Selective diminishment of virus-specific translatable mRNA in the densonucleosis virus-infected midgut of the silkworm, Bombyx mori, reared at a supraoptimal temperature

Effect of a supraoptimal temperature on the accumulation of virus-specific translatable mRNAs was examined in the larvae of the silkworm. Bombyx mori, infected with Bombyx densonucleosis virus type 2. The results showed that incubation of infected larvae at 35 degrees C resulted in a rapid and selective decrease in the virus-specific translatable mRNAs which preexisted in the infected midgut. Upon temperature-shift from 35 degrees to 25 degrees C, the virus-specific translatable mRNAs became clearly observed within 12 hr. These results indicate that a supraoptimal temperature restricts the accumulation of virus-specific translatable mRNAs. Both rapid decay and suppressed synthesis might be responsible for the selective restriction of virus-specific translatable mRNA accumulation at a supraoptimal temperature.     

Proteomic studies of lipopolysaccharide-induced polypeptides in the silkworm, Bombyx mori

Silkworm larvae at the 5th instar were injected with lipopolysaccharide from Escherichia coli and inducible polypeptides were examined within a pI range of 3-10 and a size range of 14-97 kDa by proteomics, including peptide mass fingerprinting. No polypeptides were induced in the midgut. FB1 and H1-4 polypeptides were significantly induced in fat body and hemolymph, respectively. FB1 and H1 were estimated to be antitrypsin and serpin-2 proteinase inhibitors respectively. H2 and H3 were novel polypeptides. H4 was estimated to be attacin antibacterial polypeptide with high coverage of sequence. The amounts of all the induced polypeptides decreased at 48 h after the injection.     

家蚕二分浓核病毒感染家蚕后的释放动态研究

【目的】研究家蚕二分浓核病毒(Bm BDV)感染家蚕后不同时间段内释放到环境中的病毒量,为通过蚕沙快速检测病毒提供方法和依据。【方法】选取家蚕二分浓核病毒基因组节段VD1上的ns1基因和VD2上的ns3基因中的一小部分片段,分别克隆到p MD18-T载体上,制备标准品质粒,并用实时荧光定量PCR的方法绘制ns1和ns3的标准曲线。通过检测Bm BDV感染5龄家蚕后不同时间蚕沙中的病毒量来衡量释放到环境中的病毒的变化动态。【结果】在病毒感染家蚕第3天,大量的病毒随蚕沙释放到环境中,每微克蚕沙中约含1.38×106个VD1,6.54×105个VD2;从感染后第5天开始,释放到环境中的病毒量呈指数型增加;第7-8天病毒的释放量到达一个平台期,此时每微克蚕沙中约有2.12×107个VD1,1.34×107个VD2。普通PCR结果也反应出了类似的病毒释放动态。【结论】根据Bm BDV感染家蚕后的释放动态,推测病毒的复制周期约60 h。利用实时荧光定量PCR检测蚕沙中的Bm BDV,能简单、快速、准确地诊断病毒的感染。     

Genetically determined nonsusceptibility of the silkworm,Bombyx mori,to infection with a densonucleosis virus (Densovirus)

A nonsusceptible and a highly susceptible strain of the silkworm, Bombyx mori, to peroral infection with a densonucleosis virus (DNV) were studied by probit analysis. Tests with the susceptible and nonsusceptible parent strains, their reciprocal F₁ hybrids, the F₂ hybrid, and the backcrossed hybrids to either of the parents demonstrated that the nonsusceptibility of the silkworm to DNV was inherited and controlled by a recessive gene which was not sex-linked.     

Alkaline proteases in the midgut tissue and digestive fluid of the silkworm,Bombyx mori

In larvae of Bombyx mori proteolytic activity was found in the midgut tissue and digestive fluid. The pH-activity curves of both proteases were very similar and optimal activity was about pH 11.2. The reduction in activity at 50°C, for 10 min was about 80% in digestive fluid protease, and about 40% in tissue protease. HgCl₂ and DFP strongly inhibited protease activity, and the influence was greater in digestive fluid than in the midgut tissue. Most of the tissue proteases was found to be membrane bound enzyme from results of differential centrifugation and column experiments.     

Developmental changes in midgut trehalase activity and its localization in the silkworm, Bombyx mori

The changes in trehalase activity and its localization in the midgut of the silkworm, Bombyx mori, were studied during larval-pupal-adult development. Trehalase activity in larval midgut epithelium increased with the larval growth, reached a maximum level at the middle of the fifth instar, and then decreased gradually. Trehalase activity in larval midgut was found in the epithelial tissue but not in the digestive juice or the midgut contents.The trehalase activity in the whole midgut started to rise at the onset of spinning and increased abruptly at larval-pupal ecdysis to reach an extremely high level 3 days later. This high activity was maintained throughout the subsequent pharate adult development and dropped suddenly at emergence. The midgut trehalase activity during pupal-adult development was mainly found in the midgut contents but scarcely any in the epithelium.Subcellular distribution of midgut trehalase depended upon larval-pupal-adult development. The activity was concentrated in a precipitate fraction of the epithelium until the middle of the fifth instar. During larval-pupal development, however, the activity increased in the soluble fraction with a concomitant decrease in the precipitate fraction. Almost all the trehalase activity in pupal and pharate adult midgut was recovered in the soluble fraction of the midgut contents. The data are discussed from a viewpoint of the histolysis.     

Cellular localization and proposed function of midgut trehalase in the silkworm larva, Bombyx mori

A sorbitol density gradient analysis with the aid of several marker enzymes demonstrated that midgut trehalase of the silkworm larvae. Bombyx mori, was localized in the microsomal membranes, but not in mitochondria, lysosomes and microvilli at the apical surface. Electron microscopic examination showed that trehalase-enriched membrane fraction consisted of heterogeneous mixtures of membrane vesicles derived from the endoplasmic reticulum and plasma membrane parts other than the microvillus membrane. The enzyme-histochemical stains of trehalase activity on the midgut section could be detected only at the basal surface of the epithelium against haemocoel. Such a specific localization was further confirmed by immunohistochemistry with the peroxidase-conjugated antibody technique. Thus, it is concluded that midgut trehalase of silkworm larvae is situated on the plasma membrane at the basal surface of the epithelium. An intact preparation of midgut incubated in vitro in the medium containing [(14)C]trehalose could hydrolyse trehalose into glucose and take it up into the cell, although some glucose was liberated into the medium when incubated for extended periods. These results suggest that midgut trehalase plays a physiological role in utilization of haemolymph trehalose not in nutrient absorption.     

Difference of proteins from inclusion bodies formed in the nucleus and cytoplasm of the cytoplasmic polyhedrosis virus-infected midgut in the silkworm,Bombyx mori

Strains of cytoplasmic polyhedrosis virus (CPV) of the silkworm Bombyx mori typically form proteinaceous inclusion bodies (IBs) which occlude many virions and are formed in the cytoplasm of the midgut epithelium. In contrast, an unusual strain of CPV termed “A” strain produces IBs containing no virions in the nuclei of the epithelial cells. In this case although the viruses multiply in the cytoplasm, few IBs are formed in the cytoplasm. To clarify why the A strain forms IBs in the nucleus, the structural differences on the IB proteins (IBPs) from A and a typical (H) strain were investigated. Analyses by SDS-PAGE showed A strain IBP had slightly lower electrophoretic mobility than these of H strain. When these IBPs were partially digested with Staphylococcus aureus V8 protease, one of the digested products between the two strains showed different electrophoretic mobility. Amino acid sequence analyses of peptides produced with lysylendopeptidase from IBP of A strain indicated that a histidine residue of H strain was replaced by a tyrosine residue. The carboxyl terminal regions of the two IBPs were also different; IBP of A strain was -Leu-Leu-Val-COOH, but the terminal residue of H strain could not be determined by the same method. These differences of amino acid sequence of IBPs between A and H strains may be responsible for the partitioning of them; i.e., in the case of A strain IBP may be transportable into the nucleus by the specific signal of amino acid sequence.     

Antiviral proteins in midgut of the silkworm, Bombyx mori, fed on different food sources

Midgut proteins from the silkworms, Bombyx mori, fed on an artificial diet containing dried powder of the spiral blue-green alga, Spirulina platensis, had a very low fluorescence intensity. This indicated that chlorophyll in this alga is not utilizable for synthesizing the red fluorescent protein (RFP) which has been considered as an effective antiviral protein in silkworm midgut. Many electrophoretic bands of the midgut proteins from the mulberry-fed silkworms emitted red fluorescence under UV light. It is thus concluded that the RFP as a whole is more than one midgut protein. Enzymatic analyses showed that silkworms fed on mulberry leaves and artificial diets had similar protease activities, but they had different phospholipase C activities. Therefore, it is suggested that the antiviral activity of purified RFP in the silkworm gut juice is attributable to phospholipase C.

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Résumé Les protéines de l'intestin moyen de Bombyx mori, nourri avec un aliment artificiel contenant de la poudre séchée de l'algue bleue spiralée, Spirulina pratensis, avaient une très faible fluorescence. Cesi indique que la chlorophylle a de cette algue n'est pas utilisable pour la synthèse de la protéine fluorescente rouge (RFP), considérée comme une protéine antivirale efficace de l'intestin moyen. Après séparation par électrophorèse sur gel de polyacrylamine DISC plusieurs bandes de la protéine de l'intestin moyen de B. mori élevé sur mûrier émettaient une fluorescence rouge en lumière U.V. On peut conclure que RFP est un complexe plutôt qu'une protéine particulière de l'intestin moyen. Les analyses enzymatiques ont montré que la phospholipase C et non les protéases ont une activité antivirale dans la RFP purifiée provenant de vers à soie nourris à partir de mûrier ou sur régime artificiel.

     

家蚕对浓核病毒中国株(BmDNV-3)抗性及感性品系中肠的差异蛋白质分析

【目的】通过比较家蚕Bombyx mori抗性及感性品系的中肠蛋白质表达谱,获得家蚕对家蚕浓核病毒中国株（BmDNV-3）抗性相关的蛋白。【方法】利用双向电泳(2-DE)对感性品种华八35和抗性品种秋丰接种病毒后48 h的蛋白质表达谱进行比较分析,并对其中的差异蛋白进行MALDI-TOF-TOF质谱分析,通过NCBInr和MSDB数据库进行蛋白点的鉴定和功能分析。【结果】获得重复性较好的差异蛋白点16个,其中质谱鉴定出5种蛋白,它们分别是糖基转移酶（glycosyltransferase）、糖基转移酶-S（GlcAT-S）、21.5 kD小热休克蛋白（21.5 kD small heat shock protein）、V-ATP酶（vacuolar ATP synthase）和精氨酸激酶（arginine kinase）。这5种蛋白在抗性品系秋丰中的表达量均高于感性品系华八35。【结论】糖基转移酶和糖基转移酶-S仅在抗性品系中存在,提示它们可能是与抗性有关的蛋白。此外,增强的应激反应和能量代谢也可能与家蚕对BmDNV-3的抗性产生相关。     

Storage proteins in the silkworm,Bombyx mori

In the silkworm, Bombyx mori, two storage proteins named SP-1 and SP-2 were shown to decline in concentration in the haemolymph and increase in the fat body during the larval-pupal transformation, when protein granules are formed in the fat body at the same time as the degeneration of mitochondria and endoplasmic reticulum. At the larval-pupal ecdysis, in females the two proteins account for 60% of total fat body protein (80% of the soluble protein), while males have very little SP-1 and SP-2 comprises only 20% of the total fat body protein. The concentration of protein granules in the fat body cytoplasm is much greater in females than in males, and the granules in females have partially crystalline inner zones. This is different from males where granules with non-crystalline structure are most numerous.The properties of these proteins purified from pupal fat body are similar to those of Cecropia storage proteins and calliphorin, all of which have molecular weights of around 500,000 and are composed of subunits of mol. wt about 85,000. SP-1 differs from SP-2 by having an exceptionally high content of methionine, but much less glutamate, phenylalanine and tyrosine. SP-1 resembles another female-specific protein, vitellogenin and SP-2 resembles calliphorin in amino acid composition.From these results, it is concluded that SP-1 and SP-2 have storage roles and are deposited in protein granules.     

Purification and characterization of membrane-bound alkaline proteases from midgut tissue of the silkworm, Bombyx mori

Membrane-bound alkaline proteases from the midgut epithelia of the silkworm, Bombyx mori, were solubilized with 1% Lubrol-WX, at pH 11.2. They were purified by gel filtration on Sepharose 6B and Ultrogel AcA-202 columns and a preparative polyacrylamide gel electrophoresis. Two proteases, caseinolytic (6B3-Tc) and benzoyl-arginine-p-nitroanilide-lytic (6B3-Tb) were obtained. Both enzymes were homogeneous as judged by polyacrylamide electrophoresis. These enzymes showed high pH optima, 11.2, and pI values, above 11, and were extremely stable over a wide range of pH. The Km values for 6B3-Tb and Tc were 0.476 mM and 2.5 mg/ml respectively. Hammarsten casein and mulberry leaf protein were rapidly hydrolyzed by Tc, whereas the hydrolytic activity of Tb for Azocoll was higher than that of Tc. The protease Tb was strongly inhibited by diisopropylfluorophosphate, p-chloromercuribenzoate, benzamidine, leupeptin, and soybean trypsin inhibitor; Tc was inhibited by diisopropylfluorophosphate, tosyl phenylalanine chloromethylketone and chymostatin, but not by tosyl lysine chloromethylketone, p-chloromercuribenzoate, or iodoacetamide. The molecular weights of the proteases were estimated to be 12,800 (Tb) and 13,300 (Tc) by Sephacryl S-300 gel filtration. The amino acid analyses showed that both proteases contain a large number of acidic amino acids but a relatively small number of basic amino acids.     

Expression analysis of chlorophyllid a binding protein a secretory,red fluorescence protein in the midgut of silkworm,Bombyx mori

Chlorophyllid a binding protein （ chbp） was recently characterized by its ability to bind the prosthetic group of chlorophylls and little information is known regarding its expression. In the present study, we found that chpb was expressed highly and exclusively in the midgut of silkworm, Bombyx mori. The expression level of chbp was very high in the newly molted fifth instar larvae followed by gradual decline in the same instar. Our results demonstrated that CHBP was a secretory protein and located mainly in the apical of midgut epithelial cells. Real-time polymerase chain reaction analysis results showed that chpb highly expressed in the anterior midgut, threefold and sixfold higher compared with that of the middle midgut and posterior midgut, respectively, and chpb expression declined in darkness. In addition, the expression of chbp was affected by high-dose virus or bacterium infection.     

家蚕拟浆细胞具有吞噬功能

目的：通过观察家蚕Bombyx mori吞噬细胞的微细结构,来确定拟绛色细胞是否也具有吞噬功能。方法：用荧光小球微量注射家蚕pnd pS品系的幼虫,经荧光染色剂丫啶橙和碘化丙啶染色循环血细胞后,在荧光显微镜下观察并扫描拍摄。结果：观察发现除颗粒细胞和浆血细胞外,一些原血球细胞（血干细胞）和拟绛色细胞（多酚氧化酶）也能吞噬荧光小球。在拟绛色细胞里还发现许多和颗粒细胞一样的能被丫啶橙染色的颗粒。尽管在小球细胞中没有发现被吞噬的荧光小球,但该类血球有比较多的能被丫啶橙染色的大颗粒,这表明它们可能是已经被吞噬的凋亡小体。结论：除颗粒细胞和浆血细胞外,一些原血球细胞和拟绛色细胞也能吞噬荧光小球。说明拟绛色细胞也具有吞噬功能。     

Dietary sterol preference in the silkworm, Bombyx mori

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that beta-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: beta-sitosterol > ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouth part might be different from the sterol recognition mechanism present in sterol metabolism.     

Multiple transport pathways for dibasic amino acids in the larval midgut of the silkworm Bombyx mori

The transport pathways for dibasic amino acids were investigated in brush border membrane vesicles (BBMV) from the anterior-middle (AM) and posterior (P) regions of Bombyx mori midgut. In the absence of K(+), a low-affinity saturable transport of arginine in both AM- and P-BBMV (K(m) 1.01 mM, V(max) 4.07 nmol/7s/mg protein and K(m) 1.38 mM, V(max) 2.26 nmol/7s/mg protein, respectively) was detected. Arginine influx was dependent on the membrane electrical potential (Deltapsi) and increased raising the alkalinity of the external medium from pH 7.2 to 10.6. Competition experiments indicated the following order of substrate affinity: arginine, homoarginine, N(G)-monomethylarginine, N(G)-nitroarginine>lysine>ornithine>cysteine>methionine. Leucine, valine and BCH (2-amino-2-norbornanecarboxylic acid) did not inhibit arginine influx. In the presence of external K(+), the influx of arginine as a function of arginine concentration fitted to a complex saturation kinetics compatible with both a low-affinity and a high-affinity component. The latter (K(m) 0.035 mM, V(max) 2.54 nmol/7s/mg protein) was fully characterized. The influx rate had an optimum at pH 8.8, was strongly affected by Deltapsi and was homogeneous along the midgut. The substrate affinity rank was: homoarginine>arginine, N(G)-monomethylarginine>cysteine, lysine>N(G)-nitroarginine>ornithine>methionine. Leucine and amino acids with a hydrophobic side chain were not accepted. This system is also operative in the absence of potassium, with the same order of specificity but a very low activity. Lysine influx is mediated by two more transport systems, the leucine uniport and the K(+)/leucine symport specific for amino acids with a hydrophobic side chain that recognizes lysine at extravesicular pH values (pH(out)) exceeding 9. Both the uniport and the symport differ from the cationic transport systems so far identified in mammals because they are unaffected by N-ethylmaleimide, have no significant affinity for neutral amino acids in the presence of the cation and show a striking difference in their optimum pH.