Tissues Of Silkworm Research Articles

Proteomic analysis of larval integument, trachea and adult scale from the silkworm,Bombyx mori

Multidimensional LC-tandem MS was used to investigate the protein compositions of three tissues of silkworm, Bombyx mori. A total of 162, 259, and 175 peptides from silkworm larval integument and trachea, and adult scale obtained by database search were matched to 48, 51, and 40 proteins, respectively. Forty-one cuticular proteins were identified from three tissues and covered all five cuticular protein families of silkworm. In the adult scale, all seven cuticular proteins were identified for the first time in the final pellet after SDS extraction. The majority of cuticular proteins were found in each tissue differentially, suggesting that tissue-specific cuticular proteins were involved in the building of the specialized tissues. Seventy-three non-cuticular proteins were also identified in this analysis mainly including muscular proteins, proteinases, inhibitors, transport proteins, and redox-related proteins.

Rapid determination of free d-serine with chicken d-serine dehydratase

We have developed a simple, rapid, and inexpensive method of measuring the concentration of intrinsic free d-serine in tissue samples. This method uses chicken d-serine dehydratase in an enzymatic reaction to produce pyruvate, which is detected spectrophotometrically. Pyridoxal 5′-phosphate (PLP), a cofactor of d-serine dehydratase, increased pyruvate formation by 28%. The presence of Zn 2+ or ethylenediaminetetraacetic acid (EDTA) did not have any effect on pyruvate formation under the present assay conditions. In addition, this method was not affected by the presence of a large excess of l-serine, nor by the presence of tissue extracts, and accurately determined concentrations of 2–30 μM (200 pmol–3 nmol) of d-serine. The entire assay requires only 60 min. With this method, we determined the concentration of d-serine in various silkworm tissues. The results were in agreement with high performance liquid chromatography measurements. We found high concentrations of d-serine in silkworm larvae at day 3 of the fifth instar; specifically, 509 nmol g −1 wet tissue in the midgut, 434 nmol g −1 in the ovary, and 353 nmol g −1 in the testis.

Porphyromonas gingivalis Peptidoglycans Induce Excessive Activation of the Innate Immune System in Silkworm Larvae*

Porphyromonas gingivalis, a pathogen that causes inflammation in human periodontal tissue, killed silkworm (Bombyx mori, Lepidoptera) larvae when injected into the blood (hemolymph). Silkworm lethality was not rescued by antibiotic treatment, and heat-killed bacteria were also lethal. Heat-killed bacteria of mutant P. gingivalis strains lacking virulence factors also killed silkworms. Silkworms died after injection of peptidoglycans purified from P. gingivalis (pPG), and pPG toxicity was blocked by treatment with mutanolysin, a peptidoglycan-degrading enzyme. pPG induced silkworm hemolymph melanization at the same dose as that required to kill the animal. pPG injection increased caspase activity in silkworm tissues. pPG-induced silkworm death was delayed by injecting melanization-inhibiting reagents (a serine protease inhibitor and 1-phenyl-2-thiourea), antioxidants (N-acetyl-l-cysteine, glutathione, and catalase), and a caspase inhibitor (Ac-DEVD-CHO). Thus, pPG induces excessive activation of the innate immune response, which leads to the generation of reactive oxygen species and apoptotic cell death in the host tissue.

Properties of silkworm Na+/K+-ATPase

The high Na(+) and low K(+) concentrations in mammalian blood are maintained by Na(+)/K(+)-ATPase. In contrast, the K(+) concentration is higher than the Na(+) concentration in the hemolymph of the silkworm Bombyx mori, a Lepidopterous insect. Although Na(+)/K(+)-ATPase, therefore, appears not to be in silkworm, we confirmed the presence of Na(+)/K(+)-ATPase in nerve tissues of silkworm but not in skeletal muscle or the dorsal vessel. The enzymatic properties of silkworm Na(+)/K(+)-ATPase were characterized in detail and compared with those of dog Na(+)/K(+)-ATPase. Silkworm Na(+)/K(+)-ATPase had a much lower affinity for K(+) and a somewhat higher affinity for Na(+) than dog Na(+)/K(+)-ATPase. The optimal temperature of silkworm Na(+)/K(+)-ATPase activity was lower than that of dog Na(+)/K(+)-ATPase. The optimal Mg(2+) concentration, pH and sensitivities to Ca(2+) and ouabain, a specific inhibitor of Na(+)/K(+)-ATPase, of the two ATPases were identical. These results indicate that the enzymatic properties of the silkworm Na(+)/K(+)-ATPase are suitable for its growth, despite the differences between dog and silkworm Na(+)/K(+)-ATPases. Antisera raised against dog Na(+)/K(+)-ATPase recognized only the α-subunit of silkworm Na(+)/K(+)-ATPase.

Functional and pharmacological characterization of a β-adrenergic-like octopamine receptor from the silkworm Bombyx mori

A cDNA encoding a seven-transmembrane receptor was cloned from the nervous tissues of silkworm ( Bombyx mori) larvae. Sequence analysis indicated that the gene is an ortholog of CG6989, which encodes a Drosophila β-adrenergic-like octopamine (OA) receptor (DmOctβ2R). As very little information is available regarding this class of receptors, we generated a cell line that stably expressed the gene in HEK-293 cells and we then performed functional and pharmacological studies of this receptor. [ 3H]OA-binding assays using membrane preparations of this cell line showed that the receptor possesses a higher affinity for OA than for tyramine (TA) or dopamine (DA). The cell line elicited a bell-shaped, OA concentration-dependent increase in intracellular cAMP levels, with a maximum at 100 nM. ( R)-OA was more potent than ( S)-OA. TA and DA had weak or marginal effects on cAMP production. The OA receptor agonist demethylchlordimeform elicited a similar biphasic response, although the maximum response was attained at a concentration as low as 1 nM. The rank order of potency of other agonists was as follows: naphazoline > tolazoline, clonidine. Among the antagonists tested, only chlorpromazine significantly attenuated the OA-induced increase in cAMP levels. No increase in intracellular Ca 2+ levels was observed with OA at concentrations up to 100 μM. These findings indicate that the cloned receptor is a β-adrenergic-like OA receptor with unique functional and pharmacological properties.

Cloning and characterization of the gene encoding an ubiquitin‐activating enzyme E1 domain‐containing protein of silkworm,Bombyx mori

Abstract Bombyx moricytoplasmic polyhedrosis virus (BmCPV) is one of the major viral pathogens for the silkworm. To date, the molecular mechanism of BmCPV invasion has been unclear. We cloned the full length complementary (c)DNA which encodes the ubiquitin‐activating enzyme E1‐domain containing protein1 (UbE1DC1) ofBombyx moriby using suppression subtractive hybridization (SSH) and rapid amplification of complementary (c)DNA ends (RACE). The full‐length cDNA ofUbE1DC1gene is 1 919 bp, consisting of a 100 bp 5′ untranslated region, a 637 bp 3′ untranslated region and an 1 182 bp open reading frame (ORF), encoding a 393 amino acid protein. The protein contained the THiF_MoeB_hesA_family domain, an adenosine triphosphate binding site, which belongs to the family of ubiquitin‐activating enzyme E1. Reverse transcription – polymerase chain reaction analysis from the silkworm tissues, namely silk gland, hemocyte, fat body, gonad and midgut revealed thatUbE1DC1was expressed in all the five tissues. The real‐time quantitative polymerase chain reaction analysis indicated that the relative expression ofUbE1DC1in the normal midgut was approximately 9.78‐fold of that in the BmCPV‐infected midgut. It is implicated thatUbE1DC1may play an important role in the interaction between the host and BmCPV invasion.

The nanos gene of Bombyx mori and its expression patterns in developmental embryos and larvae tissues

The nanos gene encodes a zinc-finger protein which is required for the migration and differentiation of primordial germ cells as well as for their fate maintenance. In this study, a 1913 bp nanos gene was cloned and characterized in silkworm ( Bombyx mori). RT-PCR and Western blot analysis showed that the nanos was expressed in developing embryos and various silkworm larval tissues. The expression patterns of Nanos and Vasa in silkworm larval gonads were analyzed using immunohistochemistry. It was found that, in silkworm larval ovaries, the Nanos and Vasa proteins were expressed in oocytes. While in testes, high expression of Nanos and Vasa was detected in spermatogonia and relatively weaker expression was found in spermatocytes at latter stages.

Spread of recombinant Autographa californica nucleopolyhedrovirus in various tissues of silkworm Bombyx mori determined by real-time PCR

A cassette harboring luciferase reporter driven by Bombyx mori A3 promoter was transferred to the bacmid AcΔEGT to generate the recombinant virus AcNPVA3Luc (where Ac represents Autographa californica, NPV represents nucleopolyhedrovirus, and A3Luc represents the firefly luciferase reporter cassette driven by the A3 promoter). Recombinant baculovirus was injected into the hemocoele of newly ecdysed fifth instar larvae of the silkworm. The infection of virus in various silkworm tissues was determined by real-time PCR. The profile of viral infection showed that the copy number of recombinant AcNPV (rAcNPV) increased the fastest in the hemocyte, followed by the fat body, Malpighian tubule, middle gut, and silk gland. Detecting in nonpermissive strain silkworm showed that there was no significant difference in the entry of rAcNPV into all tested tissues. The difference in viral infection reflected mainly the big difference in replication of rAcNPV in various tissues of silkworm larvae. Real-time quantitative RT–PCR showed that it was due to the different expression of genes involved in viral DNA replication.

Microarray-based gene expression profiles in multiple tissues of the domesticated silkworm, Bombyx mori

Using a genome-wide oligonucleotide microarray, gene expression was surveyed in multiple silkworm tissues on day 3 of the fifth instar, providing a new resource for annotating the silkworm genome.

Molecular cloning and heterologous expression of an α‐adrenergic‐like octopamine receptor from the silkworm Bombyx mori

A cDNA encoding an octopamine (OA) receptor (BmOAR1) was isolated from the nerve tissue of silkworm (Bombyx mori) larvae. Comparison of amino acid sequences showed that BmOAR1 is highly identical to OA receptors isolated from Periplaneta americana (Pa oa(1)), Apis mellifera (AmOA1), and Drosophila melanogaster (OAMB or DmOA1A). BmOAR1 was stably expressed in HEK-293 cells. OA above 1 microM led to an increase in intracellular cyclic AMP concentration ([cAMP](i)). The synthetic OA-receptor agonist demethylchlordimeform also elevated [cAMP](i) to the same maximal level (approximately 5-fold over the basal level) as that induced by OA. However, other biogenic amines, tyramine and dopamine, and chlordimeform were without effects. The [cAMP](i) level raised by OA was lowered by antagonists; the rank order of antagonist activity was chlorpromazine > mianserin = yohimbine. Cyproheptadine and metoclopramide had little effect. OA above 100 nM induced a transient or sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), depending on the concentration of OA. Sequence homology and functional analysis data indicate that BmOAR1 is an alpha-adrenergic-like OA receptor of B. mori.

Regulation of glutamine metabolism during the development of Bombyx mori larvae

Waste ammonia is re-assimilated into amino acids via the amide group of glutamine and the amino group of glutamate (i.e. through glutamine synthetase/glutamate synthase pathway) for silk synthesis in the silkworm, Bombyx mori, in the last larval stadium. Glutamine concentration in hemolymph gradually decreased with the progress of the fifth instar and it remained at very low levels during the spinning stage, then followed by a sharp increase at the larval–pupal ecdysis. The changes in glutamine synthetase (GS) activity in silkworm tissues were relatively small through the larval development, while the changes in glutamate synthase (GOGAT) activity, especially in the posterior silk glands, were more drastic. In addition, activities of GOGAT in the tissues were much higher than those of the other enzymes involved in glutamine utilization, suggesting that glutamine pool was regulated mainly by the changes in GOGAT activity. Western blot analysis indicated that the changes in GOGAT protein level correlated with the changes in GOGAT activity. Topical application of a juvenile hormone analogue, methoprene, induced an accumulation of glutamine in the hemolymph of the fifth instar larvae. The levels of GOGAT protein and activity in the tissues of the methoprene treated larvae were much lower than those of the control larvae, whereas the methoprene treatment had no effect on the levels of GS activity. In conclusion, GOGAT expression promoted by reduction of juvenile hormone titer is quite important for enhanced utilization of nitrogen for synthesis of silk protein during the last larval instar.

Changes in calmodulin levels during development of the silkworm, Bombyx mori

Calmodulin levels were measured in various tissues during the larval-adult development of the silkworm, Bombyx mori. In the larval period, calmodulin levels in fat body, midgut and testis were in a range of 0.3–1.7 μg/mg protein and remained almost constant during larval growth. The silk gland contained a relatively high (0.2 μg/mg protein) level of calmodulin early in the fifth instar which gradually decreased during maturation of the larva. At pupation, testis calmodulin dropped from 1.5 to 1.7 μg/mg protein to about 1 μg/mg, and remained constant thereafter. The most striking change occurred in fat body calmodulin which fell from 0.5 to 0.6 μg/mg in the larval stage to 0.01–0.03 μg/mg during pupal-adult metamorphosis. Midgut calmodulin levels were unchanged at pupation and remained constant during pupal-adult development. When expressed on per g wet weight basis, calmodulin levels in silkworm tissues were comparable to mammalian tissue levels. However, only 2–4% of the total calmodulin in silkworm tissues was in a membrane-bound form compared to 20–60% for membrane-bound calmodulin in mammals.

Inhibition of protease from muscardine fungus by the inhibitor in the silkworm haemolymph and tissues

Inhibition of protease from muscardine fungus by the inhibitor in the silkworm haemolymph and tissues

Inhibition of guanylate cyclase by guanosine 3'-phosphate and 2'-deoxyguanosine 3'-phosphate in silkworm fat body.

Guanosine 3'-phosphate and 2'-deoxyguanosine 3'-phosphate are potent inhibitors of guanylate cyclase of various tissues of the silkworm, Bombyx mori. The inhibitory activity is specific for guanine 3'-nucleotides. Thus, 2'-GMP and 5'-GMP are one order or more less potent than 3'-GMP, and the other 3'-nucleotides except 3'-IMP, i.e. 3'-AMP, 2'-deoxy-3'-AMP, 3'-CMP, and 3'-TMP, have no inhibitory activity. Guanosine and 2'-deoxyguanosine are only weakly active. The 3'-nucleotide inhibition is dependent on the concentration of Mn2+. The concentrations of 2'-deoxy-3'-GMP and 3'-GMP required for 50% inhibition are 0.12 and 0.15 mM, respectively, with 5 mM Mn2+.

Efficiency of artificial blood for preservation of viability of silkworm tissues

Efficiency of artificial blood for preservation of viability of silkworm tissues

Effects of Various Substances on Growth of Silkworm Tissues in Vitro

Cells from the ovaries of late final instal' silkworms have undergone mitosis in hanging-drop preparations for 19 days and survived for 29 days in a medium consisting of \Vyatt's solution plus 10 components of the vitamin B complex, cholesterol, and extracts of endocrine and ovarian tissues. Attempts to subculture the cells met with only partial success.

The Inhibition of Nitrate Reduction with Silkworm Tissue

In the tissue of silkworm the existence of an inhibitor of biological nitrate reduction has been demonstrated. This substance is heat-resistant and cannot be decomposed by autolysis. The inhibitor is contained, in the tissue of larvae, even in the acetone preparation, pupae, digestive juice and worm feces, but not in the blood and alimentary canal. When the worm is attacked with polyhedral virus disease or fed with nitrites, inhibiting ability of tissue is retained.This inhibitor was isolated by extraction with diluted alkaline solution following precipitation at about pH 4.2. On the basis of ultraviolet absorption spectrum and Murexide reaction, the inhibitor was deduced to be uric acid. Thence, the inhibiting action of uric acid was comparatively studied. Both the inhibitor isolated and pure uric acid diminished their inhibiting capacity by the pretreatment of mouse liver containing uricase, but not so in the case of fowl which has not such enzyme.The reducing ability of larval tissue was attested by th...