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PubMed2005

Responses of the silkworm tyramine receptor to 2-phenylethylamines and 5-phenyloxazoles.

Tyramine (TA), a biogenic amine, attenuates intracellular cAMP production by acting on its receptor in insects. Several non-biogenic amines were examined for their actions on native and heterologously expressed silkworm TA receptors. 5-(4-Hydroxyphenyl)oxazole, which showed an attenuating effect on cAMP production in silkworm-head membranes, did not attenuate forskolin-stimulated cAMP production in HEK-293 cells expressing the silkworm TA receptor, although the compound bound to the cloned receptor. 2-Phenylethylamines (2-PEAs), which showed positive and negative effects on cAMP production in silkworm-head membranes, inhibited [3H]TA binding to the cloned TA receptor. 2-Chloro-2-(4-chlorophenyl)ethylamine was the most potent inhibitor of [3H]TA binding among the 2-PEAs tested, with an IC50 of 30.4 nM. This compound acted as an antagonist and abolished TA-attenuation of forskolin-stimulated cAMP production in the cloned TA receptor. The discrepancy in the effects of the non-biogenic amines on the native and cloned TA receptors remains to be further examined. A newly synthesized 2-PEA, 2-chloro-2-(4-hydroxyphenyl)ethylamine, attenuated forskolin-stimulated cAMP production in the cloned TA receptor, indicating that the para-hydroxy group is important for the agonist action.

Animals↗
PubMed2002

Silkworm larvae as an animal model of bacterial infection pathogenic to humans.

Silkworm larvae, Bombyx mori, were examined as an animal model of human infection with pathogenic bacteria. When 3 x 10(7) cells of Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa, or Vibrio cholerae were injected into the blood of fifth instar silkworm larvae, over 90% of the larvae died within 2 days, whereas over 90% survived for 5 days after injection of the same amount of Escherichia coli. Growth of S. aureus was observed in larvae blood and tissues. Immunostaining analysis revealed that S. aureus proliferated at the surface of the midgut. Infection of silkworm larvae by methicillin-sensitive S. aureus was cured by ampicillin, oxacillin, and vancomycin, whereas infection by methicillin-resistant S. aureus was not cured by ampicillin or oxacillin, although vancomycin was effective. Disinfectants were not effective because of toxicity against the larvae. Thus, silkworm larvae are useful for evaluating antibiotics for pathogenic bacterial infection in humans.

Animals↗
PubMed2006

A new technique for producing recombinant baculovirus directly in silkworm larvae.

A new technique for the direct production of recombinant baculovirus in the silkworm larvae is described. To assess the utility of this method, a combination of Bombyx mori nucleopolyhedroviral genome, transfer vector and Lipofectin was co-injected directly into newly ecdysed fifth instar, silkworm larvae. The recombinant virus was obtained from the hemolymph of injected larvae and the hemolymph then re-injected into the larvae as an inoculum. This resulted in a high-level production of foreign protein in the silkworm larvae. This technique produces easy and rapid recombinant protein production in silkworms.

Animals↗
PubMedDate not supplied

Silkworm diapause induction activity of myotropic pyrokinin (FXPRLamide) insect neuropeptides.

A family of myotropic neuropeptides sharing the common C-terminal pentapeptide Phe-Xxx-Pro-Arg-Leu-NH2 (Xxx = Ser, Thr, Val), known as the pyrokinins, has been isolated from the cockroach Leucophaea maderae and locust Locusta migratoria of the order Orthoptera. A hormone (Bom-DH) that elicits diapause induction in the silkworm Bombyx mori (order Lepidoptera) also contains this C-terminal pentapeptide (Xxx = Gly). The orthopteran pyrokinin neuropeptides elicit significant diapause-inducing activity in the lepidopteran silkworm. Despite containing the sterically bulky, inflexible Val residue in the variable Xxx position, the locust pyrokinin Lom-PK is threefold more active than native Bom-DH as a diapause induction agent. The C-terminally truncated cockroach leucopyrokinin (LPK) fragment, Thr-Ser-Phe-Thr-Pro-Arg-NH2 [LPK(2-7)], proved virtually inactive in the silkworm assay, demonstrating the importance of an intact C-terminal pentapeptide sequence to diapause induction activity. Bom-DH also elicits significant myostimulatory activity in a cockroach hindgut assay, although at a level several orders of magnitude less than the native myotropic peptide LPK. However, the C-terminal pentapeptide of Bom-DH (Xxx = Gly) is equipotent with the LPK C-terminal pentapeptide (Xxx = Thr) as a myostimulatory agent. The cross-activity observed for the various pyrokinins suggests that the receptors that mediate the disparate physiological processes of diapause in the silkworm and hindgut contraction in the cockroach share homologous features.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Fluoride effects on the mulberry-silkworm system.

Absorption and accumulation of fluoride (F) from ambient air by mulberry leaves, and its transfer and effects on silkworm development, were examined. When the concentration of F in air exceeded 1.5 microg dm(-2). day (-1), using lime filter papers as static monitors, the F content of mulbery leaves was more than 30 ppm, the threshold for injury to silkworm larvae. Fluoride-polluted mulberry leaves inhibited growth and development of silkworms. Leaves containing more than 80 ppm F severely inhibited cocoon production. Fluoride was absorbed from the atmosphere by the mulberry leaf and was transferred to the silkworm, soil, water, and back to the atmosphere, forming a cycle.

Journal Article↗
PubMed1995

Melatonin and arylalkylamine N-acetyltransferase activity in the silkworm, Bombyx mori.

Melatonin (N-acetyl-5-methoxytryptamine) was identified in the head and hemolymph of the silkworm, Bombyx mori, using reversed-phase high-performance liquid chromatography coupled with fluorometric detection and radioimmunoassay. In addition, evidence of arylakylamine (serotonin) N-acetyltransferase (NAT) a key enzyme controlling the synthesis of melatonin in vertebrates, was found in the head of the silkworm. Melatonin levels in the head and hemolymph and the NAT activity in the head were significantly higher during the dark period than during the light period of a 12-h light/12-h dark cycle. The day-night changes persisted in constant darkness but were suppressed by constant light. The results suggest that the synthesis and release of melatonin in the silkworm head occur as a circadian rhythm that is entrained by environmental light/dark cycles, as it is in the pineal gland of vertebrates. Melatonin in the silkworm head may function as a neurochemical mediator of photoperiodic control of developmental events such as molting, eclosion and diapause.

Animals↗
PubMed2006

Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin.

In this study we produced germline transgenic silkworms that spin cocoons containing recombinant human serum albumin (rHSA) in the sericin layer. A piggyBac-based transformation vector was constructed that carried HSA cDNA driven by sericin-1 gene promoter, viral enhancer hr3, and gene encoding viral trans-activator IE1. Isolated silk glands were bombarded with the vector and transplanted into host larvae. Three days later, the transplants were immunohistochemically analyzed, which showed that middle silk gland (MSG) cells expressed rHSA and secreted it into the MSG lumen. Then, silkworm eggs were injected with the vector and developed to larvae. The obtained transgenic silkworms spun silk threads whose sericin layers contained rHSA at 3.0microg/mg of cocoons. Most (83%) of the rHSA in cocoons was extracted with phosphate buffered saline, which was then subjected to ammonium sulfate precipitation and affinity chromatography. Finally, we obtained 2.8mg of 99%-pure rHSA from 2g of cocoons. Measurements of circular dichroism spectra of rHSA, and equilibrium dissociation constants of rHSA to warfarin and naproxen indicated that rHSA was conformationally and functionally identical to natural plasma HSA. Germline transgenic silkworms will be useful for producing various recombinant proteins in the sericin layer of cocoons.

Animals↗
PubMed2002

Silkworm hemolymph as a potent inhibitor of apoptosis in Sf9 cells.

We have previously shown that silkworm hemolymph exhibits anti-apoptotic activity against baculovirus-induced Sf9 cell apoptosis. In this study, using various chemical inducers, such as actinomycin D, camptothecin, and staurosporine, we found that silkworm hemolymph inhibits insect cell apoptosis induced not only by baculovirus but also by chemical inducers. This indicates that silkworm hemolymph contains anti-apoptotic components that work directly in insect cell apoptosis without any booster expression of baculoviral genes. With the analysis of Sf-caspase-1 activity, it was found that the inhibitory effect of silkworm hemolymph works in a further upstream step than the Sf-caspase-1 activation step.

Animals↗
PubMed1997

Hydroxyindole-O-methyltransferase activity in the silkworm (Bombyx mori).

Hydroxyindole-O-methyltransferase (HIOMT; EC 2.1.1.4) catalyzes the final step in the melatonin (N-acetyl-5-methoxytryptamine) biosynthetic pathway. HIOMT-like activity was detected in the head of fifth last-instar larvae of the silkworm (Bombyx mori), and the optimum pH for this activity was 7.9. The apparent Michaelis constants (K(m)) for S-adenosyl-L-methionine and N-acetylserotonin were 87.6 microM and 96.6 microM, respectively. When the silkworms were entrained to a 12 h light/12 h dark lighting schedule, the HIOMT-like activity showed a significant diurnal variation with high levels during the dark period. The diurnal variation in the activity persisted in constant darkness, but was suppressed by constant light. These findings demonstrate that HIOMT-like activity in the silkworm head occurs as a circadian rhythm and that the rhythm entrains to environmental light/dark cycles. The melatonin rhythm in the silkworm head may therefore be regulated not only by serotonin N-acetyltransferase (EC 2.3.1.87), but also by HIOMT.

Acetylserotonin O-Methyltransferase↗
PubMed1986

Carbohydrate-binding specificity of silkworm lectin.

The binding specificity of a lectin from the hemolymph of silkworm larvae was examined quantitatively by taking advantage of the fluorospectrophotometric properties of the lectin. On excitation at 280 nm, the lectin fraction gave a fluorescence-emission spectrum centered at 336 nm, which was attributable to tryptophan residues. The fluorescence could be completely quenched by the addition of specific saccharides. The affinity constants of the silkworm lectin with specific saccharides were calculated from the changes in intensities of fluorescence-difference spectra induced by the saccharides. The silkworm lectin had the highest affinity for dermatan sulfate and hyaluronic acid, followed by protuberic acid, heparin, and chondroitin sulfate A. Among monosaccharides tested, only D-glucuronic acid and N-acetyl-neuraminic acid induced weak but significant quenching, and their affinity constants were found to be low. These results indicate that the silkworm lectin has a strong affinity for carboxyl groups, especially alpha-L-iduronic acid residues, in the saccharides. In most cases, sulfate groups on the saccharides interfere with the specific interactions.

Animals↗
PubMed2003

A new method of pebrine inspection of silkworm egg using multiprimer PCR.

Using a mixture of several PCR primers, we evaluated whether multiprimer PCR is practically useful for the early and simultaneous detection of several kinds of microsporidia that cause silkworm pebrine. When genomic DNA extracted from silkworm eggs infected with Nosema bombycis was used as the DNA template, the specific DNA sequences were amplified by multiprimer PCR. In addition, similar results were obtained even when genomic DNA extracted from silkworms infected with N. bombycis was used as the DNA template. These findings suggest that multiprimer PCR using several primers designed for this study is practically useful for pebrine inspection of silkworm eggs.

Animals↗
PubMed2003

Effect of prostaglandin F2alpha on gonadal carbohydrate metabolism of silkworm, Bombyx mori L.

Effect of prostaglandin F2alpha (PGF2alpha) on carbohydrate accumulation in gonads of the multivoltine silkworm (hybrid: Xinhang x Keming) has been studied by means of topical application to larvae of the silkworm. Increased weights of larvae and reproduction organs, as well as carbohydrate metabolism in gonads of the silkworm, Bombyx mori L. was found after treatment with prostaglandin F2alpha. The increase in weight (larvae 21.9%, testis 28.9%, and ovary 33.3%) was associated with increases in the biomolecules (20-30%) and LDH and aldolase activity (18-25%). The results suggest that the accumulation of carbohydrates denotes a higher extent of utility of the energy sources in function of the testes and ovaries, and the routine application of prostaglandin F2alpha would be helpful in improving the reproductivity and egg quality of the silkworm.

Animals↗
PubMed2005

Genetic mapping of Z chromosome and identification of W chromosome-specific markers in the silkworm, Bombyx mori.

In the silkworm, Bombyx mori, the female is the heterogametic (ZW) sex and the male is homogametic (ZZ). The female heterogamety is a typical situation in the insect order Lepidoptera. Although the W chromosome in silkworm is strongly female determining, no W-linked gene for a morphological character has been found on it. The Z chromosome carries important traits of economic value as well as genes for various phenotypic traits, but only 2% of molecular information based on its relative size is known. Studies conducted so far indicate that the Z-linked genes are not dosage compensated. In the present study, we constructed a genetic map of randomly amplified polymorphic DNA fragments (RAPD), simple sequence repeats (SSR), and fluorescent intersimple sequence repeat PCR (FISSR) markers for the Z chromosome using a backcross mapping population. A total of 16 Z-linked markers were identified, characterized, and mapped using od, a recessive trait for translucent skin as an anchor marker yielding a total recombination map of 334.5 cM. The linkage distances obtained suggested that the markers were distributed throughout the Z chromosome. Four RAPD and four SSR markers that were linked to W chromosome were also identified. The proposed mapping approach should be useful to identify and map sex-linked traits in the silkworm. The economic and evolutionary significance of Z- and W-linked genes in silkworm, in particular, and lepidopterans, in general, is discussed.

Animals↗
PubMed2001

Comparison of multilocus RFLPs and PCR-based marker systems for genetic analysis of the silkworm, Bombyx mori.

The utility of multilocus RFLPs and three PCR-based techniques, Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeat-PCR (ISSR-PCR) and simple sequence repeats (SSRs) for genetic characterization was examined using 13 diverse silkworm strains. All four approaches successfully discriminated the 13 silkworm varieties but differed in the amount of polymorphism detected. The usefulness of each system was examined in terms of number of loci revealed (effective multiplex ratio, EMR) and the amount of polymorphism detected (diversity index, DI). For example, the six multilocus RFLP probes produced 180 products of which 97% were polymorphic; 15 SSR loci gave rise to an average of 8 alleles each, of which 86% were polymorphic. The ISSR-PCR produced 39 fragments of which 76.98% were polymorphic. The highest diversity index was observed for ISSR-PCR (0.957) and the lowest for RAPDs (0.744). The RAPD, ISSR-PCR and RFLP assays clearly separated the diapausing and non-diapausing silkworm varieties. These results are discussed in terms of choice of appropriate marker technology for different aspects of silkworm genome analysis.

Animals↗
PubMed2004

Introduction of foreign genes into silkworm eggs by electroporation and its application in transgenic vector test.

Electroporation as a methodology to introduce foreign genes into silkworm eggs was systematically analyzed. The foreign gene in both the newly hatched and 3rd instar larva DNA can be detected by PCR. The amount of foreign gene in 3rd instar larva DNA was about 1/1000 of that in newly hatched larva DNA. The ratio of foreign gene entering into silkworm eggs was voltage dependent and showed significant difference between the tested silkworm strains. When the piggyBac transposon system was applied, the effect of nuclear localization signal (NLS) peptide and the in vitro transcribed transposase mRNA on the transposition rate has been measured. Results showed that the in vitro transcribed transposase mRNA facilitated transposition to take place earlier and NLS could result in higher transposition probability and earlier transposition as well. When linearized vectors containing varied length of flanking homologous sequences around a reporter gene were introduced into silkworm eggs by electroporation, the one with 2.6 kb total arm length gave higher G1 positive ratio than that with total arm length of 1.5 kb and 800 bp.

Animals↗
PubMed2005

D-Glutamic acid-induced muscle contraction in the silkworm, Bombyx mori.

Agonists for muscle contraction in silkworms were screened by injecting test solutions into the hemolymph of decapitated silkworm larvae. Kainic acid, a glutamate receptor agonist, and D-glutamic acid induced muscle contractions, and D-aspartic acid was partially effective, whereas NMDA and AMPA, representative mammalian glutamate receptor agonists, did not induce contraction. L-Glutamic acid inhibited the kainic acid or D-glutamic acid-induced contraction. Amino acid analysis revealed that 3% of the total glutamic acid in the silkworm hemolymph is D-glutamic acid. These results suggest that d-glutamic acid acts physiologically as an agonist for muscle contraction in silkworms, and that L-glutamic acid functions as an inhibitor.

Animals↗
PubMed2006

Use of silkworm larvae to study pathogenic bacterial toxins.

Injection of stationary phase culture-supernatants of Staphylococcus aureus and Pseudomonas aeruginosa into the hemolymph of silkworm larvae caused their death, whereas a culture-supernatant of a non-pathogenic strain of Escherichia coli did not. A culture-supernatant of a mutant of agr, a global virulence regulator of S. aureus that is required for exotoxin production, was much less toxic to silkworm larvae. A culture-supernatant of a disruption mutant of the S. aureus beta-toxin gene did not kill larvae, whereas one of a deletion mutant of alpha-toxin, gamma-toxin, or aureolysin killed larvae, indicating that the beta-toxin gene is required for staphylococcal supernatant-mediated killing of silkworm larvae. The 50% lethal doses (LD50) of staphylococcal alpha-toxin and beta-toxin, Pseudomonas exotoxin A and diphtheria toxin were 12 microg/g, 9 microg/g, 0.14 microg/g and 1.1 microg/g, respectively. As the purified toxins killed the larvae, silkworm larvae could be used as a model to study the actions of pathogenic bacterial toxins in animal bodies.

Animals↗
PubMed2002

In vivo expression and immunological studies of the 42-kilodalton carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 in the baculovirus-silkworm system.

The 42-kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-1(42)) is an anti-erythrocytic stage malaria vaccine candidate. In this study, MSP-1(42) was expressed by using the Bombyx mori nuclear polyhedrosis virus-silkworm expression system, and the antigenicity and immmunogenicity of the recombinant protein, Bmp42, were evaluated. The average yield of Bmp42, as determined by a sandwich enzyme-linked immunosorbent assay (ELISA), was 379 microg/ml of infected silkworm hemolymph, which was >100-fold higher than the level attainable in cell culture medium. N-terminal amino acid sequencing revealed that Bmp42 was correctly processed in silkworm cells. Data from immunoblotting, as well as from the inhibition ELISA, suggested that the conformational B-cell epitopes of MSP-1(42) were recreated in Bmp42. Immunization of rabbits with Bmp42 in complete Freund's adjuvant generated high-titer antibody responses against the immunogen. Specificity analyses of the anti-Bmp42 antibodies using several recombinant MSP-1(19) proteins expressing variant and conserved B-cell epitopes suggested that the anti-Bmp42 antibodies recognized primarily conserved epitopes on MSP-1(19). Furthermore, the anti-Bmp42 antibodies were highly effective in inhibiting the in vitro growth of parasites carrying homologous or heterologous MSP-1(42). Our results demonstrated that the baculovirus-silkworm expression system could be employed to express biologically and immunologically active recombinant MSP-1(42) at elevated levels; thus, it is an attractive alternative for producing a protective MSP-1(42) vaccine for human use.

Animals↗