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PubMed2001

Purification and characterization of fibroin from the tropical Saturniid silkworm, Antheraea mylitta.

The fibroin protein isolated from the posterior silkgland of the tropical Saturniid silkworm Antheraea mylitta, was solubilized in lithium dodecyl sulfate and purified by gel filtration. The major fraction from gel filtration was analyzed by SDS-PAGE under non-reducing and reducing conditions. One major protein band of ca 395 kDa was obtained under non-reducing conditions and a doublet band of approximately 197 kDa under reducing conditions. The appearance of a single spot in two-dimensional electrophoresis confirmed the purity of the protein indicating that it may be a homodimeric protein of two similar sized polypeptides. Amino acid composition analysis showed that, like other Saturniid fibroins, it is rich in glycine, alanine and serine amino acids. N-terminal amino acid sequence shows significant homology with other Antheraea species. The enzymatic deglycosylation analysis indicates that the fibroin protein is glycosylated and the oligosaccharides are O-linked to the protein backbone by N-acetylgalactoseamine moiety which conforms to a Core 1 mucin-type glycosylation pattern.

Amino Acid Sequence↗
PubMed2002

Identification and characterization of genes abnormally expressed in wing-deficient mutant (flügellos) of the silkworm, Bombyx mori.

The wing-deficient mutant, flügellos (fl), of the silkworm lacks four wings in the pupa and the adult, due to aberrant wing morphogenesis during metamorphosis. To elucidate the mechanisms of wing-specific deficiencies in the fl mutant, we used mRNA differential display and identified five genes abnormally expressed in the fl wing discs. Northern blot and RT-PCR analyses revealed that four genes were overexpressed, but the fifth one was not transcribed in the fl wing discs. The expression level of ribosome-associated protein p40 in the fl wing discs was elevated approximately 10 times compared to the wild-type (WT) discs. Another overexpressed gene CB10 encodes a novel wing-specific protein with a putative zinc-finger motif. Overexpression of two components of extracellular matrix, cuticle protein 18 (BMCP18) and a fibrillin-like protein AD10, may result in the abnormal wing morphogenesis in the fl mutant. In contrast, a novel member of multifunctional Ca2+-binding protein annexins, designated as annexin b13 (Anx b13), was expressed dominantly in the wing discs of WT but completely repressed in the fl tissues. Strong expression of Anx b13 in wing discs during the fourth and fifth instar indicates that ANX B13 plays an important role in wing morphogenesis.

Amino Acid Sequence↗
PubMed2002

Ca(2+) as second messenger in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori.

Measurements of Ca(2+) influx and [Ca(2+)](i) changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca(2+) as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca(2+)](i) in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca(2+)](i) levels were dependent on extracellular Ca(2+). They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca(2+) channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca(2+) channels. The trivalent cation La(3+), a non-specific blocker of plasma membrane Ca(2+) channels, eliminated the rPTTH-stimulated increase of [Ca(2+)](i) levels in PG cells and so did amiloride, an inhibitor of T-type Ca(2+) channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca(2+)](i) levels, which was also dependent on extracellular Ca(2+) and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca(2+) channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca(2+)](i) levels and one agent's mediated increase of [Ca(2+)](i) was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca(2+) release from inositol 1,4,5 trisphosphate (IP(3))-sensitive Ca(2+) stores, blocked the rPTTH-stimulated increases of [Ca(2+)](i) levels, suggesting an involvement of IP(3) in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca(2+)](i) levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca(2+)](i) levels, filling state of IP(3)-sensitive intracellular Ca(2+) stores and the PTTH-receptor's-mediated Ca(2+) influx.

Animals↗
PubMed2003

Cell-specific expression of enhanced green fluorescence protein under the control of neuropeptide gene promoters in the brain of the silkworm, Bombyx mori, using Bombyx mori nucleopolyhedrovirus-derived vectors.

Using the larvae of the silkworm, Bombyx mori, we examined the baculovirus expression vector system for the expression of the enhanced green fluorescence protein (EGFP) gene under the control of several gene promoters in vivo. To investigate the gene-delivery efficiency of the baculovirus into various larval tissues, we constructed two recombinant baculoviruses carrying the EGFP gene downstream of the Drosophila melanogaster hsp70 gene promoter from B. mori nucleopolyhedrovirus (BmNPV) and Autographa californica nucleopolyhedrovirus (AcNPV). After injection of these recombinant baculoviruses into newly ecdysed 5th instar larvae, hsp70::EGFP-BmNPV, but not hsp70::EGFP-AcNPV, caused intense expression of EGFP not only in various non-neural tissues, but also in the neural organs including the brain 5 days postinfection. To investigate the cell-specific expression in the brain, we constructed recombinant C4/B3::EGFP-BmNPV and PTTH::EGFP-BmNPV which carry the EGFP gene under the control of bombyxin B3 and prothoracicotropic hormone (PTTH) gene promoters, respectively. Injection of these recombinant baculoviruses caused specific expression of EGFP with a high gene-expression efficiency in the neurosecretory cells of the brain depending on the neurohormone gene promoters. Present results indicate that this in vivo gene-expression system mediated by the baculovirus can serve as an efficient system permitting gene delivery into neural tissues in insects.

Animals↗
PubMed2003

A GATA-binding protein expressed predominantly in the pupal ovary of the silkworm, Bombyx mori.

Preferential localization of Bkm (Banded krait minor-satellite) DNA sequences on Y/W chromosomes of higher eukaryotes, which remain highly condensed in somatic cells but undergo extensive decondensation in the germ cells during early stages of development, led to the postulation for the existence of a sex- and tissue-specific Bkm-binding protein (BBP). Accordingly, we purified and characterized a BmBBP expressed predominantly in pupal ovary of the silkworm (Bombyx mori). 2D-PAGE revealed BmBBP as moderately basic (pI 7.8-8, in the range expected for DNA-binding proteins) and Matrix Assisted Laser Desorption/Ionization Time of Flight exhibited a value of 37.5-kDa. BmBBP neither contains nor requires divalent metal ions for its DNA-binding activity, suggesting that it does not belong to the well-studied GATA-family of transcription factors. BmBBP is unusually strong in its DNA-binding characteristics to Bkm (GATA-repeats), which suggests its probable role in bringing about coordinated chromatin conformational changes to activate genes present in associated chromosomal domains. Fluorescence immuno-localization studies employing specific anti-BmBBP antibodies revealed its presence in the follicle cells and in the ooplasm, as well as the nucleus of different developmental stages of oocytes.

Animals↗
PubMed2003

Inhibition of cAMP signalling cascade-mediated Ca2+ influx by a prothoracicostatic peptide (Mas-MIP I) via dihydropyridine-sensitive Ca2+ channels in the prothoracic glands of the silkworm, Bombyx mori.

Measurements of Ca(2+) influx in Fura-2/AM loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, after application of forskolin or the cAMP analogue, 8-bromo-cAMP, showed a steady increase in [Ca(2+)](i), which was of extracellular origin and was inhibited, in both cases, by the dihydropyridine (DHP) derivative, nitrendipine. Nitrendipine also inhibited the abrupt S(-).Bay K 8644-mediated increase in [Ca(2+)](i) and its effects were mimicked by a myoinhibitory/prothoracicostatic peptide (Mas-MIP I/PTSP), which was isolated from Manduca sexta and was found to possess ecdysteroidostatic activity in Bombyx mori PGs. This peptide blocked both the forskolin and S(-).Bay K 8644-mediated increase in [Ca(2+)](i) of PG cells. It was ineffective, however, in blocking the recombinant prothoracicotropic hormone (rPTTH)-stimulated high increase in [Ca(2+)](i) of PG cells suggesting that distinct and independently regulated Ca(2+) influx mechanisms operate in the PG cells of Bombyx mori. The dependence of DHP-sensitive Ca(2+) channels on the cAMP-signalling cascade was further corroborated by the inabilitity of nitrendipine to block the thapsigargin-stimulated high increase in [Ca(2+)](i) after depletion of Ca(2+) from the intracellular stores. This, together with the inability of thapsigargin to stimulate the cAMP levels of PG cells suggest that there is a tightly regulated cross-talk mechanism between the two signalling cascades of Ca(2+) and cAMP. The combined results suggest a cAMP-mediated regulation of the opening-state of DHP-sensitive Ca(2+) channels and stimulation of [Ca(2+)](i) increases and ecdysteroid secretion by a positive feedback mechanism. Mas-MIP I/PTSP interferes with this mechanism by blocking DHP-sensitive Ca(2+) channels. This regulatory mechanism appears to be autonomously stimulating ecdysteroidogenesis by the PGs, it is regulated by Mas-MIP I/PTSPS, and it is not involved in other Ca(2+) influx mechanisms that operate within the PG cells of Bombyx mori.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMedDate not supplied

Bmmar1: a basal lineage of the mariner family of transposable elements in the silkworm moth, Bombyx mori.

We describe a transposable element, called Bmmar1, from the genome of the silkworm moth, Bombyx mori. This element has features of the Tc1-mariner superfamily of transposable elements. Bmmar1 was first detected as a fragment in the 5' region of the larval serum protein (BmLSP) gene. Six genomic clones characterized each differed from a consensus sequence by 3-5 insertions and deletions, as well as an average of 2.3% in nucleotide sequence. The genome contains approximately 2400 copies of Bmmar1. Maximum parsimony phylogenetic analysis of the relationship of Bmmar1 and other members of the Tc1-mariner superfamily, based on their encoded transposase amino acid sequences, indicates that it represents a basal lineage of the mariner family. In particular Bmmar1 encodes a D,D37D motif thought to be the catalytic domain of mariner transposases. Bmmar1 considerably increases the known diversity of this widespread family of transposons. A new naming system is proposed for members of the family.

Amino Acid Sequence↗
PubMedDate not supplied

Purification and cDNA cloning of evolutionally conserved larval cuticle proteins of the silkworm, Bombyx mori.

A specific set of structural proteins termed larval cuticle proteins (LCPs) accumulates in integuments during larval development of the silkworm, Bombyx mori. Two major larval cuticle proteins, LCP17 and LCP22, were purified from the guanidine hydrochloride extract of the larval cuticle, and specific antibodies were raised against these proteins. Immunoblot analysis revealed that both LCPs are actively synthesized during larval intermolt stages, whereas the LCP17 epitope is also slightly but significantly detectable in pupal integuments. cDNA clones for LCPs were isolated by immunoscreening of the cDNA expression library constructed from larval epidermal mRNA. Predicted amino acid sequences of LCP17 and LCP22 are homologous to cuticle proteins from other insect species, including Manduca sexta, Drosophila melanogaster and Locusta migratoria. This fact suggests that these cuticle protein genes originated from a common ancestral gene and have been conserved during evolution. Northern blot hybridization demonstrated that the expression of LCP17 as well as LCP22 mRNA is controlled in a stage-specific manner in the epidermis of the final instar larvae, suggesting a common regulatory mechanism for transcription of these two intermolt genes.

Amino Acid Sequence↗
PubMed1998

A hydrophobic peptide (VAP-peptide) of the silkworm, Bombyx mori: structure, expression and an enhancing function of diapause hormone activity.

We have recently identified a unique lipophilic peptide (VAP-peptide) with diapause egg inducing activity in the silkworm, Bombyx mori (Imai et al., 1996). The cloning and sequencing of cDNA encoding VAP-peptide have demonstrated that the deduced amino acid sequence consisted of 84 amino acid residues, from which the mature VAP-peptide of 68 amino acid residues was released by cleaving a signal sequence. Searches of the GenBank data base revealed no significant sequence similarity to other proteins including diapause hormone (DH). VAP-peptide gene was selectively expressed just before and at adult eclosion in the head and the thorax not in the abdomen. By a Western blot analysis, VAP-peptide was also localized in the head and the thorax of adults. The purified recombinant VAP-peptide could not induce diapause eggs even when injected at a high dose of 10 nmol/pupa. Whereas, injection of a mixture of VAP-peptide and DH clearly decreased a half-maximum dose (ED50 value) and a threshold dose (TD value) of DH, and these values decreased according to increasing molar ratios of VAP-peptide to DH. Thus, the VAP-peptide is concluded to be an endogenous protein acting as a potent enhancer of DH activity through interaction with DH.

Amino Acid Sequence↗
PubMed2000

Structure of the VAP-peptide (BmACP-6.7) gene in the silkworm, Bombyx mori and a possible regulation of its expression by BmFTZ-F1.

The VAP-peptide (BmACP-6.7) is a hydrophobic peptide localized in adult cuticle of the silkworm, Bombyx mori. We isolated and characterized the VAP-peptide gene as a useful marker gene to analyze molecular mechanisms of terminal differentiation processes in the adult. The gene is composed of two exons interrupted by one intron. The 5' upstream promoter region is shown to bear a nucleotide sequence similar to the cis-element that is recognized and bound by the Bombyx mori FTZ-F1 protein (BmFTZ-F1). Expression of the BmFTZ-F1 gene preceded expression of the VAP-peptide gene and injection of 20-hydroxyecdysone suppressed the expression of both genes. An in vitro binding assay indicated direct interaction of BmFTZ-F1 with the VAP-peptide gene promoter sequence. Therefore, BmFTZ-F1 is proposed to be a possible factor regulating the stage-specific expression of the VAP-peptide gene towards adult life.

Amino Acid Sequence↗
PubMed2000

Isolation of a cDNA encoding a CHH-family peptide from the silkworm Bombyx mori.

The crustacean hyperglycemic hormone (CHH) peptide family includes four types of neuropeptide in decapod and isopod crustaceans, and the ion-transport peptide in orthopteran insects. To identify a new member of this family in Insecta, a PCR-based search for cDNAs encoding CHH-family peptides was carried out in the silkworm Bombyx mori. A cDNA, named BmCHHL (Bombyx mori CHH-like protein), with an open reading frame of 110 amino acids was isolated. Sequence analyses suggested that the conceptual protein was a precursor of a peptide of 72 amino acids which was amidated at the carboxy terminus. The BmCHHL sequence exhibited significant similarities to members of the CHH family including the orthopteran ion-transport peptide. BmCHHL expression was detected in five or six cells (per hemisphere) in the frontal area of the brain in day 4 fifth instar larvae.

Amino Acid Sequence↗
PubMed2002

Photoinactivation of vesicular stomatitis virus by a photodynamic agent, chlorophyll derivatives from silkworm excreta.

The efficacy of chlorophyll derivatives from silkworm excreta (CpD) in photodynamic antimicrobial chemotherapy (PACT) was studied. An enveloped animal virus, vesicular stomatitis virus (VSV), was used as a target organism. For CpD mediated PACT, the viruses in suspensions were treated with various doses of CpD (15-60 microg/ml) and visible red light was fixed at 120 mJ/cm(2). The antiviral effect of the CpD-PACT was measured 1 h after light irradiation by the extent of suppression of plaque forming units (pfu). In cultures inoculated with PACT-treated VSV, suppression of pfu was prominent and the results were demonstrated in a dose-dependent manner. In assays of RT-PCR, a single dose of 30 microg/ml CpD and light caused complete inhibition of viral RNA synthesis in the host cells, which agreed with the complete loss of plaque forming activity observed in pfu assays. An in vitro transcription assay for viral RNA using [3H]UTP and gel electrophoresis for the level of M protein was conducted. A gradual decrease in viral RNA transcription and an immediate decrease in M protein levels were observed in cells inoculated with the CpD-PACT-treated virus. These results demonstrated that CpD is a potential photodynamic antiviral agent, which causes inactivation of the matrix protein as well as transcription mechanisms involved in VSV replication.

Animals↗
PubMed2000

Sexual attraction in the silkworm moth: structure of the pheromone-binding-protein-bombykol complex.

BACKGROUND: Insects use volatile organic molecules to communicate messages with remarkable sensitivity and specificity. In one of the most studied systems, female silkworm moths (Bombyx mori) attract male mates with the pheromone bombykol, a volatile 16-carbon alcohol. In the male moth's antennae, a pheromone-binding protein conveys bombykol to a membrane-bound receptor on a nerve cell. The structure of the pheromone-binding protein, its binding and recognition of bombykol, and its full role in signal transduction are not known. RESULTS: The three-dimensional structure of the B. mori pheromone-binding protein with bound bombykol has been determined by X-ray diffraction at 1.8 A resolution. CONCLUSIONS: The pheromone binding protein of B. mori has six helices, and bombykol binds in a completely enclosed hydrophobic cavity formed by four antiparallel helices. Bombykol is bound in this cavity through numerous hydrophobic interactions, and sequence alignments suggest critical residues for specific pheromone binding.

Amino Acid Sequence↗
PubMed2001

A homologue of the Drosophila doublesex gene is transcribed into sex-specific mRNA isoforms in the silkworm, Bombyx mori.

The doublesex (dsx) gene is known as the final gene of the sex-determining cascade in Drosophila melanogaster. We have isolated a homologue of dsx in the silkworm, Bombyx mori, which has an epistatic feminizing gene located on the W chromosome. RT-PCR analysis indicated that B. mori dsx (Bmdsx) was transcribed in all the examined tissues, and the size of the amplified products was different between males and females. In Northern blot hybridization of poly(A)(+) RNA, the Bmdsx probe also detected a band with a sex-specific size difference. The male-specific cDNA lacked the sequence between 713 and 961nt of the female-specific cDNA. An RNase protection assay indicated that this sequence was male-specifically removed from the Bmdsx pre-mRNA. Southern blot analysis showed that Bmdsx is present at a single copy in the genome. These results suggested that the primary Bmdsx transcript is alternatively spliced to yield male- and female-specific mRNA isoforms. These sex-specific isoforms encode polypeptides with a common amino-terminal sequence but sex-specific carboxyl termini. DNA binding domain (DM domain) of BmDSX has 80% identity with D. melanogaster DSX proteins. These results suggest the Bmdsx would also regulate sexual differentiation, as does the Drosophila dsx gene.

Alternative Splicing↗
PubMed2001

Differential expression of the fibroin gene in developmental stages of silkworm, Antheraea mylitta (Saturniidae).

Fibroin gene expression during the larval developmental stages of the Saturniid silkworm, Antheraea mylitta, was analyzed. Northern blot analysis of larval silk gland total RNA using the fibroin gene as a probe showed that fibroin is expressed in the intermoult stages and repressed during the moulting stages. Abundance of fibroin transcripts gradually increased from the third to fifth intermoult stage, reaching a peak in the fifth intermoult. Transcripts declined during the early spinning stage. Western blot analysis of fibroin protein production with anti-fibroin antibody confirmed the differential fibroin expression, in accordance with fibroin mRNA synthesis. Dot blot hybridization of genomic DNA isolated from each larval developmental stage with the labelled fibroin gene showed that at the genomic level, the relative concentration of the fibroin gene was constant throughout the developmental stages. Our data confirm that fibroin gene expression in A. mylitta, like in B. mori, is transcriptionally controlled and shows differential temporal variations.

Animals↗
PubMed2003

Exogenous prostaglandin F2alpha as a mediator in the regulation of silkworm growth and silk gland genome.

Prostaglandins are locally acting hormones that have remarkable variety of physiological functions. They are rapidly synthesized in several types of vertebrate cells as oxygenated metabolites of arachidonic acid in response to various stimuli. In many insect species they are biosynthesized in fat body and hemocytes mainly in response to bacterial infections. In the present study, we administered synthetic analog of prostaglandin F2alpha, the most prominent of the prostaglandins to the 48 h old fifth instar silkworm, Bombyx mori L. at a single dose of 4 microg per larva to study its effects on the larval growth pattern and silk synthesis. The possible role of PGF2alpha at altering the quantum of silk synthesis by controlling the silk gene expression was also studied. The genomic DNA was isolated from the posterior silk gland on Days 5 and 7 of the fifth instar from the prostaglandin treated and the control larvae and were random amplified with arbitrary primers. The result presented notable variation in the amplified product suggesting the participation of PGF2alpha in the silk biosynthesis controlling the silk gene expression. The feeding period of treated larvae was unaffected while the cocoon characters exhibited considerable improvement. The filament traits also were improved notably in the treated larvae. The participation of PGF2alpha analog in the silk biosynthetic process with its physiological and molecular implications are discussed.

Animals↗
PubMed1994

Assignment of disulfide bond location in prothoracicotropic hormone of the silkworm, Bombyx mori: a homodimeric peptide.

The disulfide bond location of a homodimeric peptide, prothoracicotropic hormone (PTTH) of the silkworm, Bombyx mori, was determined by a combination of partial reduction and sequence analysis of peptide fragments generated through a partial reduction of PTTH followed by alkylation and enzyme digestion. The partial reduction and S-alkylation broke the interchain disulfide bond but did not affect the intrachain disulfide bonds, generating monomeric PTTH whose intrachain disulfide bonds were kept intact. This monomeric PTTH has about one-half the biological activity of intact PTTH. Sequence analysis of the fragments generated by lysyl endopeptidase digestion of this monomeric PTTH after complete reduction and S-alkylation by another S-alkylating reagent showed that only the Cys15 residue was reduced and S-alkylated by the foregoing partial reduction, indicating that this residue formed the interchain disulfide bond. The other disulfide bonds which formed intrachain bridgings were determined by sequence and mass analyses of the fragments generated by two successive enzyme digestions of the monomeric PTTH. In conclusion, the disulfide bond location of PTTH was assigned to Cys15-Cys15' as an interchain disulfide linkage and Cys17-Cys54, Cys40-Cys96, and Cys48-Cys98 as intrachain disulfide linkages.

Amino Acid Sequence↗
PubMedDate not supplied

Rheology and dynamic light scattering of silk fibroin solution extracted from the middle division of Bombyx mori silkworm.

Dynamic light scattering (DLS) and rheological measurements were performed on aqueous silk fibroin solutions extracted from the middle division of Bombyx mori silkworm over a wide range of polymer concentration C from 0.08 to 27.5 wt %. DLS results obtained in the dilute region of C less than 1 wt % are consistent with a model that an elementary unit is a large protein complex consisting of silk fibroin and P25 with a 6:1 molar ratio. Rheological measurements in the dilute C region reveal that those units (or clusters) with the hydrodynamic radius of about 100 nm form a network extending over the whole sample volume with small pseudoplateau modulus mainly by ionic bonding between COO(-) ions of the fibroin molecules and divalent metallic ions such as Ca(2+) or Mg(2+) ions present in the sample and also that, after a yield stress is reached, steady plastic flow is induced with viscosity much lower than the zero-shear viscosity estimated from creep and creep recovery measurements by 4-6 orders of magnitude. Angular frequency omega dependencies of the storage and the loss shear moduli, G'(omega) and G' '(omega), measured in the linear viscoelastic region, indicate that all solutions possess the pseudoplateau modulus in the low omega region and samples become highly viscoleastic for C greater, similar 4.2 wt %. Above C = 11.2 wt % another plateau appears at the high omega end accompanied by a distinct maximum of G' ' in the intermediate omega region. The relaxation motion with tau = 0.5 s corresponding to the maximum of G' ' is one of characteristic properties of the fibroin solutions in the high C region. Thermorheological behaviors of the solution with C = 27.5 wt % show that the network structure formed in the MM part of the silk gland is susceptible to temperature and a more stable homogeneous network is realized by raising the temperature up to T = 65 degrees C.

Animals↗