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PubMed1999

Recycling of urea associated with the host plant urease in the silkworm larvae, Bombyx mori.

Urea concentration and urease activity in the midgut content were compared between larvae of the silkworm, Bombyx mori fed an artificial diet and those fed fresh mulberry leaves. A considerable amount of urea was found in the midgut content of the both larvae, however it was significantly lower in the larvae fed fresh mulberry leaves than in the larvae fed the artificial diet; average urea concentrations in the midgut content of the larvae fed fresh mulberry leaves and the artificial diet were 2.9 and 4.6 &mgr;mol/g, respectively. Urea in the midgut content seems to be secreted from the insect itself since the amount of urea in both diets were negligibly small. Urease activity was detected only in the midgut content of the larvae fed fresh mulberry leaves but not in other tissues of the larvae. On the other hand, no urease activity was detected in the midgut content of the larvae fed the artificial diet. Subsequently, to elucidate the role of mulberry leaf urease in the midgut lumen, larvae that had been reared on the artificial diet were switched to fresh mulberry leaves. The diet switch caused a rapid decrease in urea concentration in the midgut content and an increase in ammonia concentration in the midgut content, suggesting that secreted urea could be hydrolyzed to ammonia by mulberry leaf urease in the midgut lumen. Furthermore, to investigate the physiological significance of mulberry leaf urease on urea metabolism of the silkworm, (15)N-urea was injected into the hemocoel, and after 12 h the larvae were dissected for (15)N analysis. A considerable amount of (15)N was found to be incorporated into the silk-protein of the larvae fed fresh mulberry leaves, but there was little incorporation of (15)N into the silk-protein of the larvae fed the artificial diet. These data indicate that urea is converted into ammonia by the action of mulberry leaf urease in the midgut lumen and used as a nitrogen source in larvae fed mulberry leaves.

Journal Article↗
PubMed2000

Sorbitol as an arrester of embryonic development in diapausing eggs of the silkworm, Bombyx mori.

Recently, it was confirmed that embryos derived from diapausing eggs of the silkworm, Bombyx mori, begin their development and reach larval maturity on mulberry leaves, when the naked eggs are cultured in vitro. In this study, we found that the method of embryo culture is useful for determining the physiological regulation of diapause. We show that the development of embryos derived from diapausing eggs was strongly inhibited by the addition of either sorbitol or trehalose to the culture medium. Furthermore, this inhibitory effect disappeared when the embryos were cultured in a control medium which did not contain either sorbitol or trehalose, indicating that the inhibitory reactions caused by both substances are reversible. The minimal effective dose of either sorbitol or trehalose was approximately 0.2 M, a value similar to the in vivo concentration of sorbitol in diapausing eggs (0.2 M). Glycerol, mannitol or glucose were moderately effective for inhibition. Sorbitol present in diapausing silkworm eggs does not appear to serve as an antifreeze, but as an strong arresting factor of embryonic development. Furthermore, these results show that a decrease in sorbitol releases the embryos from diapause at the termination of diapause.

Journal Article↗
PubMed2000

Structure of insect chitin isolated from beetle larva cuticle and silkworm (Bombyx mori) pupa exuvia.

Chitin samples in a alpha-form structure were isolated from beetle larva cuticle and silkworm (Bombyx mori) pupa exuvia by treatment with 1 N HCl and 1 N NaOH. Chitosan was prepared by treating them in 40% NaOH containing NaBH(4). Chitin and chitosan were analyzed by X-ray, [13C]CP/MAS NMR, [13C]FT-NMR, and scanning electron microscopy (SEM) methods. Insect chitin degraded more readily than shrimp chitin when treated with 6 N HCl and the enzyme-chitinase. After treatment with 2 N HCl at 100 degrees C, the insect chitin crystallinity increased. N-deacetylation of insect chitin was easier than that of crustaceous chitin, and about 94% of the N-acetyl groups were removed in one treatment with 40% NaOH for 4 h at 110 degrees C. After treatment with 2 N HCl, 55% of the N-acetyl groups of silkworm chitin were removed under the same conditions. Beetle chitin showed a higher affinity for chitinase than shrimp chitin.

Animals↗
PubMed2002

Molecular cloning and expression of a Toll receptor gene homologue from the silkworm, Bombyx mori.

A novel Toll receptor gene was cloned from the bacterial artificial chromosomal (BAC) library of the silkworm, Bombyx mori, using primers for polymerase chain reactions (PCR), synthesized based on an expressed-sequence tag (EST). The open reading frame of this gene was intronless. The deduced amino acid sequence including a putative signal peptide from the nucleotide sequence of the B. mori Toll (BmToll) gene shows 53.7%, 49.0%, 42.3% and 39.2% similarity to those of Toll-7, 18 wheeler (18w), Toll-6 and Toll-8 from Drosophila melanogaster. BmToll, however, does not show marked similarity to Toll, Toll-3, Toll-4 or Toll-5. Phylogenetic insect Toll family analysis shows that BmToll is strongly related to Toll-7 and 18w. An analysis of the tissue-specific expression of the BmToll gene by reverse transcription PCR (RT-PCR) showed that the BmToll gene is constitutively expressed in the fat body but not in the Malpighian tubule, silk gland, midgut or hemocyte. Injecting lipopolysaccharide (LPS) into silkworm hemocoel strongly suppressed BmToll gene expression. The time course showed that BmToll gene expression is totally suppressed within 2 h and gene transcripts appeared again 12 h after LPS injection, suggesting strong downregulation of the BmToll gene expression by LPS.

Amino Acid Sequence↗
PubMed1996

Discovery of a new tetrahydrobiopterin-synthesizing enzyme in the lemon mutant of the silkworm Bombyx mori.

A new tetrahydrobiopterin-synthesizing enzyme, which is different from sepiapterin reductase (EC 1.1.1.153), was discovered in the integument of the lemon mutant of the silkworm Bombyx mori. This enzyme converted 6-pyruvoyltetrahydropterin to tetrahydrobiopterin, an essential cofactor in the hydroxylation of aromatic amino acids, in the presence of NADPH. The reaction proceeded via 6-lactoyltetrahydropterin and 1'-hydroxy-2'-oxopropyltetrahydropterin as intermediates. The molecular mass of this enzyme was estimated to be 40 kDa. N-Acetylserotonin, a potent inhibitor of sepiapterin reductase, slightly inhibited the enzymatic reaction. In the presence of 0.5 mM N-acetylserotonin, the formation of tetrahydrobiopterin by sepiapterin reductase purified from the normal strain silkworm was completely inhibited. However, the formation of tetrahydrobiopterin by the enzyme purified from the lemon mutant was inhibited by only about 50%. These results suggest an alternative biosynthetic pathway to tetrahydrobiopterin.

Alcohol Oxidoreductases↗
PubMed1979

Hemocytes and hemocytopoiesis in Silkworms.

A brief review is presented of the current state of ultrastructure, cytochemistry, and physiology of the hemocytes and meso- and metathoracic peri-imaginal-wing organs in silkworms. According to the accepted morphological classification, five circulating types of hemocytes are recognized in Bombyx mori as well as in Antheraea pernyi. They are prophemocytes or stem cells, plasmatocytes or pre-differentiated cells and three specialized cells, granulocytes, spherule cells and oenocytoids. During post-embryonic development the last four types are the most common in the circulating hemolymph. Plasmatocytes are considered to be pluripotent cells from which granulocytes, spherule cells and oenocytoids are derived. Contrary to the situation in most insects the plasmatocytes are not phagocytic in Antheraea. The granulocytes are efficient phagocytes. Both plasmatocytes and granulocytes are involved in pinocytosis. Another possible function of the granulocytes is hemolymph coagulation. The function of the spherule cells which contain a paracrystalline material (muco- or glycoproteins) is by no means clear. The phenoloxidase activity found within the cytosol of oenocytoids appears effective against the natural monophenol and diphenol substrates. The involvement of oenocytoids in the complex metabolism of phenols and particularly in the production of plasma phenolases has been reported. The mitotic division of five circulating hemocyte types is well known and was long regarded as the only mechanism of postembryonic hemocyte production. We present for silkworms, experimental evidence of the hemocytopoietic function of the meso- and metathoracic organs surrounding the imaginal wing discs. Ablation experiments demonstrate that the mitotic activity of free hemocytes is unable to maintain the normal hemocytogram in the absence of the two paris of organs. These organs are typically divided into cell islets ensheathed by a connective tissue membrane. Two types of islets may be classified by the disposition of the cells : the compact islets or aggregations of stem cells and the reticulate islets which are mainly composed of hemocytes at different steps of differentiation. The relative number of prohemocytes in the total hemocyte population ranges from 84 to 97 p. cent in organs of Antheraea pernyi. This well-defined cell type appears to be the major hemocyte type in hemocytopoietic organs. In Antheraea, the mitotic index (the relative number of mitotic hemocytes in the total cell population) varies from 0.5 to about 3 p. cent. Finally, our data direct attention to cyclic functional changes such as mitotic divisions and hemocyte differentiation which run parallel to the molting cycle.

Animals↗
PubMed1998

Expression of grass carp growth hormone by baculovirus in silkworm larvae.

A total of five recombinant Bombyx mori nuclear polyhedrosis viruses (BMNPV) carrying the grass carp (Ctenopharyngodon idellus) growth hormone (GH) cDNA were constructed in this study. Two of them were able to express the hormone up to a level of 12 microgram/ml medium when cultured B. mori cells were infected for 4 days. Inoculation of the viruses into silkworm (B. mori) host significantly increased the level of GH achievable. The amount of hormone produced per larva was estimated to be around 1 mg. The recombinant grass carp GH had immunological and biological activities similar to the native hormone. The N-terminal sequence of the recombinant hormone was the same as the native one, indicating that the fish signal peptide was correctly processed by the insect cells. Silkworm powder prepared from larvae infected with the recombinant virus was used as food supplement for fish. Compared with the control, this dietary supplement was effective in increasing the growth rate of juvenile carp.

Animals↗
PubMed1998

Molecular cloning of a cDNA encoding a silkworm protein that contains the conserved BH regions of Bcl-2 family proteins.

To search for a Bcl-2 family homologue in the posterior silk gland of Bombyx mori, Western blot analysis was performed with the anti-rat Bcl-XL antiserum preabsorbed with a XL1-Blue MRF' lysate. The antiserum was shown to cross-react specifically with a silkworm protein of 80000 mol. wt (BmP80). The level of BmP80 increased dramatically during the spinning stage and decreased rapidly after the formation of a cocoon, implying that the silkworm protein was involved in histolysis of posterior silk gland as a stimulator. Screening a B. mori cDNA library with the same preabsorbed antiserum, a cDNA clone contaiing a cDNA fragment that is presumably large enough to encode the entire BmP80 protein was identified. The cDNA fragment contained 127 nucleotides (nt) of untranslated sequence at the 5' end, 2895nt of presumptive coding sequence and 625nt of untranslated sequence including a poly(A) tail at the 3' end. The calculated mol. wt of the presumptive protein (BmP109) was 108800. BmP109 shared sequence homology with the antiapoptotic proteins within the four conserved regions, BH1, BH2, BH3 and BH4, which were located at the C-terminal half of the protein and resided in the same characteristic order as Bcl-2 family proteins. Comparison of amino acid content revealed that BmP109 contained much more cysteine and lysine but less glycine and arginine than the antiapoptotic proteins. Northern blot analysis indicated that the mRNA for BmP109 is about 4.0kb. Reverse transcription-polymerase chain reaction experiments showed that the mRNA level for BmP109 increased dramatically during the spinning stage and decreased rapidly after formation of a cocoon, suggesting the involvement of transcriptional regulation.

Amino Acid Sequence↗
PubMed2001

Esterase isozyme polymorphism, specific and nonspecific esterase, syngenic lines development and natural occurrence of a thermostable esterase in the tropical silkworm Bombyx mori L.

Esterase isozyme polymorphism was documented for digestive juice and haemolymph of the tropical multivoltine silkworm, Bombyx mori L., breed CB5 (GP) and its syngenic lines (CB5Lm(e)-1, CB5Lm-2 and CB5Lm-5) using alpha- and beta-naphthylacetate separately as nonspecific substrates (Ogita, Z., Kasai, T., 1965. Genetico-biochemical analysis of specific esterases in Musca domestica. Jpn. J. Genet. 40, 173-184). Polymorphism existed in the isozyme pattern of alpha-esterase with two or three bands in digestive juice and three to five bands in haemolymph. No polymorphism was observed in beta-esterase isozyme pattern having four bands in digestive juice and two bands in haemolymph. During the course of esterase isozyme studies, the presence of some specific alpha-esterase bands (Est-1, 4 and 5) in haemolymph and beta-esterase bands (Est-1, 2 and 3) in digestive juice were observed. But both alpha- and beta-esterase bands Est-3 and 4 in digestive juice and Est-2 and 3 in haemolymph were found to be nonspecific. Nonspecific beta-esterase band (Est-3) in haemolymph of CB5 (GP) and its syngenic lines withstood a temperature up to 80+/-1 degrees C for 10 min. No thermostable band was observed in the isozyme zymogram of alpha-esterase in digestive juice and haemolymph or beta-esterase in digestive juice. Overall, this study discusses the presence of esterase heterogeneity in the CB5 (GP) genepool, syngenic lines development, occurrence of specific alpha- and beta-esterase bands in digestive juice and haemolymph and thermostable beta-esterase band Est-3 in haemolymph in tropical silkworm Bombyx mori L.

Animals↗
PubMed2003

Mutations of the silkworm molybdenum cofactor sulfurase gene, og, cause translucent larval skin.

Normal silkworms (Bombyx mori) have opaque larval skin due to uric acid accumulation in the epidermis while a mutant, og, is translucent owing to a deficiency in xanthine dehydrogenase (XDH), which synthesizes uric acid. Molybdenum cofactor (MoCo) sulfurase is responsible for XDH activation in various organisms. A silkworm MoCo sulfurase gene was cloned and found to be on the og locus, whose mutant alleles, og(k) and og(t), show premature stop codons, proving that og is the MoCo sulfurase gene. It was observed that a miniature inverted-repeat transposable element (MITE), named Organdy, when inserted in an og(t) mutant allele exon, causes unstable splicing of a downstream intron leading to incomplete open reading frames.

Amino Acid Sequence↗
PubMedDate not supplied

Purification and characterization of a protease degrading 30 kDa yolk proteins of the silkworm, Bombyx mori.

The second major yolk proteins, 30 kDa proteins (30kPs) of the silkworm, Bombyx mori, which have been provided during oogenesis, are kept continuously unused during embryogenesis and are utilized just before larval hatching. The crude extracts of newly hatched larvae cleaved 30kPs in an in vitro incubation system. A protease was highly purified from newly hatched larvae using ammonium sulfate precipitation, gel filtration and ionic exchange chromatography, and non-denaturing-polyacrylamide gel electrophoresis (ND-PAGE). The protease shared the NH2-terminal amino acid sequence conserved in many serine proteases, and the apparent molecular mass was estimated to be approximately 600 kDa by gel filtration column chromatography. The enzymatic activity was strongly inhibited by elastatinal and diisopropyl fluorophosphate (DFP), indicating that this protease is an elastase-like serine protease. The protease selectively hydrolysed 30kP-1 and 30kP-4 between Ser6 and Ala7, but could not attack other 30kPs such as 30kP-2, 30kP-3 and 30kP-5. Consequently, the protease characterized in the present study is a unique protease which may be specialized for the selective degradation of yolk proteins in silkworm eggs.

Amino Acid Sequence↗
PubMed1998

Purification and characterization of NADH-dependent glutamate synthase from the silkworm fat body (Bombyx mori).

NADH-dependent glutamate synthase (Nadh-Gogat; EC 1.41.14) was purified 766-fold from the fat body of 5th instar larvae of the silkworm with a final specific activity of 13.8 units/mg protein by a produce including ammonium sulfate fraction, Q-Sepharose HP ion exchange column chromatography, Blue Sepharose FF affinity column chromatography and Superdex 200 HR gel filtration. The purified enzyme yielded a single band corresponding to a molecular mass of 195kDa by SDS-polyacrylamide gel electrophoresis. Molecular mass of the native enzyme was estimated to be 190 kDa by Superdex 200HR gel filtration, suggesting that the enzyme is composed of a monomeric polypeptide. The enzyme showed an absorption spectrum with maximum values at 272, 375, and 435 nm, suggesting the presence of a flavin prosthetic group in the enzyme. The N-terminal amino acid sequence showed a high similarity to those of other GOGATs from plants, yeast and bacteria, but no similarity to other known proteins was detected. The enzyme exhibited a strong specificity to the electron donor and substrates; NADH as electron donor, 2-oxoglutarate as amino acceptor and glutamine as amino donor were essential for the catalytic activity. The optimum pH was around 7.5, at which Km values for 2-oxoglutarate, glutamine and NADH were 17, 220 and 5.7 micro M, respectively. Azaserine, 6-diazo-5-oxonorleucine and p-chloromercuribenzoic acid were strong inhibitors of the activity. These result show that NADH-GOGAT in the silkworm fat body strongly resembles other eukaryotic NADH-GOGATs, suggesting that it plays a significant role in ammonia assimilation in the same manner as the other enzymes.

Amino Acid Sequence↗
PubMed2003

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein in the silkworm, Bombyx mori.

We have cloned the full length of a novel cDNA named Bombyx mori cuticle protein that contains an AlaAlaProAla/Val-repeat (BMCPA) from a cDNA library of integument in the larval silkworm. Both a typical tandem repeat (A-A-P-A/V) for cuticle protein and a unique tandem repeat with Ser, Ala, Gly, Pro, Val, Tyr and Thr were observed in the predicted amino acid sequence of the cDNA encoding BMCPA. Approximately 80% of the amino acids in BMCPA were composed of Ser, Ala, Gly, Pro, Val and Tyr. Northern-hybridization analysis indicated that BMCPA mRNA is expressed only in the larval epidermis and that the expression pattern of the BMCPA gene in the developmental stage was observed mainly at the larval stage. We propose BMCPA may be a novel component of cuticle, and may play an important role in the integument of the larval silkworm.

Amino Acid Sequence↗
PubMed1980

Stereochemical importance of fucosterol epoxide in the conversion of sitosterol into cholesterol in the silkworm Bombyx mori.

Studies on the stereochemistry of 24,28-epoxy-24-ethylcholesterol, a key intermediate in sitosterol to cholesterol conversion in insects, were undertaken. In nutritional experiments, the 24R,28S and 24S,28R stereoisomers (isofucosterol epoxides) were unable to support growth and development of silkworm larvae. Incubation of 3 alpha-3H-labeled (24R,28R)- and (24S,28S)-epoxide (fucosterol epoxides) with a cell-free preparation from silkworm guts resulted in effective conversion into desmosterol and cholesterol; (24R,28R)-epoxide was the slightly better substrate. Fucosterol incubation yielded (24R,28R)-epoxide and the 24S,28S isomer in approximately equal amounts, while slightly preferential formation of the 24R,28R isomer was observed in in vivo experiments. These results indicate that both the formation of the epoxide from fucosterol and its conversion to desmosterol proceed with a low degree of stereospecificity.

Animals↗
PubMed2007

Some observations on the structure and function of the spinning apparatus in the silkworm Bombyx mori.

Silkworm silk has outstanding mechanical properties despite being spun at room temperature and from aqueous solution. Although it has been proposed that fiber formation is mainly induced by shearing and extensional flow in the spinneret, the detailed structure and function of the spinning apparatus of Bombyx mori silkworms are still not fully elucidated. In this paper we describe three aspects of the functional microanatomy of the spinning apparatus: changes in the diameter of the silk gland duct with distance along the duct, how the birefringence of the fibroin changes as it flows down the duct, and the detailed three-dimensional structure of the silk press and related structures. The existence of a double escaped nematic liquid crystal texture in the fibroin in a region of the duct is described. After this region the birefringence suddenly disappeared until the start of an internal draw down taper which commenced just before the silk press. In the internal draw down taper the birefringence increased dramatically to an asymptotic value as a thread was drawn from the fibroin gel. The structure of the silk press suggests that it acts as a restriction die whose diameter can be regulated.

Animals↗
PubMed2005

Effect of metallic ions on silk formation in the Mulberry silkworm, Bombyx mori.

A protein conformation transition from random coil and/or helical conformation to beta-sheet is known to be central to the process used by silk-spinning spiders and insects to convert concentrated protein solutions to tough insoluble threads. Several factors including pH, metallic ions, shear force, and/or elongational flow can initiate this transition in both spiders and silkworms. Here, we report the use of proton induced X-ray emission (PIXE), inductively coupled plasma mass spectroscopy (ICP-MS) and atomic adsorption spectroscopy (AAS) to investigate the concentrations of six metal elements (Na, K, Mg, Ca, Cu, and Zn) at different stages in the silk secretory pathway in the Bombyx mori silkworm. We also report the use of Raman spectra to monitor the effects of these six metallic ions on the conformation transition of natural silk fibroin dope and concentrated regenerated silk fibroin solution at concentrations similar to the natural dope. The results showed that the metal element contents increased from the posterior part to the anterior part of silk gland with the exception of Ca which decreased significantly in the anterior part. We show that these changes in composition can be correlated with (i) the ability of Mg2+, Cu2+, and Zn2+ to induce the conformation transition of silk fibroin to beta-sheet, (ii) the effect of Ca2+ in forming a stable protein network (gel), and (iii) the ability of Na+ and K+ to break down the protein network.

Animals↗
PubMed1992

Characterization of silkworm chlorophyll metabolites as an active photosensitizer for photodynamic therapy.

Silkworm excreta containing chlorophyll metabolites have long been used for their medicinal activities in the Far East. The major chlorophyll derivative (CpD) fraction in the Me2CO extract from the silkworm excreta exhibits strong photodynamic action on mice and human tumor cells in vitro. The main CpD component was isolated, purified, and subjected to extensive structural and characterization studies to assess its photodynamic therapeutic activity. According to the spectral analyses, the structure of the major unknown CpD was determined to be 10-hydroxypheophytin a [1]. The CpD component possessed a high quantum efficiency (50%) for the photosensitized production of singlet molecular oxygen, thus providing a molecular basis for its photodynamic therapeutic efficiency. A further advantage is that the "photooxidation/degradation" product(s) from this CpD retains all the characteristics of a photodynamic sensitizer.

Animals↗
PubMed2004

Method for purifying porcine mature interleukin-18 from silkworm haemolymph.

Recombinant porcine mature interleukin-18 (rPomIL-18) has been purified from the haemolymph of silkworms. After co-infection of two recombinant baculoviruses (BmAcpVL1392-IL-18-His and BmAcpVL1392-casp-1) into the silkworm, rPomIL-18 was produced and secreted into the haemolymph. Polyethylene glycol (PEG) 6000 was added to the haemolymph at 8% (v/w) to precipitate storage proteins which would otherwise bind non-specifically to the metal chelating column and the supernatant then was applied to Sepharose bonded with Ni2+. rPomIL-18 was eluted from the column using 100 mM imidazole buffer at pH 8 with a purity of 93.6%. Approximately 5.3 mg purified rPomIL-18 was obtained from 22 ml haemolymph. It could induce interferon-gamma formation from porcine peripheral blood mononuclear cells.

Animals↗