of the different classes of carotenoids of lipophorin identified the presence of lutein as the major chromophore (62-77%), followed by beta-carotenes (22-38%). Although lutein and beta-carotene content of mutants' lipophorin differed significantly, the ratio of lutein to beta-carotene of 3:1 was not different among mutants. Similarly, lipid compositions of mutant silk glands were not significantly different, but carotenoid contents were. The significantly high concentration of lutein in the Y+I mutant silk gland represented more than 160-fold increase compared to +Y+I mutant (p<0.001). In this report, we conclude that lipid metabolism in the mutants is not defected and that the molecular basis for colorless hemolymph and cocoons is a defect in the cellular uptake of lutein associated with the Y-gene recessive mutants.

Animals↗

PubMed2004

Overexpression in Escherichia coli and purification of recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm.

Present research provided an efficient approach to obtain large quantities of active recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm, Bombyx mori. The cDNA encoding mature CI-b1 was cloned into pDEST17 vector. Recombinant protein with hexa-histidine tag attached to the N-terminal of CI-b1 was expressed in Escherichia coli Origami B cells. It can be purified to homogeneity via the gel filtration chromatography on a Sephacryl S-200 column followed the affinity chromatography on a Ni-NTA column. The two sequential purification procedures yielded 4.3mg purified (His)(6)-tagged CI-b1 from 200ml of culture medium. Studies on (His)(6)-tagged CI-b1 revealed that three disulfide bonds were formed in the recombinant CI-b1 and the inhibitory properties of recombinant CI-b1 against alpha-chymotrypsin were similar to those of native CI-b1. Recombinant CI-b1 immobilized on Ni-NTA resin was used to detect the interactions occurring between the CI-b1 and its target factors.

Animals↗

PubMed2004

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↗

PubMed2004

Proteomic studies of lipopolysaccharide-induced polypeptides in the silkworm, Bombyx mori.

Silkworm larvae at the 5th instar were injected with lipopolysaccharide from Escherichia coli and inducible polypeptides were examined within a pI range of 3-10 and a size range of 14-97 kDa by proteomics, including peptide mass fingerprinting. No polypeptides were induced in the midgut. FB1 and H1-4 polypeptides were significantly induced in fat body and hemolymph, respectively. FB1 and H1 were estimated to be antitrypsin and serpin-2 proteinase inhibitors respectively. H2 and H3 were novel polypeptides. H4 was estimated to be attacin antibacterial polypeptide with high coverage of sequence. The amounts of all the induced polypeptides decreased at 48 h after the injection.

Animals↗

PubMed2004

Utility of dry gel from two-dimensional electrophoresis for peptide mass fingerprinting analysis of silkworm proteins.

We compared the use of wet and dry two-dimensional electrophoresis (2-DE) gels for in-gel tryptic digestion and subsequent analysis by mass spectrometry, first using bovine serum albumin (BSA) as a model protein and then using unknown proteins from an extract of the silkworm midgut. The gel was either dried at 80 degrees C or left wet. Upon analysis of BSA, there was little difference in peptide recovery from 2-DE or in mass spectrum between the dry and the wet gels. The midgut extract was resolved into more than 1,100 protein spots by 2-DE, and 40 of these spots were sampled for further analysis. For all of the 40 proteins, the results obtained from dry and wet gels were quite similar in mass spectra and protein identification, although the relative amounts of peptides from tryptic digestion ranged from 45 to 146%. Based on these results, we confirmed the utility of dry electrophoretic gels for proteomics of insect extracts.

Animals↗