A possible mechanism for low affinity of silkworm Na+/K+-ATPase for K.

The affinity for K+ of silkworm nerve Na+/K+-ATPase is markedly lower than that of mammalian Na+/K+-ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K+ affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na+/K+-ATPase α and ß subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and ß subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na+/K+-ATPase α1 subunit. On the other hand, the amino acid identity of the ß subunit with mammalian counterparts was as low as 30%. Cloned α and ß cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na+/K+-ATPase. Na+/K+-ATPase expressed in the cultured cells showed a low affinity for K+ and a high affinity for Na+, characteristic of the silkworm nerve Na+/K+-ATPase. These results suggest that the ß subunit is responsible for the affinity for K+ of Na+/K+-ATPase.

Clone and functional analysis of Seryl-tRNA synthetase and Tyrosyl-tRNA synthetase from silkworm, Bombyx mori.

Aminoacyl-tRNA synthetases are the key enzymes for protein synthesis. Glycine, alanine, serine and tyrosine are the major amino acids composing fibroin of silkworm. Among them, the genes of alanyl-tRNA synthetase (AlaRS) and glycyl-tRNA synthetase (GlyRS) have been cloned. In this study, the seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) genes from silkworm were cloned. Their full length are 1709 bp and 1868 bp and contain open reading frame (ORF) of 1485 bp and 1575 bp, respectively. RT-PCR examination showed that the transcription levels of SerRS, TyrRS, AlaRS and GlyRS are significantly higher in silk gland than in other tissues. In addition, their transcription levels are much higher in middle and posterior silk gland than in anterior silk gland. Moreover, treatment of silkworms with phoxim, an inhibitor of silk protein synthesis, but not TiO2 NP, an enhancer of silk protein synthesis, significantly reduced the transcription levels of aaRS and content of free amino acids in posterior silk gland, therefore affecting silk protein synthesis, which may be the mechanism of phoxim-silking disorders. Furthermore, low concentration of TiO2 NPs showed no effect on the transcription of aaRS and content of free amino acids, suggesting that TiO2 NPs promotes silk protein synthesis possibly by increasing the activity of fibroin synthase in silkworm.

Identification and characterization of an atypical 2-cys peroxiredoxin from the silkworm, Bombyx mori.

Peroxiredoxins (Prxs) play an important role in the protection of insects against the toxicity of reactive oxygen species. Here, we identified and characterized a novel, atypical 2-cysteine (Cys) peroxiredoxin (BmPrx3) from an expressed sequence tag database in a lepidopteran insect, Bombyx mori. The BmPrx3 cDNA contained an open reading frame of 684 bp that encodes a 228-amino-acid protein with a calculated molecular mass of 25 kDa. Sequence comparison revealed that BmPrx3 belongs to the atypical 2-Cys Prxs. Quantitative real-time PCR revealed that BmPrx3 can be detected in all tissues and developmental stages. Recombinant BmPrx3 purified from Escherichia coli exhibited antioxidant activity that removed hydrogen peroxide and protected DNA from oxidative damage. Disc diffusion and viability assays revealed that recombinant BmPrx3 increased bacterial survival under H2 O2 -mediated oxidative stress. In addition, quantitative real-time PCR analysis indicated that BmPrx3 transcription levels were significantly increased in response to various oxidative stresses. Furthermore, BmPrx3 transcription levels in the midgut were regulated by bacterial infection. Taken together, these results suggest that BmPrx3 acts as an antioxidant enzyme to protect the silkworm from various oxidative stresses.

Plant phenolics are detoxified by prophenoloxidase in the insect gut.

Plant phenolics are a group of important secondary metabolites that are toxic to many animals and insects if ingested at high concentrations. Because most insects consume plant phenolics daily, they have likely evolved the capacity to detoxify these compounds. Here, we used Drosophila melanogaster, Bombyx mori and Helicoverpa armigera as models to study the metabolism of plant phenolics by prophenoloxidases. We found that insect foreguts release prophenoloxidases into the lumen, and that the survival of prophenoloxidase-deletion mutants was impaired when fed several plant phenolics and tea extracts. Using l-DOPA as a model substrate, biochemical assays in large Lepidopteran insects demonstrated that low levels of l-DOPA are rapidly metabolized into intermediates by phenoloxidases. Feeding with excess l-DOPA showed that the metabolic intermediate 5,6-dihydroxyindole reached the hindgut either by passing directly through the midgut, or by transport through the hemolymph. In the hindgut, 5,6-dihydroxyindole was further oxidized by prophenoloxidases. Intermediates exerted no toxicity in the hemocoel or midgut. These results show that plant phenolics are not toxic to insects unless prophenoloxidase genes are lost or the levels of phenolics exceed the catalytic activity of the gut prophenoloxidases.

Effect of Venom from the Jellyfish Nemopilema nomurai on the Silkworm Bombyx mori L.

The silkworm Bombyx mori L. (B. mori) has a significant impact on the economy by producing more than 80% of the globally produced raw silk. The exposure of silkworm to pesticides may cause adverse effects on B. mori, such as a reduction in the production and quality of silk. This study aims to assay the effect of venom from the jellyfish Nemopilema nomurai on growth, cuticle and acetylcholinesterase (AChE) activity of the silkworm B. mori by the leaf dipping method. The experimental results revealed that the four samples caused neither antifeeding nor a lethal effect on B. mori. The sample SFV inhibited B. mori growth after 6 days of treatment in a dose-dependent manner. The samples SFV, DSFV and Fr-1 inhibited the precipitation and synthesis of chitin in the cuticle after 12 and 14 days of treatment. In the case of the four samples, the AChE was significantly improved after 14 days of treatment.

Comparative analyses of the cholinergic locus of ChAT and VAChT and its expression in the silkworm Bombyx mori.

The cholinergic locus, which encodes choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), is specifically expressed in cholinergic neurons, maintaining the cholinergic phenotype. The organization of the locus is conserved in Bilateria. Here we examined the structure of cholinergic locus and cDNA coding for ChAT and VAChT in the silkworm, Bombyx mori. The B. mori ChAT (BmChAT) cDNA encodes a deduced polypeptide including a putative choline/carnitine O-acyltransferase domain and a conserved His residue required for catalysis. The B. mori VAChT (BmVAChT) cDNA encodes a polypeptide including a putative major facilitator superfamily domain and 10 putative transmembrane domains. BmChAT and BmVAChT cDNAs share the 5'-region corresponding to the first and second exon of cholinergic locus. Polymerase chain reaction analyses revealed that BmChAT and BmVAChT mRNAs were specifically expressed in the brain and segmental ganglia. The expression of BmChAT was detected 3 days after oviposition. The expression level was almost constant during the larval stage, decreased in the early pupal stage, and increased toward eclosion. The average ratios of BmChAT mRNA to BmVAChT mRNA in brain-subesophageal ganglion complexes were 0.54±0.10 in the larvae and 1.92±0.11 in adults. In addition, we examined promoter activity of the cholinergic locus and localization of cholinergic neurons, using a baculovirus-mediated gene transfer system. The promoter sequence, located 2kb upstream from the start of transcription, was essential for cholinergic neuron-specific gene õexpression. Cholinergic neurons were found in several regions of the brain and segmental ganglia in the larvae and pharate adults.

Molecular cloning and functional characterization of the dual oxidase (BmDuox) gene from the silkworm Bombyx mori.

Reactive oxygen species (ROS) from nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and their related dual oxidases are known to have significant roles in innate immunity and cell proliferation. In this study, the 5,545 bp cDNA of the silkworm Bombyx mori dual oxidase (BmDuox) gene containing a full-length open reading frame was cloned. It was shown to include an N-terminal signal peptide consisting of 28 amino acid residues, a 240 bp 5'-terminal untranslated region (5'-UTR), an 802 bp 3'-terminal region (3'-UTR), which contains nine ATTTA motifs, and a 4,503 bp open reading frame encoding a polypeptide of 1,500 amino acid residues. Structural analysis indicated that BmDuox contains a typical peroxidase domain at the N-terminus followed by a calcium-binding domain, a ferric-reducing domain, six transmembrane regions and binding domains for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD). Transcriptional analysis revealed that BmDuox mRNA was expressed more highly in the head, testis and trachea compared to the midgut, hemocyte, Malpighian tube, ovary, fat bodies and silk glands. BmDuox mRNA was expressed during all the developmental stages of the silkworm. Subcellular localization revealed that BmDoux was present mainly in the periphery of the cells. Some cytoplasmic staining was detected, with rare signals in the nucleus. Expression of BmDuox was induced significantly in the larval midgut upon challenge by Escherichia coli and Bombyx mori nucleopolyhedrovirus (BmNPV). BmDuox-deleted larvae showed a marked increase in microbial proliferation in the midgut after ingestion of fluorescence-labeled bacteria compared to the control. We conclude that reducing BmDuox expression greatly increased the bacterial load, suggesting BmDuox has an important role in inhibiting microbial proliferation and the maintenance of homeostasis in the silkworm midgut.

Evaluation of drug-induced tissue injury by measuring alanine aminotransferase (ALT) activity in silkworm hemolymph.

BACKGROUND: Our previous studies suggest silkworms can be used as model animals instead of mammals in pharmacologic studies to develop novel therapeutic medicines. We examined the usefulness of the silkworm larvae Bombyx mori as an animal model for evaluating tissue injury induced by various cytotoxic drugs. Drugs that induce hepatotoxic effects in mammals were injected into the silkworm hemocoel, and alanine aminotransferase (ALT) activity was measured in the hemolymph 1 day later. RESULTS: Injection of CCl4 into the hemocoel led to an increase in ALT activity. The increase in ALT activity was attenuated by pretreatment with N-acetyl-L-cysteine. Injection of benzoic acid derivatives, ferric sulfate, sodium valproate, tetracycline, amiodarone hydrochloride, methyldopa, ketoconazole, pemoline (Betanamin), N-nitroso-fenfluramine, and D-galactosamine also increased ALT activity. CONCLUSIONS: These findings indicate that silkworms are useful for evaluating the effects of chemicals that induce tissue injury in mammals.

Cloning and characterization of the Bombyx mori ecdysone oxidase.

The physiological titer of molting hormones in insects depends on relative activities of synthesis and degradation pathways. Ecdysone oxidase (EO) is a key enzyme in the inactivation of ecdysteroid. However, there are only a few reports on ecdysteroid inactivation and its enzymes in silkworm. In this study, we cloned and characterized the Bombyx mori EO (BmEO). The BmEO cDNA contains an ORF of 1,695 bp and the deduced protein sequence contains 564 amino acid residues. The deduced protein sequence contains two functional domains of glucose-methanol-choline oxidoreductase in N-terminal and C-terminal. Comparing the expression levels of BmEO in different tissues, high transcription was mainly present in hemocytes. Reduced expression of this enzyme is expected to lead to pathological accumulation of ecdysone in the hemolymph of silkworm larvae or pupae. Our data show that RNA inference of BmEO transcripts resulted in the accumulation of ecdysteroid and death of larvae or pupae. We infer that EO is a crucial element in the physiology of insect development.

Identification of two arginases generated by alternative splicing in the silkworm, Bombyx mori.

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of arginine to ornithine and urea. Here, we have cloned two arginase cDNAs from the silkworm, Bombyx mori. The analysis of exon/intron structures showed that the two mRNAs named bmarg-r and bmarg-f were generated from a single gene by alternative usage of exons. The bmarg-r and bmarg-f were predicted to encode almost the same amino acid sequences, except that the latter had additional ten N-terminal residues. Recombinant bmARG-r and bmARG-f in Escherichia coli cell lysates were roughly similar to each other in enzymatic characteristics, which did not show large difference from those of arginases assayed by using tissue extracts. Differential RT-PCR experiments and tissue distribution analyses of arginase activity indicated that the bmarg-r gene is expressed in the male reproductive organs, especially in the glandula lacteola and vesicular seminalis, from which it is secreted to the seminal fluid and transferred to the female during copulation, whereas the bmarg-f gene is expressed in the larval and adult nonreproductive organs including the fat body and muscle, where the produced arginase proteins are considered to stay in the cells. Thus, the two silkworm arginase isoforms may have a difference in whether or not the product is excreted out of the cells in which it is synthesized.

Effect of induced oxidative stress and herbal extracts on acid phosphatase activity in lysosomal and microsomal fractions of midgut tissue of the silkworm, Bombyx mori.

Lysosomal and microsomal acid phosphatase activity was estimated in midgut tissue of silkworm larvae, Bombyx mori L. (Lepidoptera: Bombycidae), after induced oxidative stress by D-galactose. The larvae were simultaneously were treated with ethanolic extracts of Bacopa monniera and Lactuca sativa to study their antioxidant properties. Lipid peroxidation and fluorescence was measured to analyze extent of oxidative stress. The ethanolic extract of Lactuca sativa was found to be more effective in protecting membranes against oxidative stress than Bacopa monniera.

Identification and characterization of an arginine kinase as a major allergen from silkworm (Bombyx mori) larvae.

BACKGROUND: The silkworm, Bombyx mori, is an important insect in the textile industry and its pupa are used in Chinese cuisine and traditional Chinese medicine. The silk, urine and dander of silkworms is often the cause of allergies in sericulture workers and the pupa has been found to be a food allergen in China. Recent studies have focused on reporting cases of silkworm allergies, but only a few studies have addressed the specific allergens present in the B. mori silkworm. METHODS: We collected sera from 10 patients with a positive skin prick test to silkworm crude extract (SCE) and analyzed these samples by Western blot and ELISA. The cDNA of arginine kinase from the B. mori silkworm was also cloned and expressed in high yield in Escherichia coli. Allergenicity and cross-allergenicity of the recombinant B. mori arginine kinase (rBmAK) were investigated by ELISA inhibition assay. RESULTS: Collected sera all reacted to a 42-kDa protein in a Western blot with SCE as the antigen. Preincubation of sera with rBmAK eliminated the reactivity of the patients' sera to this 42-kDa band. All patient sera also exhibited positive reactivity to SCE in an ELISA assay. BmAK also demonstrated cross-reactivity with a recombinant AK from cockroach. CONCLUSION: Arginine kinase from the B. mori silkworm is a major allergen and crossreacts with cockroach AK.

Biochemical characterization of rab proteins from Bombyx mori.

The small GTPases known as Rab proteins are key regulators of membrane trafficking. We used RT-PCR to isolate cDNA clones of insect-specific Rab proteins (BRabN1 and BRabN2) showing low homology with known Rab proteins from other animals, from mRNA of Bombyx mori. These 2 Rabs were produced in Escherichia coli and purified. BRabN1 bound [(3)H]-GDP and [(35)S]-GTPgammaS with dissociation constants of 0.087 x 10(-6) M and 1.02 x 10(-6) M, respectively, whereas those of BRabN2 were 0.546 x 10(-6) M and 1.02 x 10(-6) M, respectively. Binding of [(35)S]-GTPgammaS to BRabN1 and N2 was inhibited by GDP and GTP. The GTP-hydrolysis activities of BRabN1 and N2 were 154 and 35.5 mmol/min/mole, respectively, and bound [(35)S]-GTPgammaS was exchanged efficiently with GTP. BRabN1 also showed ATPase activity and exchange of [(35)S]-GTPgammaS with ATP. Monoclonal antibodies against BRabN1 and N2 did not recognize any other Rab proteins, and Western blotting using the anti-BRabN1 antibody revealed a single band in the testis of B. mori. These results suggest that BRabN1 and N2 of B. mori bind GTP, convert from the GTP-bound state to the GDP-bound state by intrinsic GTP hydrolysis activity, and return to the GTP-bound state with the exchange, and that BRabN1 is specifically expressed in testis. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.

Silkworm as a model animal to evaluate drug candidate toxicity and metabolism.

To evaluate the feasibility of using the silkworm as a model animal for screening drug candidates, we examined whether the lethal dose of cytotoxic chemicals in silkworm, Bombyx mori, were consistent with those in mammals, and compared the metabolic pathways of these drugs between silkworms and mice. The lethal dose levels of cytotoxic chemicals in silkworms were consistent with those in mammals. We examined the fate of model drugs, 4-methyl umbelliferone, umbelliferone, and 7-ethoxycoumarine, in silkworm larvae. The half-life of 4-methyl umbelliferone in the silkworm larvae hemolymph was 7.0+/-0.1 min, similar to that in mouse blood. In silkworm larvae, 4-methyl umbelliferone was conjugated with glucose, whereas in mammals it is conjugated with glucuronate or sulfate. These results are consistent with a previous report that UDP-glucosyltransferase catalyzes the conjugation of 4-methyl umbelliferone. The glucose-conjugation reaction of 4-methyl umbelliferone was observed in microsomal fractions of fat bodies isolated from silkworms. Furthermore, most umbelliferone and 7-ethoxycoumarine injected into the hemolymph of silkworms was eliminated through the feces in the glucose-conjugated form. These findings suggest that chemicals are metabolized through a pathway common to both mammals and silkworms: reaction with cytochrome P450, conjugation with hydroxylated compounds, and excretion.

Induction of tyrosine hydroxylase gene expression by bacteria in the fat body of eri-silkworm, Samia cynthia ricini.

A cDNA clone encoding tyrosine hydroxylase (TH) was isolated from larval fat body of immunized Samia cynthia ricini. In naive larvae, the TH gene was expressed only in the brain, but strongly induced in fat body and hemocytes after injecting UV-killed bacteria. The induction of the gene was rather short-lived compared to that of antibacterial protein genes, reaching the maximum levels 6h after bacterial challenge, and then quickly diminished. A strong induction of the gene expression was caused by both Gram-negative and positive bacteria and zymosan, but little if any by soluble peptidoglycan or lipopolysaccharide. A possible role of TH in the fat body of bacteria-challenged larvae would be to supply catecholamines as the substrate for phenoloxidase leading to melanization, working together with dopa decarboxylase.

Bombyx mori pyridoxal kinase cDNA cloning and enzymatic characterization.

Pyridoxal kinase (PLK) (EC 2.7.1.35) catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal-5.-phosphate (PLP), an important cofactor for many enzymatic reactions. Bombyx mori, similar to mammals, relies on a nutritional source of vitamin B6 to synthesize PLP. This article describes how a cDNA encoding PLK was cloned from Bombyx mori using the PCR method (GenBank accession number: DQ452397). The cDNA has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.1 kDa. The amino acid sequence shares 48.6% identity with that of human PLK, and it also contains signature conserved motifs of the PLK family. However, the protein is 10 or more amino acids shorter than the PLK from mammals and plants, and several amino acid residues conserved in the PLK from mammals and plants are changed in the protein. The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter/T7 RNA polymerase expression system, and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity with a value of 30 nmol/min/mg, confirming that the cDNA encodes the functional PLK of Bombyx mori. This is the first identification of a gene encoding PLK in insects.

Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system.

Superoxide dismutase (SODs) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and, thus, form a crucial part of the cellular antioxidant defense mechanism. In this paper, we used the total fat body RNA of silkworm, Bombyx mori L. to clone and sequence a 648-bp Mn-SOD cDNA fragment through RT-PCR. Furthermore, a newly established Bac-to-Bac/BmNPV Baculovirus expression system was used to overexpress the recombinant Mn-SOD enzyme in silkworm larvae. The hemolymph was collected from the infected larvae 96 h post-infection and subjected to a 12 % SDS-PAGE and Western blotting. A 18.0-kDa protein was visualized after rBacmid/BmNPV/SOD infection. The SOD enzyme activity was determined with a tetrazolium salt for detection of superoxide radicals generated by xanthine and xanthine oxidase and its peak appeared in 96 h post-infection with 2.7 times of the control larvae. The availability of large quantities of SOD that the silkworm provides should greatly facilitate the future research and testing of this protein for potential application in medicine.

Juvenile hormone diol kinase, a calcium-binding protein with kinase activity, from the silkworm, Bombyx mori.

Juvenile hormone (JH) diol kinase (JHDK) is an important enzyme involved in the JH degradation pathway. Bombyx mori (Bommo)-JHDK cDNA (637bp) contains an open reading frame encoding a 183-amino acid protein, which reveals a high degree of identity to the two previously reported JHDKs. JHDK is similar to GTP-binding proteins with three conserved sequence elements involved in purine nucleotide binding, contains eight alpha-helices and three EF-hand motifs, and resembles the three-dimensional model of 2SCP and some other calcium-binding proteins. The Bommo-JHDK gene has only a single copy in the silkworm haploid genome, contains only one exon, and its 5'-upstream sequence does not have a JH response element. Although Bommo-JHDK is highly expressed in the gut of the silkworm, its mRNA expression remains at a constant level during larval development suggesting this enzyme is constitutive and not regulated by JH, at least at the transcriptional level. Recombinant Bommo-JHDK catalyzed the conversion of 10S-JH diol into JH diol phosphate, confirming its enzymatic function. Recombinant enzyme formed a dimer and had biochemical characteristics similar to other JHDKs. Bommo-JHDK, a calcium-binding protein with kinase activity, provides unique insights on how JH levels are regulated in the silkworm.

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.

A deleted portion of one of the two xanthine dehydrogenase genes causes translucent larval skin in the oq mutant of the silkworm (Bombyx mori).

A silkworm mutant, oq, has translucent larval skin because it is deficient in xanthine dehydrogenase (XDH) activity and is unable to synthesize uric acid, which is normally accumulated in the larval epidermis and makes the skin white and opaque. Two XDH bands were found in zymograms of the silkworm fat body: an intense band (XDHalpha) and a faint one (XDHbeta). The oq mutant lacks only XDHalpha, which seemed to be the major source of XDH activity in the fat body. An 8-bp deletion found in BmXDH1, a silkworm XDH gene, generates a premature stop codon. The resulting truncated BmXDH1 protein lacks three molybdenum cofactor-binding domains necessary for enzyme activity. BmXDH2, the other XDH gene, does not show any apparent deficiencies. BmXDH1 expressed in yeast cells yielded an activity band with the same mobility as that of XDHalpha in zymograms. BmXDH1 of the oq mutant did not yield active XDH in yeast, while the activity was restored by filling in the deleted sequence. These results showed that BmXDH1 deletion in the oq mutant is responsible for the absence of significant XDH activity, resulting in the translucent larval skin of the mutant phenotype.