Overexpression of celB gene coding for beta-glucosidase from Pyrococcus furiosus using a baculovirus expression vector system in silkworm, Bombyx mori

beta-Glucosidase is a member of the glycosyl hydrolases that specifically catalyze the hydrolysis of terminal nonreducing beta-D-glucose residues from the end of various oligosaccharides with the release of beta-D-glucose. CelB gene, encoding the thermostable beta-glucosidase, was amplified from the Pyrococcus furiosus genome and then cloned into the baculoviral transfer vector under the control of the polyhedrin gene promoter. After co-transfection with the genetically modified parental Bombyx mori nucleopolyhedrovirus (BmNPV), the recombinant virus containing celB gene was used to express beta-glucosidase in silkworm. The recombinant beta-glucosidase was purified to about 81% homogeneity in a single heat-treatment step. The optimal activity of the expressed beta-glucosidase was obtained at pH 5.0 and about 105 degrees C; divalent cations and high ionic strength did not affect the activity remarkably. This suggested that the enzymatic characteristics of recombinant beta-glucosidase were similar to the native counterpart. The expressed beta-glucosidase accounted for more than 10% of silkworm total haemolymph proteins according to the protein quantification and densimeter scanning. The expression level reached 10,199.5 U per ml haemolymph and 19,797.4 U per silkworm larva, and the specific activity of the one-step purified crude enzyme was 885 U per mg. It was demonstrated to be an attractive approach for mass production of thermostable beta-glucosidase using this system.     

Morphogenetic mechanisms of coelom formation in the direct-developing sea urchin <Emphasis Type="Italic">Heliocidaris erythrogramma</Emphasis>

Indirect development via a feeding pluteus larva represents the ancestral mode of sea urchin development. However, some sea urchin species exhibit a derived form of development, called direct development, in which features of the feeding larva are replaced by accelerated development of the adult. A major difference between these two developmental modes is the timing of the formation of the left coelom and initiation of adult development. These processes occur much earlier in developmental and absolute time in direct developers and may be underlain by changes in morphogenetic processes. In this study, we explore whether differences in the cellular mechanisms responsible for the development of the left coelom and adult structures are associated with the change in the timing of their formation in the direct-developing sea urchin Heliocidaris erythrogramma. We present evidence that left coelom formation in H. erythrogramma, which differs in major aspects of coelom formation in indirect developers, is not a result of cell division. Further, we demonstrate that subsequent development of adult structures requires cell division.     

Changes in growth and lipid profiles of silk gland,mid-gut biochemical composition of silkworm,Bombyx mori L. on exposure to prostaglandin F2alpha

The growth of the silkworm is influenced by the outside and inside environment. Among them, the category of various endocrine hormone of inside is the main factors that adjust the characters such as growth and propagate. In this experiment, we applied different dosage of prostaglandin to the fourth and fifth instar silkworm to observe the effects of prostaglandin F2alpha (PGF2alpha) on silk gland growth, mid-gut biochemical constituents and the lipid profiles of silkworm larva, Bombyx mori L. The weight of the posterior silk gland increased significantly (P < 0.001) by 20-24% after treatment with PGF2alpha. The increase in the lipid profiles except lipase activity suggests that the silk gland had more synthetic activity that might reflect in active spinning of silkworm larva. The changes of total proteins, free amino acids and alkaline phosphatase in mid-gut of control and PGF2alpha treated silkworm, B. mori L. indicate that PGF2alpha favored stimulatory effect on physiology of digestion, absorption and transportation of nutrients which might influence on the growth and development of larva.     

Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm <Emphasis Type="Italic">Bombyx mori</Emphasis>

To construct an efficient system for the production of recombinant proteins in silkworm (Bombyx mori), we investigated the promoter activity of the silkworm sericin 1, 2, and 3 genes (Ser1, Ser2, and Ser3) using a GAL4/UAS binary gene expression system in transgenic silkworm. The promoter activity of the upstream region of Ser1 was strong, yielding high expression of an enhanced green fluorescent protein (EGFP) transgene in the middle and posterior regions of the middle silk gland (MSG) after day 2 of the fifth instar. The Ser3 upstream region exhibited moderate promoter activity in the anterior MSG, but the Ser2 upstream region did not exhibit any promoter activity. Since the strongest promoter activity was observed for Ser1, we devised a system for the production of recombinant proteins using a GAL4–Ser1 promoter construct (Ser1-GAL4). Transgenic silkworms harboring both the Ser1-GAL4 construct and the previously reported upstream activating sequence (UAS)–EGFP construct, which contains the TATA box region of the Drosophila hsp70 gene, yielded approximately 100 μg EGFP per larva. When we then analyzed the TATA box region, signal peptide, and intron sequences for their effects on production from the UAS-EGFP construct, we found that the optimization of these sequences effectively increased production to an average of 500 μg EGFP protein per transgenic larva. We conclude that this binary system is a useful tool for the mass production of recombinant proteins of biomedical and pharmaceutical interest in silkworm.     

Development of Serotonergic Neurons in Embryos of the Sea Urchin, Strongylocentrotus purpuratus

The development of the serotonergic component of the nervous system of larvae of S. purpuratus is traced using indirect immunofluorescence with a polyclonal antibody against the neurotransmitter serotonin. Initially one or two neuroblasts can be detected in the thickened epithelium of the animal plate of late gastrulae (56 hr). The number of immunoreactive cells increases to about eight during formation of the pluteus (85–90 hr). Immunoreactive axons appear simultaneously from all neuroblasts present in the 79 hr prism stage larva and form the apical ganglion. It is proposed that this component of the larval nervous system is derived from a small number of ectodermal cells associated with the apical tuft.     

How does Trichinella spiralis make itself at home?

The nurse cell-parasite complex of Trichinella spiralis is unlike anything else in Nature. It is derived from a normal portion of striated skeletal muscle cell and develops in a matter of 15 to 20 days after the larva invades that cell type. What are the molecular mechanisms at work that result in this unique relationship? Here, Dickson Despommier presents a hypothesis to account for its formation, in which secreted tyvelosylated proteins of the larva play a central role. These proteins are always present in the intracellular niche of the larva from Day 7 after infection and may be responsible for redirecting host genomic expression, leading to nurse cell formation.     

High-level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L.) using recombinant baculovirus

A hybrid of Autographa californica nuclear polyhedrosis virus and Bombyx mori nuclear polyhedrosis virus, which is infectious to both Spodoptera frugiperda and Bombyx mori, was prepared in our previous study. Two recombinant hybrid baculoviruses, carrying cDNAs of human acidic and basic fibroblast growth factors, respectively, were successfully constructed in this study, for the large-scale production of human aFGF and bFGF using silkworm as host. These recombinant viruses were used to inoculate silkworm larvae. After the infection, the recombinant proteins were not found in the hemolymph. Such nonsecretion from cells has also been observed in the established insect cell lines, Sf21 and Tn-5. Tissue distribution analysis indicated that the expressed products were mainly located in fat body and the production of the recombinant aFGF and bFGF was maximal at around 80 h postinfection. Therefore, silkworm larvae infected with recombinant viruses were dissected and fat bodies were collected for the purification of recombinant aFGF and bFGF. The expression levels in both cases were estimated to be as high as approximately 600-700 microg per larva. Furthermore, the recombinant proteins were characterized and their biological activities were evaluated by in vitro bioassay using cell culture.     

D‐3‐phosphoglycerate dehydrogenase from the silkworm Bombyx mori: Identification,functional characterization,and expression

D‐3‐phosphoglycerate dehydrogenase (PHGDH) is a key enzyme involved in the synthesis of l ‐serine. Despite the high serine content in silk proteins and the crucial role of PHGDH in serine biosynthesis, PHGDH has not been described in silkworms to date. Here, we identified PHGDH in the silkworm Bombyx mori and evaluated its biochemical properties. On the basis of the amino acid sequence and phylogenetic tree, this PHGDH has been categorized as a new type and designated as bmPHGDH. The recombinant bmPHGDH was overexpressed and purified to homogeneity. Kinetic studies revealed that PHGDH uses NADH as a coenzyme to reduce phosphohydroxypyruvate. High expression levels of bmphgdh messenger RNA (mRNA) were observed in the middle part of the silk gland and midgut in a standard strain of silkworm. Moreover, a sericin‐deficient silkworm strain displayed reduced expression of bmphgdh mRNA. These findings indicate that bmPHGDH might play a crucial role in the provision of l ‐serine in the larva of B. mori.     

Effect of juvenile hormone treatment on caste differentiation in the honeybee, Apis mellifera

Synthetic juvenile hormone (methyl trans,trans,cis-10-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate, 1 μg/μl acetone per animal) (JH) was topically applied to 2- to 3-day-old worker honeybee larvae in the hive. Eighty per cent of the hormone-treated larvae were removed from their brood cell before pupation. Only 1 out of 42 adults showed characteristics of an intercaste. Fifty per cent of the control larvae (1 μl acetone) developed to adults, all of which were workers.After topical application of JH and feeding on royal jelly under in vitro conditions, the rate of survival is high (up to 85 per cent adults), but up to 67 per cent of queens and 44 per cent of workers exhibit eye malformations with characteristics of somatic mutation. Formation of a more solid web by the spinning larvae, shortening of the diapause by 1 to 2 days, and unusual shapes of mandibles, legs, and abdomen are a consequence of hormone treatment. The effects are less marked after application of 0·1 instead of 1 μg hormone or after its addition to the food (2 μg/g royal jelly). Treatment of the 2- to 3-day-old worker larvae and subsequent rearing on royal jelly is followed by a shift in caste differentiation from queens and workers to intercastes. In no case, are more queens developed after juvenile hormone treatment.Queen bee determinator, partially purified from royal jelly, induces a concentration-dependent shift from workers to queen differentiation. A threefold increase in the natural determinator concentration of royal jelly results in an almost exclusive (98 per cent) queen formation from 2- to 3-day-old worker larvae. In contrast to this direct effect, the influence of JH is explained as an indirect morphogenetic effect not directly coupled with honeybee caste differentiation.     

Introduction of foreign genes into silkworm eggs by electroporation and its application in transgenic vector test

Up to now, the most practical method for introducingforeign gene into insect eggs is the microinjection as thatwas utilized in Drosophila melanogaster [1–3]. The dis-covery of transposable elements including: P, Minos,Hermes, mariner, hobo, piggyBac, etc…     

Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield

Spider dragline silk is a unique fibrous protein with combination of tensile strength and elasticity, but the isolation of large amount of silk from spiders is not feasible. In this paper, we used a newly established Bac-to-Bac/BmNPV Baculovirus expression system to express the recombinant spider (Nephila clavata) dragline silk protein (MaSp1) fused EGFP in BmN cells and larvae of silkworm. A 70 kDa fusion protein was visualized after rBacmid/BmNPV/drag infection by SDS-PAGE and immunoblotting analysis. Fusion protein expressed in the BmN cells probably occupied five percent of the cell total protein; In a silkworm larva, approximately 6 mg fusion proteins were expressed. Solubility analysis of the expressed spider dragline silk protein indicated that 60% fusion protein is insoluble. EGFP fluorescence showed that fusion protein is tend to form aggregate by self assemblage. The results indicated the solubility is the primary limit for spider dragline proteins yield. It also suggested that directly produce fibrous spider silk in the secreting-silk organs of the transgenic silkworm larvae might be a better method.     

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.     

The chemistry of insect hemolymph; organic components of the hemolymph of the silkworm,Bombyx mori,and two other species

1. Hemolymph was collected for analysis from the silkworm, Bombyx mori, in a series of developmental stages ranging from the second molt to the late pupa. The mean pH of larval hemolymph after collection was found to be 6.45, that of pupal hemolymph, 6.57; in vivo values may be slightly lower. Total dry solids ranged from 5.4 to 10.6 per cent. Total protein ranged from 1.2 to 5.3 per cent, increasing rapidly during the fifth instar. 2. Free amino acids were separated chromatographically and estimated. Of 19 amino acids identified, amounting collectively to 823 to 1497 mg. per 100 ml., glutamine, histidine, and lysine generally occurred in greatest amount. Tryptophan was not detected, and cystine (or cysteine) was found in only one sample. The total free amino acids account for 35 to 55 per cent of the non-protein nitrogen of the plasma. 3. Free sugars, estimated semiquantitatively on chromatograms, comprise glucose, fructose, and sucrose in total amount ranging from about 5 to 40 mg. per 100 ml. Total acid-soluble, ultrafiltrable carbohydrate, estimated as glucose by the anthrone reaction, ranged from 166 to 635 mg. per 100 ml., indicating the presence of low molecular weight sugar derivatives. 4. Inorganic phosphate amounted to 5 to 15 mg. per 100 ml., and acid-soluble organic phosphate to 100 to 200 mg. per 100 ml. The latter fraction includes several substances, of which one was tentatively identified as glucose-6-phosphate and the remainder are as yet unidentified. 5. Single samples of hemolymph were also taken from larvae of the wax moth, Galleria mellonella, and the spruce sawfly, Diprion hercyniae. These contained even higher concentrations of solutes than the silkworm samples, but with a generally similar distribution. The proportions of the free amino acids were different in each species.     

RH-5992--an ecdysone agonist on model system of the silkworm Bombyx mori

RH-5992 is a novel synthetic non-steroidal ecdysteroid agonist with a high selectivity towards Lepidopteran species. The effect of ecdysone agonist RH-5992 on larval period, larval weight, silk gland weight and haemolymph protein profile were examined in the model organism, the larvae of silkworm, Bombyx mori. The LD50 values were found to be 16.21 and 12.01 micrograms/larva for 72 and 96 hr respectively. In the present study, three sublethal concentrations of 1/5th, 1/10th and 1/20th of LD50 at 72 hr were selected and applied on the mid-dorsal line of the silkworm B. mori. The maximum mortality of 35% was observed in the group which received the highest (3.2 micrograms/larva) concentration of RH-5992. The mortality rate was found to be dose dependent as well as time dependent. Interesting results were observed in haemolymph profile of the RH-5992 treated larvae as staining intensity of 30 kDa protein decreased significantly whereas the effect was not marked on other major proteins like storage proteins and vitellogenin polypeptides. From the results, it is confirmed that RH-5992 causes changes in larval characters and protein profile of silkworm B. mori. It is proposed that RH-5992 may cause negative effect specifically on reproductive characters like development of ovary and egg production due to decrease in 30 kDa protein.     

Effect of Cordyceps, Paecillomyces sp., Cordyceps pruinosa and Paecillomyces japonica, on the Development of Domestic Silkworm, Bombyx mori

ABSTRACT This study was carried out to investigate pathogenicity of cordyceps on the silkworm larva, Bombyx mori . For experiment, Cordyceps, Pacillomyces sp. strain collected from Chiak mountain area in Gangwon Province and Pacillomyces japonica collected from dead silkworm larva at the rearing room in Sangju National University were used. Spores of cordyceps were propagated on sterile PDA (potato dextrose agar 39g, water 1,000mL) at 25°C for 21 days. Spores of cordyceps isolated from media were inoculated on integuments of 3rd instar newly exuviated silkworm for pathogenicity on the silkworm larvae. The cordyceps used in this study was highly infectious to the silkworms. Virulence of cordyceps was different depending on species. Pacillomyces japonica was the most efficacious with 70% mortality on silkworm larvae. Cordyceps had adverse effect on the overall rearing from larval period to mounting of matured silkworm larvae, cocoon making, pupation and moth emergence. The result suggests that cordyceps used in this study may be useful for the tracking of biocontrol.     

Fibroin silk proteins from the nonmulberry silkworm Philosamia ricini are biochemically and immunochemically distinct from those of the mulberry silkworm Bombyx mori

Silk proteins were isolated from the cocoons of the nonmulberry silkworm, Philosamia ricini. Three polypeptides of 97, 66, and 45 kDa were identified. The 66-kDa molecule represented sericin, whereas the 97-kDa and the 45-kDa polypeptides linked together through a disulfide bond constituted the fibroin protein. Antibodies raised against the 97-kDa P. ricini fibroin heavy chain reacted specifically with this molecule and did not recognize fibroin heavy chain from another nonmulberry silkworm, Antheraea assama or from the mulberry silkworm, Bombyx mori, suggesting the presence of P. ricini species-specific determinants in this heavy chain. Antibodies generated against fibroin light chain of P. ricini also showed similar reactivity pattern. Immunoblot analysis with proteins isolated from the silk glands of P. ricini at different stages of larval development showed that the expression of fibroin heavy chain was developmentally and spatially regulated. The protein was most abundant in the 5th instar larva, and could be detected in the middle and the posterior but not the anterior silk glands. The amino acid composition of the 97-kDa fibroin protein showed abundance of glutamic acid and did not contain (Gly-Ala)(n) motifs, a characteristic feature of B. mori fibroin heavy chain. Our study reveals significant differences between the nonmulberry silkworm P. ricini and the mulberry silkworm B. mori in the biochemical composition and immunochemical characteristics of fibroin heavy chain. These differences might be responsible for the differences seen in the quality of silk produced by these two silkworms.     

Bilateral Hernias in the Female

An experience with 216 bilateral hernias in female patients is reviewed. The condition is rare, occurring only once in every 250 patients admitted for a hernia repair. Bilateral primary indirect inguinal hernias were the most frequent type. Bilateral primary femoral hernias were quite rare while bilateral primary direct inguinal hernias were even more uncommon. Other rare bilateral combinations are briefly described. The incidence in children is given.Etiological factors are discussed, emphasizing the strong posterior wall of the inguinal canal in females.Two per cent of patients developed a recurrent hernia; one per cent of hernias recurred. No recurrence following a bilateral primary indirect inguinal hernia repair and no “femoral” recurrence following inguinal repair were recorded.     

PROTEIN SYNTHESIS IN THE MEDULLA OF THE RAT BRAIN. REPRODUCIBILITY OF THE RESULTS

Abstract— [¹⁴C]Leucine was injected intracranially into the brainstem reticular formation at the level of the upper medulla by the stereotaxic method. Subcellular fractions prepared 3 hr after injection showed that the specific activities of leucine-incorporated proteins decreased in the order soluble, microsomal, nuclear and mitochondrial fractions. Specific activities of proteins in the sera were 2·2 per cent of whole homogenate proteins.
The results from 27 experiments showed that 66·6 per cent of the mean specific activities of proteins extracted from whole homogenates fell within ·1 s.d . and 100 per cent within ± 2 s.d . (close to a normal distribution). The coefficients of variation were between 40 and 50 per cent for whole homogenates, sera and all subcellular fractions. Reproducibility of results and factors concerned with possible errors in the technique are discussed.     

Some Effects of Yellow Rust (Puccinia striiformis) on the Growth and Yield of a Spring Wheat

Yellow Rust had a considerable effect on the yield of the springwheat Jufy I, that of severely infected plants being reducedby as much as 64.5 per cent. This was due to a reduction inthe number of florets per ear, the number of grains per ear,and in some cases the weight of individual grains. There wasalso a marked effect on the general growth and vigour of theplant, the growth of leaves being retarded and the final lengthand breadth greatly reduced. The roots were more seriously affectedthan any other part of the plant with reductions in dry weightof as much as 78 per cent in fully infected plants. The relatively direct effect of a late rust infection greatlyreduced the yield, while the effects of an early infection,although indirect, still caused large reductions. The variedeffects of rust on the several factors contributing to the yield,and the relative importance of the effects on these factorsare discussed.     

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen. International Journal for Parasitology4: 173–176. Third stage larvae of N. brasiliensis were frozen over liquid nitrogen and after storage for 7 days were thawed rapidly and inoculated subcutaneously into rats. When ensheathed larvae were used, none survived freezing as judged by motility and infectivity trials. Separate vials of exsheathed larvae survived freezing in proportions ranging from 10 to 64 per cent. Female worms, derived from frozen exsheathed larvae, had a normal complement of eggs in the uterus and both male and female worms had a normal histological appearance. Exsheathed larvae frozen in the presence of 10 per cent dimethylsulphoxide had the same survival rate as those frozen without the addition of cryoprotectant. The addition of 10 per cent glycerol adversely effected the survival of frozen exsheathed larvae.