Occurrence of sepiapterin deaminase in the normal type silkworm, Bombyx mori

Sepiapterin deaminase was prepared from the normal strain of the silkworm, Bombyx mori. After inhibition experiments, this enzyme was found to have the same properties as that isolated from the lemon mutant strain. Several new inhibitors and their Ki values are described for the deaminase.     

Evaluation of biological effects of three neodymium compounds on silkworm,Bombyx mori

Neodymium (Nd) is one of the most famous members of rare earth elements family. With the increasing global demand for Nd, it can inevitably enter the environment and affect the ecological environment through the food chain and other ways. Therefore, it is very important to study the biological effects of Nd. In this study, the biological effects of 3 kinds of Nd compounds, i.e. neodymium oxide (Nd₂O₃), neodymium chloride (NdCl₃) and neodymium nitrate (Nd(NO₃)₃), were evaluated by using silkworm models through the method of intravascular injection. Each of the three Nd compounds was prepared into 6 groups at different concentrations (40, 50, 60, 70, 80, 90 μg/μL). The results show that within the certain concentration range, Nd compounds show great damage to the growth of silkworms since high death rate is observed: the death rate from Nd₂O₃ group is floating between 30% and 80%, the one from NdCl₃ group is between 17% and 33% and that from Nd(NO₃)₃ group is between 23% and 37%. It also reveals that Nd compounds have some negative effects on the body length and weight of silkworms. However, the histophysiological study shows that the three Nd compounds cause no obvious negative effects on the cell morphologies in midgut, fat body and silk gland. The XRD and FTIR spectra show that the secondary and the crystalline structures of silks are not destroyed, the TGA and DTG data exhibit that the thermal stability of silks is not damaged, and the tensile test shows that the mechanical properties of silks are not affected significantly by the intake of Nd compounds. In general, the intake of Nd compounds has significant inhibitory effects on silkworm growth but little negative impact on silk structures or cell morphology, the study can provide useful toxicological data for the safe application of Nd compounds in various fields.     

A composite genetic map of the parasitoid wasp Trichogramma brassicae based on RAPD markers

OBJECTIVES: Our objectives were to identify and define a minimum set of variables for interventional cardiology that carried the most statistical weight for predicting adverse outcomes. Though "gaming" cannot be completely avoided, variables were to be as objective as possible and reproducible and had to be predictive of outcome in current databases. BACKGROUND: Outcomes of percutaneous coronary interventions depend on patient risk characteristics and disease severity and acuity. Comparing results of interventions has been difficult because definitions of similar variables differ in databases, and variables are not uniformly tracked. Identifying the best predictor variables and standardizing their definitions are a first step in developing a universal stratification instrument. METHODS: A list of empirically derived variables was first tested in eight cardiac databases (158,273 cases). Three end points (in-hospital death, in-hospital coronary artery bypass graft surgery, Q wave myocardial infarction) were chosen for analysis. Univariate and multivariate regression models were used to quantify the predictive value of the variable in each database. The variables were then defined by consensus by a panel of experts. RESULTS: In all databases patient demographics were similar, but disease severity varied greatly. The most powerful predictors of adverse outcome were measures of hemodynamic instability, disease severity, demographics and comorbid conditions in both univariate and multivariate analyses. CONCLUSIONS: Our analysis identified 29 variables that have the strongest statistical association with adverse outcomes after coronary interventions. These variables were also objectively defined. Incorporation of these variables into every cardiac dataset will provide uniform standards for data collected. Comparisons of outcomes among physicians, institutions and databases will therefore be more meaningful.     

Behavior of centrioles during meiosis in the male silkworm, Bombyx mori (Lepidoptera)

The behavior of centrioles during eupyrene and apyrene meiosis was examined in the silkworm, Bombyx mori, by transmission electron microscopy and indirect immunofluorescence for tubulin. In eupyrene spermatocytes the centrioles, accompanied by axonemes, attached temporarily to the nucleus at diplotene, then detached from the nucleus in diakinesis. After the separation, a beret-shaped structure consisting of a double membrane covered the proximal region of the pair of centrioles. The structure disappeared after breakdown of the nuclear membrane. The centriole, with the axoneme, reattached to the nucleus at telophase I. The process was repeated during meiosis II until the centrioles maintained their nuclear attachment in newly developed spermatids. In stark contrast to their eupyrene counterparts, apyrene spermatocytes were conspicuously devoid of any attachment of the centrioles to the nucleus. These eupyrene-specific and apyrene-specific relationships were consistently and repeatedly found between the nuclear membrane and centrioles, giving rise to suspicion that the behavioral phenomena may be related to differentiation of the dimorphic sperm types.     

Application of NMR microscopy to the morphological study of the silkworm, Bombyx mori, during its metamorphosis

Zero (ZQ) and double (DQ) quantum 2D chemical shift selective and spin-echo 3D NMR imaging at microscopy resolution, has been applied to the morphological study of silkworm, Bombyx mori, during its metamorphosis. Attention had been focused on the evolution of the internal structure of the insect during its postembryonal life occurring through the larval, pupal and adult development. A major objective of this work was the characterization of the silk glands, responsible for the synthesis and secretion of fibroin and sericin, through the changes of distribution and mobility of water, by imaging the water protons during postembryonal life stages. Moreover, alanine deriving from silk gland proteins was imaged during the last life stage of Bombyx mori.     

Induction of gene expression of antibacterial proteins by chitin oligomers in the silkworm, Bombyx mori

A 71-year-old male with advanced gastric carcinoma with paraaortic lymph node metastases underwent distal gastrectomy. Cisplatin (CDDP) 50 mg/body was administered intravenously (i.v.) on day 1 followed by the administration of 5-fluorouracil 500 mg/body/day i.v. on day 2 through day 7. After two courses of this regimen, further enlargement of paraaortic lymph nodes was revealed by CT scan, and chemotherapy was suspended. Multiple liver and lung metastases were diagnosed 6 months after initial diagnosis, and mitomycin C (MMC) 10 mg/body i.v. was administered on day 1 followed by CDDP 50 mg/body i.v. on day 2. After three courses of this regimen, partial response of the liver metastases and complete response of the lung metastases were observed, and the general condition was markedly improved without any adverse effect except slight nausea. Though the patient died of brain metastases one year after initial diagnosis, the combination chemotherapy with MMC and CDDP was nevertheless thought to improve his quality of life.     

Transovarian transmission of a foreign gene in the silkworm, Bombyx mori, by Autographa californica nuclear polyhedrosis virus

We introduced a firefly luciferase gene, expressed under control of Drosophila heat shock protein gene promoter, into Autographa californica nuclear polyhedrosis virus (AcNPV). When the 5th instar larvae of the silkworm, Bombyx mori, were inoculated with the recombinant virus, luciferase activities were detected in the virus-infected larvae and pupae, and in the newly hatched larvae of the next generation. PCR amplification and Southern blot hybridization analysis demonstrated that the luciferase gene was transmitted through at least the F2 generation. In addition, the V-cathepsin gene, encoding a cysteine protease of AcNPV, was also detected in the DNA of all individuals of the F2 generation. These results show that AcNPV can be utilized as vector for the transovarian transmission of foreign genes in the silkworm.     

In vitro phagocytosis of Escherichia coli and release of lipopolysaccharide by adhering hemocytes of the silkworm, Bombyx mori

A primary culture containing adhering hemocytes mainly granular cells from the silkworm, Bombyx mori, was used to investigate in vitro phagocytosis of Escherichia coli. Phagocytosis was confirmed to occur in this system by microscopic observation. Lipopolysaccharide (LPS) concentration in the culture medium was measured by a Limulus test and a higher LPS concentration was detected in phagocytosis-occurred samples than in control samples, which omitted either E. coli cells or adhering hemocytes. Moreover, it was found that LPS containing sample but not control samples strongly induces gene expression of cecropin B, an antibacterial protein. These results suggest that bacterial cell wall components like LPS released by phagocytosis play an important role in the induction of insect antibacterial proteins.     

Protein A linked latex antisera test for detection of infectious flacherie of silkworm, Bombyx mori L. caused by B. mori infectious flacherie virus

Protein A linked latex antisera (PALLAS) test was used for detection of B. mori infectious flacherie virus (BmIFV) in silkworms. The test was found superior to direct latex agglutination (DLA) test for detection of BmIFV in infected larval and faecal matter extracts and purified preparations. Latex beads sensitized with anti-BmIFV IgG were able to detect up to 75 ng/ml BmIFV in PALLAS test and 300 ng/ml in DLA test. BmIFV was detected in diluted extracts of infected larvae (up to 1:200) and their faecal matter (up to 1:500). BmIFV progressive infection was detected at 12 and 24 hr of post infection in PALLAS and DLA tests, respectively in both larval and faecal matter extracts. PALLAS tests provides a simple, specific and highly sensitive test for detection of infectious flacherie virus.     

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.     

A walk in the chorion locus of Bombyx mori

We have recovered overlapping clones that represent in the aggregate a contiguous segment of chromosomal DNA 270 kb in length, or probably one third of the chorion locus of Bombyx mori. Approximately 70 genes have been identified, the majority of which are arranged in coordinately expressed pairs. The nonidentical genes expressed in the late period of choriogenesis are clustered within a single, 130 kb region, which is flanked by regions containing genes that are active during the middle developmental period. The late genes encode two families of high-cysteine proteins; the evolutionarily persistent clustering of these families contrasts sharply with the extensive sequence diversification of the structural genes and their flanking DNA elements. We discuss the possible regulatory significance of the clustered arrangement, as well as certain features of multigene family evolution.     

Behaviors of nucleus, basal bodies and microtubules during eupyrene and apyrene spermiogenesis in the silkworm, Bombyx mori (Lepidoptera)

Immunofluorescent studies with anti-2A antisera, raised specifically against a synthetic C-terminal peptide of native murine P70, the testes-specific cognate heat shock protein 70, demonstrated that the rat homologue of P70 is expressed on the surface of testicular cells. The murine hsp 70.2 gene, encoding P70, was cloned and expressed in Escherichia coli. The recombinant P70 (rP70) protein with a 6Xhistidine affinity tag at its amino terminus was purified from E. coli via nickel affinity column chromatography. Monoclonal anti-hsp70 antisera and anti-2A antisera cross-reacted with purified rP70. Binding of rP70 was specific for sulfogalactosylceramide (SGC) and sulfogalactosyglycerolipid (SGG). Binding was not inhibited by the sugar, galactose 3'sulfate, nor was binding observed to desulfated derivatives of SGC and SGG, to other negatively charged lipids or other sulfated lipids. Furthermore, rP70 bound to an SGC-column and was eluted only at high salt in combination with high pH. These results show rP70 to possess a specific sulfatide binding site. Since the biochemical properties and immunoreactivity of rP70 are indistinguishable from native P70 and SLIP1 (testicular sulfoglycolipid immobilized protein 1) rP70 can be employed to examine the role of hsp70-mediated sulfatide binding in fertilization.     

Small GTP-binding proteins in the brain-corpus cardiacum-corpus allatum complex of the silkworm, Bombyx mori: involvement in the secretion of prothoracicotropic hormone

BACKGROUND: Proteolysis, modulated in part by intramural fibrinolytic system proteins and their inhibitors, appears to influence vascular smooth muscle cell (SMC) migration and proliferation and remodeling of extracellular matrix (ECM). Alterations of fibrinolysis in circulating blood and of proteolysis within vessel walls in experimental animals and patients with diabetes have been associated with accelerated vascular disease. Hyaluronan, a prominent component of ECM in normal vessels, is increased in the tunica media of macroscopically normal arterial vessels from patients with type 2 diabetes. OBJECTIVE: To determine whether hyaluronan alters the expression of the fibrinolytic system protein, plasminogen activator inhibitor type-1 (PAI-1), in human vascular SMCs, thereby potentially accelerating vascular disease in patients with type 2 diabetes. METHODS: Urokinase-type and tissue-type plasminogen activators (uPA and tPA) and PAI-1 were assayed in vascular SMC conditioned media and in cell lysates, using enzyme-linked immunosorbent assay and western blotting. RESULTS: Hyaluronan increased the 24-h release of PAI-1 into conditioned media in a concentration-dependent and time-dependent manner (1.8-fold compared with control with 1 mg/ml hyaluronan; n = 9, P < 0.01). Although the accumulation of uPA in conditioned media tended to increase also, uPA content was reduced in cell lysates (64% of control with 0.1 mg/ml hyaluronan at 24 h; n = 9, P < 0.01) without any change in PAI-1. Concentrations of tPA in conditioned media and cell lysates were unchanged. Digestion of hyaluronan with hyaluronidase (50 turbidity reducing units (TRU)/ml) or exposure of the smooth muscle cells to antihuman CD44 antibody (1 microgram/ml) that binds to the hyaluronan cell surface receptor obviated the effects of hyaluronan. CONCLUSION: Our results indicate that increases in hyaluronan increase vascular SMC expression of PAI-1, a phenomenon that may alter the balance between proteolysis and its inhibition in vessels of patients with type 2 diabetes, thereby contributing to the acceleration of macroangiopathy.     

Studies on the posterior silk gland of the silkworm Bombyx mori. VI. Distribution of microtubules in the posterior silk gland cells

There are two microtubule systems in the posterior silk gland cells. One is a radial microtubule system in which the microtubules run radially from the basal to the apical cytoplasm and in which fibroin globules (secretory granules of fibroin) and mitochondria are arranged along these microtubules, thus composing a "canal system" which is assumed to be responsible for the intracellular transport of fibroin globules. The other is a circular microtubule system in the apical cytoplasm which is composed of bundles of microtubules and microfilaments running in a circular arrangement around the glandular lumen at an interval of approximately 4 mum at the end of the fifth instar. This system is presumably concerned with secretion and/or intraluminal transport of fibroin.     

Effect of ether anesthesia on the circulatory rhythm of adult lepidoptera, Bombyx mori

Long marketed antiemetics, which are still used in medicine or veterinary medicine and whose sites of actions were either assumed to be peripheral or unknown, were subjected to evaluation as antiemetics, against oral copper sulfate emesis. In cats, ethyl aminobenzoate 0.5 g/head, cerium oxalate 0.1 g/head, pinelliae tuber 0.5 g/head and gelatin 2.0 g/head were not effective. In dogs, ethyl aminobenzoate 0.5 g/head, pinelliae tuber 2.0 g/head and hange-syasintoo 2.0 g/head were not effective. Cerium oxalate 0.1 g/head was found to have a slight antiemetic action.     

A comparative analysis of Ungulata species by different molecular genetic markers (proteins, RAPD-PCR)]

The investigation of genetic interrelation between a number of Artiodactyla and Perissodactyla species with the use of different types of molecular-genetic markers (proteins, RAPD-PCR) were carried out. The marker-specific features of interspecific relations and their similarities on the groups of markers of both types were revealed. The distinctions between interspecies genetic relations and ones estimated from the phylogeny on the determined group of different types of markers were observed. It was supposed that these discrepancies may be related with common selection factors and involving this marker group in selection in some species.     

The molecular karyotype of the megabase chromosomes of Trypanosoma brucei and the assignment of chromosome markers

We present the molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock TREU927/4 (927). We have identified 11 diploid chromosomes ranging in size from 1 to 5.2 Mb approximately and pairs of homologues differ in size by up to 15%. A total of 401 cDNA probes were hybridised to T. brucei stock 927 chromosomes and 168 chromosome-specific markers were defined. Most of these markers were hybridised to the separated chromosomal DNA of two other cloned field isolates and four F1 progeny clones from a laboratory cross. The chromosomes vary in size by up to two and a half times between stocks and the DNA content of the 11 pairs of homologues varies by up to 33% in different stocks. Stock 927 contains the smallest chromosomes and the least nuclear genomic DNA. Nevertheless, all 11 syntenic groups of cDNA probes are maintained in all stocks. In the F1 hybrids only we have identified one extra PFG band to which none of our probes hybridise. We have shown that probes thought to be specific for the bloodstream-form variant surface glycoprotein expression sites hybridise to different chromosomes in different stocks and may hybridise to either one or both of a homologous pair of chromosomes. We have also determined the chromosomal location of the ribosomal RNA gene arrays.     

The lipopolysaccharide-binding protein participating in hemocyte nodule formation in the silkworm Bombyx mori is a novel member of the C-type lectin superfamily with two different tandem carbohydrate-recognition domains

We recently isolated and characterized the lipopolysaccharide (LPS)-binding protein, BmLBP, from the larval hemolymph of the silkworm Bombyx mori. BmLBP is a pattern recognition molecule that recognizes the lipid A portion of LPS and participates in a cellular defense reaction. This paper describes the cDNA cloning of BmLBP. The deduced amino acid sequence of BmLBP revealed that BmLBP is a novel member of the C-type lectin superfamily with a unique structural feature that consists of two different carbohydrate-recognition domains in tandem, a short and a long form.