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The Origin and Dispersal of the Domesticated Chinese Oak Silkworm, Antheraea pernyi, in China: A Reconstruction Based on Ancient Texts
PubMed Central
Sericulture is one of the great inventions of the ancient Chinese. Besides the mulberry silkworm (Bombyx mori), Chinese farmers developed rearing of the Chinese oak silkworm (Antheraea pernyi) about 400 years ago. In this paper, the historic records of the origins and dispersal of the domesticated Chinese oak silkworm in China are summarized. The first document with clearly recorded oak silkworm artificial rearing appeared in 1651, although Chinese oak silkworm was documented in about 270 AD. All of the evidence in the available historic records suggests that the domesticated Chinese oak silkworm originated in central and southern areas of Shandong Province in China around the 16th century, and then was introduced directly and indirectly by human commerce into the present habitations in China after the late 17th century. The results strongly support the hypothesis that only one geographically distinct event occurred in domestication of the modern Chinese oak silkworm.
Liu, Yanqun; Li, Yuping; Li, Xisheng; Qin, Li
Overexpression of small heat shock protein 21 protects the Chinese oak silkworm Antheraea pernyi against thermal stress.
Small heat shock proteins (sHSPs) usually act as molecular chaperones to prevent proteins from being denatured in extreme conditions. We first report the sHSP21 gene, named as Ap-sHSP21, in the Chinese oak silkworm Antheraea pernyi (Lepidoptera: Saturniidae). The full-length cDNA of Ap-sHSP21 is 976 bp, including a 5'-untranslated region (UTR) of 99 bp, a 3'-UTR of 316 bp and an open reading frame (ORF) of 561 bp encoding a polypeptide of 186 amino acids. The deduced A. pernyi sHSP21 protein sequence reveals the percent identity is 82-93% in comparison to other sHSPs from insects. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis shows that Ap-sHSP21 expression is higher in testis than that in other examined tissues and significantly up-regulated after heat shock. In addition, prokaryotic expression and purification of the Ap-sHSP21 protein were performed. SDS-PAGE and Western blot analysis demonstrated that a 25 kDa recombinant protein was successfully expressed in Escherichia coli cells and the purified recombinant protein was also confirmed to protect restriction enzymes from thermal inactivation. The expression of Ap-sHSP21 was significantly down-regulated after RNA interference, which was confirmed by qRT-PCR and Western blot analysis. All together, these results suggest that Ap-sHSP21 play a key role in thermal tolerance. PMID:23763950
Liu, Qiu-Ning; Zhu, Bao-Jian; Dai, Li-Shang; Fu, Wei-Wei; Lin, Kun-Zhang; Liu, Chao-Liang
Molecular cloning and characterization of two 12 kDa FK506-binding protein genes in the Chinese oak silkworm, Antheraea pernyi.
Two 12 kDa FK506-binding protein (FKBP12) genes were isolated and characterized from Chinese oak silkworm Antheraea pernyi , an important agricultural and edible insect, designated ApFKBP12 A and B, respectively. Both ApFKBP12 A and B contained 108 amino acids with 82% sequence identity. Phylogenetic analysis showed that FKBP12 B sequences of A. pernyi, Bombyx mori , and Danaus plexippus were clearly separated from FKBP12 A sequences of these three species, suggesting that insect FKBP12 A and B may have been evolving independently. RT-PCR analyses revealed that two ApFKBP12 genes were expressed during the four developmental stages and in all tested tissues, and that the mRNA expression level of the ApFKBP12 A gene was significantly higher than that of the ApFKBP12 B gene. After heat shock treatment, expressions of the two FKBP12 genes were up-regulated, but at different time points. The results suggested that each paralogue of the FKBP12 genes may play a distinct functional role in the development of A. pernyi. PMID:23617895
Chen, Mo; Chen, Miao-Miao; Yao, Rui; Li, Yan; Wang, Huan; Li, Yu-Ping; Liu, Yan-Qun
A novel fibrin(ogen)olytic trypsin-like protease from Chinese oak silkworm (Antheraea pernyi): purification and characterization.
A novel fibrin(ogen)olytic protease from Antheraea pernyi (important economically insect), named cocoonase, was isolated by a combination of ion-exchange chromatography and gel filtration. Furthermore, the characterization of cocoonase was investigated using fibrin(ogen)olytic, thrombolysis, and hemorrhagic assays. The NH2-terminal sequence (IVGGY SVTID KAPYQ) was established by Edman degradation. Based on the N-terminal sequencing, cocoonase cDNA has been cloned by means of RT-PCR and 5'RACE. It is composed of 261 amino acid residues and possesses the structural features of trypsin-like serine protease. The purified cocoonase showed specific esterase activity on N-?-benzoyl-l-arginine ethyl (BAEE), and the kinetic constants, Km and Vmax were 2.577 � 10(-3)mol/L and 4.09 � 10(-3)?mol/L/s, respectively. Cocoonase showed strong activities on both fibrin and fibrinogen, preferentially hydrolyzed A? and B? chains followed by ?-chains of fibrinogen. Cocoonase exhibited a thrombolysis activity both in vitro (blood-clot lysis activity assay) and in vivo (carrageenan-induced thrombosis model). These findings indicate that A. pernyi cocoonase ia a novel fibrin(ogen)olytic enzyme and may have a potential clinical application as an antithrombotic agent. PMID:24491553
Geng, Peng; Lin, Lan; Li, Yuan; Fan, Qi; Wang, Naihong; Song, Lixin; Li, Wenli
Diapause-associated protein3 functions as Cu/Zn superoxide dismutase in the Chinese oak silkworm (Antheraea pernyi).
To better understand the molecular mechanism underlying of diapause in Antheraea pernyi (A. pernyi), we cloned a novel diapause-associated protein 3 (DAP3) gene from A. pernyi by reverse transcription-polymerase chain reaction (RT-PCR) and studied the biological functions. Sequence analysis revealed that this gene encodes 171 amino acids and has a conserved domain of Copper/Zinc superoxide dismutase (Cu/Zn-SOD). Western blot and qRT-PCR results showed that DAP3 was mainly expressed in the pupal stage, and gradually decreased as diapause development. DAP3 was also expressed in 1st and 5th instar larvae of A. pernyi. In tissues of 5th instar larvae of A. pernyi, DAP3 was mainly expressed in the epidermis, followed by the head, hemolymph and fat body. To identify the SOD activity of DAP3, we constructed a prokaryotic expression vector by inserting the coding region sequence into plasmid pET-28a (+) and obtained the purified rHIS-DAP3 fusion protein by Ni-NTA affinitive column. Importantly, we found the SOD activity of DAP3 fusion protein was approximately 0.6674 U/�g. To further confirm the SOD activity of DAP3 in vivo, we induced the oxidative stress model of pupae by UV irradiation. The results showed that both the mRNA and protein level of DAP3 significantly increased by UV irradiation. Furthermore, the SOD activity of the total lysate of pupae increased significantly at 10 min post UV irradiation and transiently returned to normal level afterwards. These results suggested that DAP3 might be a novel protein with SOD activity getting involved in regulation of diapause in A. pernyi. PMID:24613963
Bi, Zhenle; Yang, Xiaoli; Yu, Wei; Shu, Jianhong; Zhang, Yaozhou
Purification and characterization of digestive amylase from the tasar silkworm, Antheraea mylitta (Lepidoptera: Saturniidae)
Microsoft Academic Search
Digestive amylase was purified from larvae of Indian tasar silkworm, Antheraea mylitta using ammonium sulphate precipitation, glycogen complex precipitation and gel filtration chromatography. Specific activity increased from 0.673 AU\\/mg in the crude digestive juice to 94.80 AU\\/mg in the final purified sample. Activity of the purified enzyme was 15-fold less than that of the digestive amylase of silkworm. Bombyx mori.
J. Nagaraju; E. G. Abraham
Isolation, purification and characterization of silk protein sericin from cocoon peduncles of tropical tasar silkworm, Antheraea mylitta
A high molecular weight water-soluble glue protein, sericin was identified in the cocoon peduncle (a strong thread connecting the cocoons to the branches of the tree with a ring) of the tropical tasar silkworm, Antheraea mylitta. The sericin was isolated by 8M urea containing 1% sodium dodecyl sulfate and ?-mercaptoethenol (2%) or by 1% sodium chloride. The protein was purified
Rupesh Dash; Soumen Mukherjee; S. C. Kundu
Influence of Thyroxine on Different Ion-Dependent ATPase Activities in Fat Body of Tasar Silkworm, Antheraea mylittaD
The activities of Na+-K+-, Ca2+-, and Mg2+-ATPase of tasar silkworm,Antheraea mylittaD. fat body were investigated from fifth larval stage to adult emergence after injection of various doses (0.5, 1.0, 2.0, and 5.0 ?g\\/g) of mammalian thyroxine (T4) to 1-hr-old fifth instar larvae. In normal silkworms, both sexes exhibited maximum enzyme activity before spinning (Day 12). Na+-K+-, Ca2+-, and Mg2+-ATPase activities
K. Damodar Reddy; A. Chaudhuri; K. Thangavelu
Identification of RAPD and SCAR markers associated with yield traits in the Indian tropical tasar silkworm Antheraea mylitta drury.
The tropical tasar silkworm, Antheraea mylitta, is a semi-domesticated vanya silk-producing insect of high economic importance. To date, no molecular marker associated with cocoon and shell weights has been identified in this species. In this report, we identified a randomly amplified polymorphic DNA (RAPD) marker and examined its inheritance, and also developed a stable diagnostic sequence-characterized amplified region (SCAR) marker. Silkworms were divided into groups with high (HCSW) and low (LCSW) cocoon and shell weights, and the F(2) progeny of a cross between these two groups were obtained. DNA from these silkworms was screened by PCR using 34 random primers and the resulting RAPD fragments were used for cluster analysis and discriminant function analysis (DFA). The clustering pattern in a UPGMA-based dendogram and DFA clearly distinguished the HCSW and LCSW groups. Multiple regression analysis identified five markers associated with cocoon and shell weights. The marker OPW16(905 bp) showed the most significant association with cocoon and shell weights, and its inheritance was confirmed in F(2) progeny. Cloning and sequencing of this 905 bp fragment showed 88% identity between its 134 nucleotides and the Bmc-1/Yamato-like retroposon of A. mylitta. This marker was further converted into a diagnostic SCAR marker (SCOPW 16(826 bp)). The SCAR marker developed here may be useful in identifying the right parental stock of tasar silk-worms for high cocoon and shell weights in breeding programs designed to enhance the productivity of tasar silk. PMID:23271934
Dutta, Suhrid R; Kar, Prasanta K; Srivastava, Ashok K; Sinha, Manoj K; Shankar, Jai; Ghosh, Ananta K
Elevation of Different Ion-Specific ATPase Activities by L-Thyroxine (T4) in Different Tissues of Tasar Silkworm, Antheraea mylitta (Lepidoptera: Saturniidae) during Developmental Stages
Tissue-specific age-dependent changes were observed in Na+K+-, Ca2+-, and Mg2+-ATPase activities in tropical tasar silkworm, Antheraea mylitta Drury. Maximum enzyme activity was recorded in all the tissues on day 12 (before spinning) in control group of animals. In testis, Na+K+-, Ca2+-, and Mg2+-ATPase activities gradually increased from day 2 to day 12 during fifth larval age and level was maintained
K. Damodar Reddy; A Chaudhuri; K Thangavelu
Analysis of Transcripts Expressed in One-Day-Old Larvae and Fifth Instar Silk Glands of Tasar Silkworm, Antheraea mylitta
Antheraea mylitta is one of the wild nonmulberry silkworms, which produces tasar silk. An EST project has been undertaken to understand the gene expression profile of A. mylitta silk gland. Two cDNA libraries, one from the whole bodies of one-day-old larvae and the other from the silkglands of fifth instar larvae, were constructed and sequenced. A total of 2476 good-quality ESTs (1239 clones) were obtained and grouped into 648 clusters containing 390 contigs and 258 singletons to represent 467 potential unigenes. Forty-five sequences contained putative coding region, and represented potentially novel genes. Among the 648 clusters, 241 were categorized according to Gene Ontology hierarchy and showed presence of several silk and immune-related genes. The A. mylitta ESTs have been organized into a freely available online database �AmyBASE�. These data provide an initial insight into the A. mylitta transcriptome and help to understand the molecular mechanism of silk protein production in a Lepidopteran species.
Maity, Samita; Goel, Sagar I.; Roy, Sobhan; Ghorai, Suvankar; Bhattacharyya, Swati; Venugopalan, Aravind; Ghosh, Ananta K.
Genetic characterization of a novel Iflavirus associated with vomiting disease in the Chinese oak silkmoth Antheraea pernyi.
Larvae of the Chinese oak silkmoth (Antheraea pernyi) are often affected by AVD (A. pernyi vomiting disease), whose causative agent has long been suspected to be a virus. In an unrelated project we discovered a novel positive sense single-stranded RNA virus that could reproduce AVD symptoms upon injection into healthy A. pernyi larvae. The genome of this virus is 10,163 nucleotides long, has a natural poly-A tail, and contains a single, large open reading frame flanked at the 5' and 3' ends by untranslated regions containing putative structural elements for replication and translation of the virus genome. The open reading frame is predicted to encode a 3036 amino acid polyprotein with four viral structural proteins (VP1-VP4) located in the N-terminal end and the non-structural proteins, including a helicase, RNA-dependent RNA polymerase and 3C-protease, located in the C-terminal end of the polyprotein. Putative 3C-protease and autolytic cleavage sites were identified for processing the polyprotein into functional units. The genome organization, amino acid sequence and phylogenetic analyses suggest that the virus is a novel species of the genus Iflavirus, with the proposed name of Antheraea pernyi Iflavirus (ApIV). PMID:24637949
Geng, Peng; Li, Wenli; Lin, Lan; de Miranda, Joachim R; Emrich, Scott; An, Lijia; Terenius, Olle
Comparative Study of Total Hydroperoxides and Antioxidant Defense System in the Indian Tropical Tasar Silkworm, Antheraea mylitta, in Diapausing and Non-Diapausing Generations
In the present study, the total hydroperoxides, catalase, glutathione-s-transferase, and ascorbic acid contents were determined in different developmental stages of the non-diapause and the diapause generation of the tropical tasar silkworm, Antheraea mylitta Drury (Lepidoptera: Saturniidae). The results showed stage-specific significantly higher levels of total hydroperoxides, catalase, and ascorbic acid contents in the non-diapause as compared to the diapause generation (p < 0.05). However, a significantly enhanced level of glutathione-S-transferase activity was observed in mature 5th instar larvae of the diapause generation (p < 0.05). In the case of pupae, significantly higher levels of total hydroperoxides, catalase, and glutathione-s-transferase activity were observed in the non-diapause generation (p < 0.05). These results could be the effect of intensive metabolic transformation that takes place in tissues of the non-diapause generation and causes increased production of reactive oxygen species, such as hydroperoxides. The results suggest that antioxidants play an important role in protecting cells against reactive oxygen species.
Jena, Karmabeer; Kar, Prasanta K.; Babu, Chittithoti S.; Giri, Shantakar; Singh, Shyam S.; Prasad, Bhagwan C.
Construction of a full-length cDNA Library from Chinese oak silkworm pupa and identification of a KK-42-binding protein gene in relation to pupa-diapause termination.
In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, Antheraea pernyi, the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the diapause stage. The titer of the library was 5 x 10(5) cfu/ml and the proportion of recombinant clones was approximately 95%. Expressed sequence tag (EST) analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes, 97 (55.4%) were known genes but only five from A. pernyi, 37 (21.2%) were known ESTs without function annotation, and 41 (23.4%) were novel ESTs. By EST sequencing, a gene coding KK-42-binding protein in A. pernyi (named as ApKK42-BP; GenBank accession no. FJ744151) was identified and characterized. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18, and contained two putative conserved domains, abhydro_lipase and abhydrolase_1, suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of diapause stage, suggesting it may also be involved in pupa-diapause termination. PMID:19564928
Li, Yu-Ping; Xia, Run-Xi; Wang, Huan; Li, Xi-Sheng; Liu, Yan-Qun; Wei, Zhao-Jun; Lu, Cheng; Xiang, Zhong-Huai
Analysis of the genomic sequence of Philosamia cynthia nucleopolyhedrin virus and comparison with Antheraea pernyi nucleopolyhedrin virus
Background Two species of wild silkworms, the Chinese oak silkworm (Antheraea pernyi) and the castor silkworm Philosamia cynthia ricini, can acquire a serious disease caused by Nucleopolyhedrin Viruses (NPVs) (known as AnpeNPV and PhcyNPV, respectively). The two viruses have similar polyhedral morphologies and their viral fragments share high sequence similarity. However, the physical maps of the viral genomes and cross-infectivity of the viruses are different. The genome sequences of two AnpeNPV isolates have been published. Results We sequenced and analyzed the full-length genome of PhcyNPV to compare the gene contents of the two viruses. The genome of PhcyNPV is 125, 376 bp, with a G + C content of 53.65%, and encodes 138 open reading frames (ORFs) of at least 50 amino acids (aa) (GenBank accession number: JX404026). Between PhcyNPV and AnpeMNPV-L and -Z isolates, 126 ORFs are identical, including 30 baculovirus core genes. Nine ORFs were only found in PhcyNPV. Four genes, cath, v-chi, lef 10 and lef 11, were not found in PhcyNPV. However, most of the six genes required for infectivity via the oral route were found in PhcyNPV and in the two AnpeNPV isolates, with high sequence similarities. The pif-3 gene of PhcyNPV contained 59 aa extra amino acids at the N-terminus compared with AnpeNPV. Conclusions Most of the genes in PhcyNPV are similar to the two AnpeNPV isolates, including the direction of expression of the ORFs. Only a few genes were missing from PhcyNPV. These data suggest that PhcyNPV and AnpeNPV might be variants of each other, and that the differences in cross-infection might be caused by gene mutations.
Cloning and sequence analysis of the Antheraea pernyi nucleopolyhedrovirus gp64 gene.
Frequent outbreaks of the purulence disease of Chinese oak silkworm are reported in Middle and Northeast China. The disease is produced by the pathogen Antheraea pernyi nucleopolyhedrovirus (AnpeNPV). To obtain molecular information of the virus, the polyhedra of AnpeNPV were purified and characterized. The genomic DNA of AnpeNPV was extracted and digested with HindIII. The genome size of AnpeNPV is estimated at 128 kb. Based on the analysis of DNA fragments digested with HindIII, 23 fragments were bigger than 564 bp. A genomic library was generated using HindIII and the positive clones were sequenced and analysed. The gp64 gene, encoding the baculovirus envelope protein GP64, was found in an insert. The nucleotide sequence analysis indicated that the AnpeNPV gp64 gene consists of a 1,530 nucleotide open reading frame (ORF), encoding a protein of 509 amino acids. Of the eight gp64 homologues, the AnpeNPV gp64 ORF shared the most sequence similarity with the gp64 gene of Anticarsia gemmatalis NPV, but not Bombyx mori NPV. The upstream region of the AnpeNPV gp64 ORF encoded the conserved transcriptional elements for early and late stage of the viral infection cycle. These results indicated that AnpeNPV belongs to group I NPV and was far removed in molecular phylogeny from the BmNPV. PMID:16388134
Wang, Wenbing; Zhu, Shanying; Wang, Liqun; Yu, Feng; Shen, Weide
Silk fibroin protein from mulberry and non-mulberry silkworms: cytotoxicity, biocompatibility and kinetics of L929 murine fibroblast adhesion
Silks fibers and films fabricated from fibroin protein of domesticated mulberry silkworm cocoon have been traditionally utilized\\u000a as sutures in surgery and recently as biomaterial films respectively. Here, we explore the possibility of application of silk\\u000a fibroin protein from non-mulberry silkworm cocoon as a potential biomaterial aid. In terms of direct inflammatory potential,\\u000a fibroin proteins from Antheraea mylitta and Bombyx
Chitrangada Acharya; Sudip K. Ghosh; S. C. Kundu
The expression of GFP under the control of fibroin promotor in primary ovarian cells of Antheraea pernyi.
The fibroin promoter can stably express foreign gene in lepidopteran cells. Total RNA was extracted from the gland of silkworm, Antheraea pernyi and the transcription initiation site of fibroin gene of A. pernyi was identified by RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). The expression vector (pGFP-N2/Fib) was constructed by use of replacing the CMV promoter with the fibroin promoter. The results of visual screening under a fluorescent inverted microscope and Western blot analysis indicated that the GFP gene was expressed in the primary cells of ovary origins from A. pernyi. PMID:14660868
Li, Wenli; Jin, Liji; Bu, Pengcheng; An, Lijia
Extraction conditions of Antheraea mylitta sericin with high yields and minimum molecular weight degradation.
Although the technique for extracting the Bombyx mori sericin has been extensively known, the extraction of sericin from wild-silkworm cocoons is not yet standardized. The aim of this study was to find the optimal conditions for the extraction of sericin from Antheraea mylitta cocoons, with high yields and minimum degradation. We attempted to apply various protocols for the extraction of the A. mylitta sericin (AmS). Among these, we found that the extraction of AmS with a sodium carbonate solution exhibited the highest yield except the conventional soap-alkali extraction. To find the optimal conditions for the AmS extraction with the sodium carbonate, we changed the concentration of sodium carbonate and the treatment time. With an increase in the sodium carbonate concentration and the extraction time, the yield of AmS increased, but the molecular weight (MW) of AmS decreased. Considering the yield, molecular weight distribution (MWD) and amino acid composition of AmS, we suggest that the optimal conditions for the AmS extraction require treatment with 0.02 M sodium carbonate and boiling for 60 min. PMID:23026092
Yun, Haesung; Oh, Hanjin; Kim, Moo Kon; Kwak, Hyo Won; Lee, Jeong Yun; Um, In Chul; Vootla, Shyam Kumar; Lee, Ki Hoon
Copy number variations among silkworms
Background Copy number variations (CNVs), which are important source for genetic and phenotypic variation, have been shown to be associated with disease as well as important QTLs, especially in domesticated animals. However, little is known about the CNVs in silkworm. Results In this study, we have constructed the first CNVs map based on genome-wide analysis of CNVs in domesticated silkworm. Using next-generation sequencing as well as quantitative PCR (qPCR), we identified ~319 CNVs in total and almost half of them (~ 49%) were distributed on uncharacterized chromosome. The CNVs covered 10.8�Mb, which is about 2.3% of the entire silkworm genome. Furthermore, approximately 61% of CNVs directly overlapped with SDs in silkworm. The genes in CNVs are mainly related to reproduction, immunity, detoxification and signal recognition, which is consistent with the observations in mammals. Conclusions An initial CNVs map for silkworm has been described in this study. And this map provides new information for genetic variations in silkworm. Furthermore, the silkworm CNVs may play important roles in reproduction, immunity, detoxification and signal recognition. This study provided insight into the evolution of the silkworm genome and an invaluable resource for insect genomics research.
Materials: Surprising strength of silkworm silk
Commercial silkworm silk is presumed to be much weaker and less extensible than spider dragline silk, which has been hailed as a 'super-fibre'. But we show here that the mechanical properties of silkworm silks can approach those of spider dragline silk when reeled under controlled conditions. We suggest that silkworms might be able to produce threads that compare well with
Zhengzhong Shao; Fritz Vollrath
Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus
Background Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of Reoviridae family, infects Indian non-mulberry silkworm, Antheraea mylitta, and contains 11 segmented double stranded RNA (S1-S11) in its genome. Some of its genome segments (S2 and S6-S11) have been previously characterized but genome segments encoding viral capsid have not been characterized. Results In this study genome segments 1 (S1) and 3 (S3) of AmCPV were converted to cDNA, cloned and sequenced. S1 consisted of 3852 nucleotides, with one long ORF of 3735 nucleotides and could encode a protein of 1245 amino acids with molecular mass of ~141 kDa. Similarly, S3 consisted of 3784 nucleotides having a long ORF of 3630 nucleotides and could encode a protein of 1210 amino acids with molecular mass of ~137 kDa. BLAST analysis showed 20-22% homology of S1 and S3 sequence with spike and capsid proteins, respectively, of other closely related cypoviruses like Bombyx mori CPV (BmCPV), Lymantria dispar CPV (LdCPV), and Dendrolimus punctatus CPV (DpCPV). The ORFs of S1 and S3 were expressed as 141 kDa and 137 kDa insoluble His-tagged fusion proteins, respectively, in Escherichia coli M15 cells via pQE-30 vector, purified through Ni-NTA chromatography and polyclonal antibodies were raised. Immunoblot analysis of purified polyhedra, virion particles and virus infected mid-gut cells with the raised anti-p137 and anti-p141 antibodies showed specific immunoreactive bands and suggest that S1 and S3 may code for viral structural proteins. Expression of S1 and S3 ORFs in insect cells via baculovirus recombinants showed to produce viral like particles (VLPs) by transmission electron microscopy. Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein. Conclusion Our results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3 encoded viral structural proteins can self assemble to form viral outer capsid and S1 encoded protein remains associated with it as inner capsid to maintain the stability. Further studies will help to understand the molecular mechanism of capsid formation during cypovirus replication.
Silk fibroin protein from mulberry and non-mulberry silkworms: cytotoxicity, biocompatibility and kinetics of L929 murine fibroblast adhesion.
Silks fibers and films fabricated from fibroin protein of domesticated mulberry silkworm cocoon have been traditionally utilized as sutures in surgery and recently as biomaterial films respectively. Here, we explore the possibility of application of silk fibroin protein from non-mulberry silkworm cocoon as a potential biomaterial aid. In terms of direct inflammatory potential, fibroin proteins from Antheraea mylitta and Bombyx mori are immunologically inert and invoke minimal immune response. Stimulation of murine peritoneal macrophages and RAW 264.7 murine macrophages by these fibroin proteins both in solution and in the form of films assayed in terms of nitric oxide and TNFalpha production showed comparable stimulation as in collagen. Kinetics of adhesion of L929 murine fibroblasts, for biocompatibility evaluation, monitored every 4 h from seeding and studied over a period of 24 h, reveal A. mylitta fibroin film to be a better substrate in terms of rapid and easier cellularization. Cell viability studies by MTT assay and flow cytometric analyses indicate the ability of fibroin matrices to support cell growth and proliferation comparable to collagen for long-term culture. This matrix may have potential to serve in those injuries where rapid cellularization is essential. PMID:18322779
Acharya, Chitrangada; Ghosh, Sudip K; Kundu, S C
Phylogeny and evolutionary history of the silkworm.
The silkworm, Bombyx mori, played an important role in the old Silk Road that connected ancient Asia and Europe. However, to date, there have been few studies of the origins and domestication of this species using molecular methods. In this study, DNA sequences of mitochondrial and nuclear loci were used to infer the phylogeny and evolutionary history of the domesticated silkworm and its relatives. All of the phylogenetic analyses indicated a close relationship between the domesticated silkworm and the Chinese wild silkworm. Domestication was estimated to have occurred about 4100 years ago (ya), and the radiation of the different geographic strains of B. mori about 2000 ya. The Chinese wild silkworm and the Japanese wild silkworm split about 23600 ya. These estimates are in good agreement with the fossil evidence and historical records. In addition, we show that the domesticated silkworm experienced a population expansion around 1000 ya. The divergence times and the population dynamics of silkworms presented in this study will be useful for studies of lepidopteran phylogenetics, in the genetic analysis of domestic animals, and for understanding the spread of human civilizations. PMID:22744178
Sun, Wei; Yu, Hongsong; Shen, Yihong; Banno, Yutaka; Xiang, Zhonghuai; Zhang, Ze
Isolation of mammalian pathogenic bacteria using silkworms.
We developed a method to predict bacterial pathogenicity against mammals by measuring bacterial virulence in silkworms at 37�C, human body temperature. One hundred and twenty-two strains of bacteria were isolated from the intestines of fish and shellfish and tested for their virulence against silkworms. Overnight cultures of 50 strains killed at least 50% of the silkworms when injected into the hemolymph. Of 10 strains that showed the most potent pathogenicity against silkworms, 8 also killed mice within 4 days after injection, including Staphylococcus simiae and Staphylococcus pasteuri, neither of which was previously reported to be pathogenic against mammals. These findings suggest that bacterial pathogenicity against mammals can be predicted based on measurements of silkworm-killing activity. PMID:22466142
Kaito, C; Usui, K; Kyuma, T; Sekimizu, K
Growth of Insect Cells (Antheraea Eucalypti) in Hemolymph-Free Medium.
National Technical Information Service (NTIS)
Antheraea eucalypti (moth) cells were grown in suspension in a medium containing 10% fetal bovine serum but no insect hemolymph. Cultures were grown in 25-ml quantities of medium in 250-ml plastic tissue culture flasks at 26 to 28 C on a rotary shaker ope...
S. C. Nagle W. C. Crothers N. L. Hall
Period Protein Is Necessary for Circadian Control of Egg Hatching Behavior in the Silkmoth Antheraea pernyi
We examined the molecular basis of the circadian control of egg hatching behavior in the silkmoth Antheraea pernyi. Egg hatching is rhythmically gated, persists under constant darkness, and can be entrained by light by midembryogenesis. The time of appearance of photic entrainment by the silkmoth embryo coincides with the appearance of Period (PER) and Timeless (TIM) proteins in eight cells
Ivo Sauman; Tony Tsai; Alfred L Roca; Steven M Reppert
Complete sequence and organization of Antheraea pernyi nucleopolyhedrovirus, a dr-rich baculovirus
BACKGROUND: The completion and reporting of baculovirus genomes is extremely important as it advances our understanding of gene function and evolution. Due to the large number of viral genomes now sequenced it is very important that authors present significantly detailed analyses to advance the understanding of the viral genomes. However, there is no report of the Antheraea pernyi nucleopolyhedrovirus (AnpeNPV)
Zuo-Ming Nie; Zhi-Fang Zhang; Dan Wang; Ping-An He; Cai-Ying Jiang; Li Song; Fang Chen; Jie Xu; Ling Yang; Lin-Lin Yu; Jian Chen; Zheng-Bing Lv; Jing-Jing Lu; Xiang-Fu Wu; Yao-Zhou Zhang
Cyclic GMP Levels and Guanylate Cyclase Activity in Pheromone Sensitive Antennae of the Silkmoths Antheraea polyphemus and Bombyx mori
Female sex pheromones applied to freshly isolated, living antennae of male Antheraea polyphemus and Bombyx mori led to an increase of cGMP. A 1:l mixture of 2 pheromone components of Antheraea polyphemus blown for 10 set in physiological concentrations over their antenna1 branches raised cGMP levels about 1.34-fold (kO.08 SEM, n = 23) from a basal level of 3.0 f
G. Ziegelberger; M. J. van den Berg; K.-E. Kaissling; S. Klumpp; J. E. Schultz
Biomimetic Nucleation of Hydroxyapatite Crystals Mediated by Antheraea pernyi Silk Sericin Promotes Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stem Cells.
Biomacromolecules have been used as templates to grow hydroxyapatite crystals (HAps) by biomineralization to fabricate mineralized materials for potential application in bone tissue engineering. Silk sericin is a protein with features desirable as a biomaterial, such as increased hydrophilicity and biodegradation. Mineralization of the silk sericin from Antheraea pernyi (A. pernyi) silkworm has rarely been reported. Here, for the first time, nucleation of HAps on A. pernyi silk sericin (AS) was attempted through a wet precipitation method and consequently the cell viability and osteogenic differentiation of BMSCs on mineralized AS were investigated. It was found that AS mediated the nucleation of HAps in the form of nanoneedles while self-assembling into ?-sheet conformation, leading to the formation of a biomineralized protein based biomaterial. The cell viability assay of BMSCs showed that the mineralization of AS stimulated cell adhesion and proliferation, showing that the resultant AS biomaterial is biocompatible. The differentiation assay confirmed that the mineralized AS significantly promoted the osteogenic differentiation of BMSCs when compared to nonmineralized AS as well as other types of sericin (B. mori sericin), suggesting that the resultant mineralized AS biomaterial has potential in promoting bone formation. This result represented the first work proving the osteogenic differentiation of BMSCs directed by silk sericin. Therefore, the biomineralization of A. pernyi silk sericin coupled with seeding BMSCs on the resultant mineralized biomaterials is a useful strategy to develop the potential application of this unexplored silk sericin in the field of bone tissue engineering. This study lays the foundation for the use of A. pernyi silk sericin as a potential scaffold for tissue engineering. PMID:24666022
Yang, Mingying; Shuai, Yajun; Zhang, Can; Chen, Yuyin; Zhu, Liangjun; Mao, Chuanbin; Ouyang, Hongwei
The Diapause Hormone of the Silkworm, Bombyx mori
IN 1924, Watanabe1 postulated that silkworm eggs overwinter as a result of receiving an `inhibitory' substance from the mother moths, but the existence of the substance has not hitherto been substantiated experimentally. On the other hand, I have found2 that the suboesophageal ganglion of the silkworm is responsible for the hibernation of silkworm eggs; but the organ that furnishes the
Kinsaku Hasegawa
Neofunctionalization in an ancestral insect desaturase lineage led to rare ?6 pheromone signals in the Chinese tussah silkworm.
The Chinese tussah silkworm, Antheraea pernyi (Lepidoptera: Saturniidae) produces a rare dienoic sex pheromone composed of (E,Z)-6,11-hexadecadienal, (E,Z)-6,11-hexadecadienyl acetate and (E,Z)-4,9-tetradecadienyl�acetate, and for which the biosynthetic routes are yet unresolved. By means of gland composition analyses and in vivo labeling we evidenced that pheromone biosynthesis towards the immediate dienoic gland precursor, the (E,Z)-6,11-hexadecadienoic acid, involves desaturation steps with ?(6) and ?(11) regioselectivity. cDNA cloning of pheromone gland desaturases and heterologous expression in yeast demonstrated that the 6,11-dienoic pheromone is generated from two biosynthetic routes implicating a ?(6) and ?(11) desaturase duo albeit with an inverted reaction order. The two desaturases first catalyze the formation of the (E)-6-hexadecenoic acid or (Z)-11-hexadecenoic acid, key mono-unsaturated biosynthetic intermediates. Subsequently, each enzyme is able to produce the (E,Z)-6,11-hexadecadienoic acid by accommodating its non-respective mono-unsaturated product. Besides elucidating an unusually flexible pheromone biosynthetic pathway, our data provide the first identification of a biosynthetic ?(6) desaturase involved in insect mate communication. The occurrence of this novel ?(6) desaturase function is consistent with an evolutionary scenario involving neo-functionalization of an ancestral desaturase belonging to a gene lineage different from the ?(11) desaturases commonly involved in moth pheromone biosynthesis. PMID:20691782
Wang, Hong-Lei; Li�nard, Marjorie A; Zhao, Cheng-Hua; Wang, Chen-Zhu; L�fstedt, Christer
Combination solar hothouse and silkworm breeding house
SciTech Connect
The basic arrangement is shown for a combination silkworm breeding house and solar hothouse with subsoil irrigation and accumulation of heat; it employs a semicylindrical film covering. The process of accumulation of solar heat in the subsoil pebble stores, in water-heater banks, and in the soil is described.
Vardiashvili, A.B.; Muradov, M.; Kim, V.D.
SilkDB: a knowledgebase for silkworm biology and genomics
The Silkworm Knowledgebase (SilkDB) is a web- based repository for the curation, integration and study of silkworm genetic and genomic data. With the recent accomplishment of a 6X draft genome sequence of the domestic silkworm (Bombyx mori), SilkDB provides an integrated representation of the large-scale, genome-wide sequence assembly, cDNAs, clusters of expressed sequence tags (ESTs), transposable elements (TEs), mutants, single
Jing Wang; Qingyou Xia; Ximiao He; Mingtao Dai; Jue Ruan; Jie Chen; Guo Yu; Haifeng Yuan; Yafeng Hu; Ruiqiang Li; Tao Feng; Chen Ye; Cheng Lu; Jun Wang; Songgang Li; Gane Ka-shu Wong; Huanming Yang; Jian Wang; Zhonghuai Xiang; Zeyang Zhou; Jun Yu
Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties
The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers.
Teule, Florence; Miao, Yun-Gen; Sohn, Bong-Hee; Kim, Young-Soo; Hull, J. Joe; Fraser, Malcolm J.; Lewis, Randolph V.; Jarvis, Donald L.
Nucleotide Diversity and Selection Signature in the Domesticated Silkworm, Bombyx mori, and Wild Silkworm, Bombyx mandarina
To investigate the patterns of nucleotide diversity in domesticated silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) and its wild relative, Chinese wild silkworm, Bombyx mandarina Moore, we sequenced nine nuclear genes. Neutrality test and coalescent simulation for these genes were performed to look at bottleneck intensity and selection signature; linkage disequilibrium (LD) within and between loci was employed to investigate allele association. As a result, B. mori lost 33�49% of nucleotide diversity relative to wild silkworm, which is similar to the loss levels found in major cultivated crops. Diversity of B. mori is significantly lower than that of B. mandarina measured as ?total (0.01166 vs. 0.1741) or ?W(0.01124 vs. 0.02206). Bottleneck intensity of domesticated silkworm is 1.5 (in terms of k = Nb/d, Nb-bottleneck population size; d-bottleneck duration) with different durations. Gene DefA showed signature of artificial selection by all analysis methods and might experience strong artificial selection in B. mori during domestication. For nine loci, both curves of LD decay rapidly within 200 bp and drop slowly when distance is > 200 bp, although that of B. mori decays slower than B. mandarina at loci investigated. However, LD could not be estimated at DefA in B. mori and at ER in both silkworms. Elevated LD observed in B. mori may be indicator of selection and demographic events.
Guo, Yi; Shen, Yi-Hong; Sun, Wei; Kishino, Hirohisa; Xiang, Zhong-Huai; Zhang, Ze
Complete sequence and organization of Antheraea pernyi nucleopolyhedrovirus, a dr -rich baculovirus
Background��The completion and reporting of baculovirus genomes is extremely important as it advances our understanding of gene function\\u000a and evolution. Due to the large number of viral genomes now sequenced it is very important that authors present significantly\\u000a detailed analyses to advance the understanding of the viral genomes. However, there is no report of theAntheraea pernyinucleopolyhedrovirus (AnpeNPV) genome.\\u000a \\u000a \\u000a \\u000a \\u000a Results��The genome
The pathway of ammonia assimilation in the silkworm, Bombyx mori
Ammonia can easily be assimilated into amino acids and used for silk-protein synthesis in the silkworm, Bombyx mori. To determine the metabolic pathway of ammonia assimilation, silkworm larvae were injected with methionine sulfoximine (MS), a specific inhibitor of glutamine synthetase (GS). Activity of GS in the fat body 2h after treatment with 400?g MS decreased to less than 10% of
Chikara Hirayama; Kotaro Konno; Hiroshi Shinbo
Microarray-based gene expression profiles of silkworm brains
BACKGROUND: Molecular genetic studies of Bombyx mori have led to profound advances in our understanding of the regulation of development. Bombyx mori brain, as a main endocrine organ, plays important regulatory roles in various biological processes. Microarray technology will allow the genome-wide analysis of gene expression patterns in silkworm brains. RESULTS: We reported microarray-based gene expression profiles in silkworm brains
Ling Gan; Xilong Liu; Zhonghuai Xiang; Ningjia He
Inhibition of human cell apoptosis by silkworm hemolymph.
Many studies on preventing apoptosis have been carried out from the viewpoint of anti-apoptotic cloned-gene expressions inside cells, whereas in this study, we investigated the inhibition of apoptosis by the addition of silkworm hemolymph, a natural compound, from outside of the cells. In a previous study, we reported the inhibition effect of silkworm hemolymph on the baculovirus-induced insect cell apoptosis. Using the vaccinia virus-HeLa cell system as a model system in this study, we found that silkworm hemolymph, the insect serum, inhibits apoptosis not only in the insect cell system but also in the human cell system. The vaccinia virus-induced HeLa cell apoptosis was analyzed using DNA electrophoresis, TUNEL, and flow cytometry, and the resulting data confirmed that silkworm hemolymph inhibits human cell apoptosis. The inhibition of apoptosis due to silkworm hemolymph was not caused by an inhibition of virus binding and internalization steps, nor did silkworm hemolymph interfere with the virus production. The inhibition of apoptosis by silkworm hemolymph decreased the cell detachment from an adhering surface. With these characteristics, silkworm hemolymph can be effectively used to minimize cell death in commercial animal cell culture. PMID:12153324
Choi, Shin Sik; Rhee, Won Jong; Park, Tai Hyun
An Amplified Fragment Length Polymorphism Map of the Silkworm
The silkworm (Bombyx mori L.) is a lepidopteran insect with a long history of significant agricultural value. We have constructed the first amplified fragment length polymorphism (AFLP) genetic linkage map of the silkworm B. mori at a LOD score of 2.5. The mapping AFLP markers were genotyped in 47 progeny from a backcross population of the cross no. 782 3
Yuan-De Tan; Chunling Wan; Yufang Zhu; Chen Lu; Zhonghuai Xiang; Hong-Wen Deng
Odour-source localization system mimicking behaviour of silkworm moth
A new method for localizing odour sources by mimicking the behaviour of silkworm moths is proposed. A male silkworm moth is able to localize its female counterpart by tracking airborne sexual pheromone. Through the observation of this behaviour, we have confirmed that wing vibrations are effective in enhancing the directivity of the odour stimulus. An artificial system with this mechanism
H. Ishida; K. Hayashi; M. Takakusaki; T. Nakamoto; T. Moriizumi; R. Kanzaki
Postintegration stability of the silkworm piggyBac transposon.
The piggyBac transposon is the most widely used vector for generating transgenic silkworms. The silkworm genome contains multiple piggyBac-like sequences that might influence the genetic stability of transgenic lines. To investigate the postintegration stability of piggyBac in silkworms, we used random insertion of the piggyBac [3�נp3 EGFP afm] vector to generate a W chromosome-linked transgenic silkworm, named W-T. Results of Southern blot and inverse PCR revealed the insertion of a single copy in the W chromosome of W-T at a standard TTAA insertion site. Investigation of 11 successive generations showed that all W-T females were EGFP positive and all males were EGFP negative; PCR revealed that the insertion site was unchanged in W-T offspring. These results suggested that endogenous piggyBac-like elements did not affect the stability of piggyBac inserted into the silkworm genome. PMID:24727025
Jiang, Liang; Sun, Qiang; Liu, Weiqiang; Guo, Huizhen; Peng, Zhengwen; Dang, Yinghui; Huang, Chunlin; Zhao, Ping; Xia, Qingyou
The Effect of W Chromosome Origin on Sex-Chromosome Pairing in ZZWW Tetraploid Females of the Domesticated Silkworm, Bombyx Mori , and the Congenic Wild Silkworm, Bombyx Mandarina
To analyze the degree of pairing of the Z and W chromosomes in ZZWW tetraploid female silkworms that have the W chromosomes of the domesticated silkworm, Bombyx mori, and those of the wild silkworm, Bombyx mandarina, we induced two types of ZZWW tetraploid female silkworms (Cr4n, Wr4n) through cold treatment of the eggs. The Wr4n female is congenic to the
N. Tanaka; T. Yokoyama; H. Abe; O. Ninagi; T. Oshiki
Kinetic properties of a sex pheromone-degrading enzyme: the sensillar esterase of Antheraea polyphemus.
Behavioral and electrophysiological evidence has suggested that sex pheromone is rapidly inactivated within the sensory hairs soon after initiation of the action-potential spike. We report the isolation and characterization of a sex-pheromone-degrading enzyme from the sensory hairs of the silkmoth Antheraea polyphemus. In the presence of this enzyme at physiological concentration, the pheromone [(6E,11Z)-hexadecadienyl acetate] has an estimated half-life of 15 msec. Our findings suggest a molecular model for pheromone reception in which a previously reported pheromone-binding protein acts as a pheromone carrier, and an enzyme acts as a rapid pheromone inactivator, maintaining a low stimulus noise level within the sensory hairs.
Vogt, R G; Riddiford, L M; Prestwich, G D
Antiviral activity in the mulberry silkworm, Bombyx mori L
The silkworm Bombyx mori is exploited both as a powerful biological model system and also as a tool to convert leaf protein into silk. Silkworm larvae\\u000a often suffer from viral infections causing heavy losses to the economy of the silk industry. Insects exhibit both humoral\\u000a and cellular immune responses that are effective against various pathogens like bacteria, fungi, protozoa, etc.,
Hui-peng Yao; Xiao-feng Wu; K. Gokulamma
Genetics of insect hemolymph ?-mannosidase in the silkworm, Bombyx mori
The genetics of hemolymph ?-mannosidase was investigated in the silkworm, Bombyx mori. By selecting individuals showing either high or low enzyme activities, homozygotes were separated, with activities varying about five-fold. No differences in the activities of ?-galactosidase and ?-N-acetylglucosaminidase were observed. Thus, it seems that high- and low-enzyme silkworms (High and Low lines) share the same genetic background except for
Shigeru Kimura I
Detection of peptidoglycan and ?-glucan with silkworm larvae plasma test
A method to detect peptidoglycan and (1 ? 3)-?-d-glucan with silkworm larvae plasma (SLP) derived from the hemolymph of the silkworm, Bombyx mori was developed. SLP contains all of the factors of the pro-phenol oxidase cascade, an important self-defense mechanism of insects. Peptidoglycan or (1 ? 3)-?-d-glucan initiates the cascade, in which pro-phenol oxidase is finally activated to phenol oxidase.
Masakazu Tsuchiya; Nobuo Asahi; Fukiko Suzuoki; Masaaki Ashida; Shuji Matsuura
Effects of fluoride in mulberry leaves on the growth and development of silkworm
The effects of fluorides on mulberry and silkworm were investigated. The results had shown that polluted mulberry leaves which contain more than 30 parts per million fluorides (dry wt.) may induce acute damage to silkworm. 6 tables.
Wang Chia-hsi; Qian Da-fu; Li Zheng-fang; Gao Xu-ping
Use of silkworms for identification of drug candidates having appropriate pharmacokinetics from plant sources
BACKGROUND: We use silkworms to evaluate therapeutic effects of drug candidates. Our previous reports have revealed that there are common mechanisms of pharmacokinetics of chemicals in silkworms and mammals. In this report, we attempt to establish a method by using silkworms to identify chemicals from plant extracts which are absorbed from intestine and also stably exist in body fluids. RESULTS:
Yukihiro Asami; Ryo Horie; Hiroshi Hamamoto; Kazuhisa Sekimizu
Genome segment 5 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes a bona fide guanylyltransferase
Background Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of Reoviridae family, infects non mulberry Indian silk worm, Antheraea mylitta, and contains eleven segmented double stranded RNA in its genome (S1-S11). Some of its genome segments (S1-S3, and S6-S11) have been previously characterized but genome segment encoding the viral guanylyltransferase which helps in RNA capping has not been characterized. Results In this study genome segment 5 (S5) of AmCPV was converted to cDNA, cloned and sequenced. S5 consisted of 2180 nucleotides, with one long ORF of 1818 nucleotides and could encode a protein of 606 amino acids with molecular mass of ~65�kDa (p65). Bioinformatics analysis showed presence of KLRS and HxnH motifs as observed in some other reoviral guanylyltransferase and suggests that S5 may encodes viral guanylyltransferase. The ORF of S5 was expressed in E. coli as 65�kDa his tagged fusion protein, purified through Ni-NTA chromatography and polyclonal antibody was raised. Immunoblot analysis of virion particles with the purified antibody showed specific immunoreactive band and suggests p65 as a viral structural protein. Functional analysis showed that recombinant p65 possesses guanylyltransferase activity, and transfers GMP moiety to the 5' diphosphate (A/G) ended viral RNA after the formation of p65-GMP complex for capping. Kinetic analysis showed Km of this enzyme for GTP and RNA was 34.24 uM and 98.35 nM, respectively. Site directed mutagenesis at K21A in KLRS motif, and H93A or H105A in HxnH motif completely abolished the autoguanylylation activity and indicates importance of these residues at these sites. Thermodynamic analysis showed p65-GTP interaction was primarily driven by enthalpy (?H?=?-399.1?�?4.1�kJ/mol) whereas the p65-RNA interaction by favorable entropy (0.043?�?0.0049�kJ/ mol). Conclusion Viral capping enzymes play a critical role in the post transcriptional or post replication modification in case of RNA virus. Our results of cloning, sequencing and functional analysis of AmCPV S5 indicates that S5 encoded p65 through its guanylyltransferase activity can transfer guanine residue to the 5' end of viral RNA for capping. Further studies will help to understand complete capping process of cypoviral RNA during viral replication within the viral capsid.
A new mapping method for quantitative trait loci of silkworm
Background Silkworm is the basis of sericultural industry and the model organism in insect genetics study. Mapping quantitative trait loci (QTLs) underlying economically important traits of silkworm is of high significance for promoting the silkworm molecular breeding and advancing our knowledge on genetic architecture of the Lepidoptera. Yet, the currently used mapping methods are not well suitable for silkworm, because of ignoring the recombination difference in meiosis between two sexes. Results A mixed linear model including QTL main effects, epistatic effects, and QTL � sex interaction effects was proposed for mapping QTLs in an F2 population of silkworm. The number and positions of QTLs were determined by F-test and model selection. The Markov chain Monte Carlo (MCMC) algorithm was employed to estimate and test genetic effects of QTLs and QTL � sex interaction effects. The effectiveness of the model and statistical method was validated by a series of simulations. The results indicate that when markers are distributed sparsely on chromosomes, our method will substantially improve estimation accuracy as compared to the normal chiasmate F2 model. We also found that a sample size of hundreds was sufficiently large to unbiasedly estimate all the four types of epistases (i.e., additive-additive, additive-dominance, dominance-additive, and dominance-dominance) when the paired QTLs reside on different chromosomes in silkworm. Conclusion The proposed method could accurately estimate not only the additive, dominance and digenic epistatic effects but also their interaction effects with sex, correcting the potential bias and precision loss in the current QTL mapping practice of silkworm and thus representing an important addition to the arsenal of QTL mapping tools.
Functional analysis of the inhibitor of apoptosis genes in Antheraea pernyi nucleopolyhedrovirus.
The inhibitor of apoptosis proteins (IAP) plays an important role in cell apoptosis. We cloned two novel IAP family members, Ap-iap1 and Ap-iap2, from Antheraea pernyi nucleopolyhedrovirus (ApNPV) genome. Ap-IAP1 contains two baculoviral IAP repeat (BIR) domains followed by a RING domain, but Ap-IAP2 has only one BIR domain and RING. The result of transient expression in Spodoptera frugiperda (Sf21) showed that Ap-iap1 blocked cell apoptosis induced by actinomycin D treatment and also rescued the p35 deficient Autographa californica nucleopolyhedrovirus (AcNPV) to replicate in Sf9 cells, while Ap-iap2 does not have this function. Several Ap-IAP1 truncations were constructed to test the activity of BIRs or RING motif to inhibit cell apoptosis. The results indicated that BIRs or RING of Ap-IAP1 had equally function to inhibit cell apoptosis. Therefore deletion of above both of the above domains could not block apoptosis induced by actinomycin D or rescue the replication of AcMNPV Delta p35. We also screened two phage-display peptides that might interact with Ap-IAP1. PMID:20437152
Yan, Feng; Deng, Xiaobei; Yan, Junpeng; Wang, Jiancheng; Yao, Lunguang; Lv, Songya; Qi, Yipeng; Xu, Hua
Antheraea pernyi silk fibroin for targeted gene delivery of VEGF165-Ang-1 with PEI.
Vascularization is a crucial challenge in tissue engineering. One solution for this problem is to implant scaffolds that contain functional genes that promote vascularization by providing angiogenic growth factors via a gene delivery carrier. Poly(ethylenimine) (PEI) is a gene delivery carrier with high transfection efficiency but with cytotoxicity. To solve this problem, we utilized Antheraea pernyi silk fibroin (ASF), which has favorable cytocompatibility and biodegradability, RGD sequences and a negative charge, in conjunction with PEI, as the delivery vector for vascular endothelial growth factor (VEGF) 165-angiopoietin-1 (Ang-1) dual gene simultaneous expression plasmid, creating an ASF/PEI/pDNA complex. The results suggested that the zeta potential of the ASF/PEI/pDNA complex was significantly lower than that of the PEI/pDNA complex. Decreased nitrogen and increased oxygen on the surface of the complex demonstrated that the ASF had successfully combined with the surface of the PEI/pDNA. Furthermore, the complexes resisted digestion by nucleic acid enzymes and degradation by serum. L929 cells were cultured and transfected in vitro and improved cytotoxicity was found when the cells were transfected with ASF/PEI/pDNA compared with PEI/pDNA. In addition, the transfection efficiency and VEGF secretion increased. In general, this study provides a novel method for decreasing the cytotoxicity of PEI gene delivery vectors and increasing transfection efficiency of angiogenesis-related genes. PMID:24867887
Ma, Caili; Lv, Linlin; Liu, Yu; Yu, Yanni; You, Renchuan; Yang, Jicheng; Li, Mingzhong
Pheromone receptors in Bombyx mori and Antheraea pernyi. II. Morphometric analysis.
Sensilla trichodea of the silk moths, Antheraea pernyi and Bombyx mori, were reconstructed from serial sections after freeze substitution. The volume and surface area of the different sensillar cells were calculated from the area and circumference of consecutive section profiles. A. pernyi and B. mori differ largely in the size of the sensory hair and the larger outer dendritic segments as well as in the volume of the receptor lymph within the hair, while there are only small differences regarding inner dendritic segments, receptor-cell somata, trichogen and tormogen cells and the volume of the receptor lymph below the hair base. In each sensillum the two (or three) receptor-cell somata, dendrites, and initial axonal segments differ significantly in volume and surface. The apical cell membranes of the trichogen and tormogen cells, which border the receptor-lymph cavity and which are the presumed site of electrogenic cation pumps, are deeply invaginated and enlarged by microlamellae and microvilli, so that their area is twice that of the remaining basolateral cell membrane. In contrast to mechanoreceptors, the trichogen cell is the largest auxiliary cell and has the largest apical membrane surface. The morphometric data are discussed with regard to recent electrophysiological observations. PMID:6697382
Gnatzy, W; Mohren, W; Steinbrecht, R A
KAIKObase: An integrated silkworm genome database and data mining tool
Background The silkworm, Bombyx mori, is one of the most economically important insects in many developing countries owing to its large-scale cultivation for silk production. With the development of genomic and biotechnological tools, B. mori has also become an important bioreactor for production of various recombinant proteins of biomedical interest. In 2004, two genome sequencing projects for B. mori were reported independently by Chinese and Japanese teams; however, the datasets were insufficient for building long genomic scaffolds which are essential for unambiguous annotation of the genome. Now, both the datasets have been merged and assembled through a joint collaboration between the two groups. Description Integration of the two data sets of silkworm whole-genome-shotgun sequencing by the Japanese and Chinese groups together with newly obtained fosmid- and BAC-end sequences produced the best continuity (~3.7 Mb in N50 scaffold size) among the sequenced insect genomes and provided a high degree of nucleotide coverage (88%) of all 28 chromosomes. In addition, a physical map of BAC contigs constructed by fingerprinting BAC clones and a SNP linkage map constructed using BAC-end sequences were available. In parallel, proteomic data from two-dimensional polyacrylamide gel electrophoresis in various tissues and developmental stages were compiled into a silkworm proteome database. Finally, a Bombyx trap database was constructed for documenting insertion positions and expression data of transposon insertion lines. Conclusion For efficient usage of genome information for functional studies, genomic sequences, physical and genetic map information and EST data were compiled into KAIKObase, an integrated silkworm genome database which consists of 4 map viewers, a gene viewer, and sequence, keyword and position search systems to display results and data at the level of nucleotide sequence, gene, scaffold and chromosome. Integration of the silkworm proteome database and the Bombyx trap database with KAIKObase led to a high-grade, user-friendly, and comprehensive silkworm genome database which is now available from URL: .
Shimomura, Michihiko; Minami, Hiroshi; Suetsugu, Yoshitaka; Ohyanagi, Hajime; Satoh, Chikatada; Antonio, Baltazar; Nagamura, Yoshiaki; Kadono-Okuda, Keiko; Kajiwara, Hideyuki; Sezutsu, Hideki; Nagaraju, Javaregowda; Goldsmith, Marian R; Xia, Qingyou; Yamamoto, Kimiko; Mita, Kazuei
Burst expansion, distribution and diversification of MITEs in the silkworm genome
Background Miniature inverted-repeat transposable elements (MITEs) are widespread in plants and animals. Although silkworm (Bombyx mori) has a large amount of and a variety of transposable elements, the genome-wide information of the silkworm MITEs is unknown. Results We used structure-based and homology approaches to search for MITEs in the silkworm genome. We identified 17 MITE families with a total of 5785 members, accounting for ~0.4% of the genome. 7 of 17 MITE families are completely novel based on the nucleotide composition of target site duplication (TSD) and/or terminal inverted repeats (TIR). Silkworm MITEs were widely and nonrandom distributed in the genome. One family named BmMITE-2 might experience a recent burst expansion. Network and diversity analyses for each family revealed different diversification patterns of the silkworm MITEs, reflecting the signatures of genome-shocks that silkworm experienced. Most silkworm MITEs preferentially inserted into or near genes and BmMITE-11 that encodes a germline-restricted small RNA might silence its the closest genes in silkworm ovary through a small RNA pathway. Conclusions Silkworm harbors 17 MITE families. The silkworm MITEs preferred to reside in or near genes and one MITE might be involved in gene silence. Our results emphasize the exceptional role of MITEs in transcriptional regulation of genes and have general implications to understand interaction between MITEs and their host genome.
Analysis of bacteria-challenged wild silkmoth, Antheraea mylitta (lepidoptera) transcriptome reveals potential immune genes
Background In the recent years a strong resemblance has been observed between the insect immune system and the mammalian innate immune mechanisms suggesting their common origin. Among the insects, only the dipterans (Drosophila and various mosquito species) have been widely investigated for their immune responses towards diverse pathogens. In the present study we constructed and analysed the immune transcriptome of the lepidopteran Antheraea mylitta, an economically important Indian tasar silkmoth with a view to unravel the potential immune-related genes and pathways. Results An expressed sequence tag (EST) library was constructed from mRNA obtained from fat bodies of A. mylitta larvae that had been challenged by infection with Escherichia coli cells. We identified 719 unique ESTs from a total of 1412 sequences so generated. A third of the transcriptome showed similarity with previously characterized immune-related genes that included both the known and putative immune genes. Of the four putative novel defence proteins (DFPs) annotated by PSI-BLAST three showed similarity to extracellular matrix proteins from vertebrates implicated in innate immunity, while the fourth was similar to, yet distinct from, the anti-microbial protein cecropin. Finally, we analysed the expression profiles of 15 potential immune-related genes, and the majority of them were induced more prominently with E. coli compared to Micrococcus luteus. We also identified several unknown proteins, some of which could have probable immune-related functions based on the results of the ProDom analysis. Conclusion The present study has identified many potential immune-related genes in A. mylitta some of which are vertebrate homologues and others are hitherto unreported putative defence proteins. Several genes were present as members of gene families, as has also been observed in other insect species.
Gandhe, Archana S; Arunkumar, KP; John, Serene H; Nagaraju, J
Carotenoids of mulberry leaves and of silkworm excreta
At the present time, great attention is being devoted to the search for new cheap sources of carotene [i]. The raw material for this may be certain industrial wastes and, in particular, silkworm excreta (SWE) the complex study of the composition of which has already been carried on over a number of years [2]. In the present communication we present
D. U. Uzakova; A. A. Kolesnik; Yu. L. Zherebin; I. K. Sarycheva
Cytological evidence for holocentric chromosomes of the silkworms, Bombyx mori and B. mandarina, (Bombycidae, Lepidoptera)
The nature of the centromere and the orientation in meiosis of silkworm chromosomes were investigated using the trivalent of the F1 hybrid between the wild and domestic silkworm and X-ray-induced aberrant chromosomes as well as normal silkworm chromosomes. The results of the experiments were as follows: (1) Pro-metaphase chromosomes showed no distinct primary constriction even after treatment with hypotonic solution,
Akio Murakami; Hirotami T. Imai
A Genome-Wide Survey for Host Response of Silkworm, Bombyx mori during Pathogen Bacillus bombyseptieus Infection
Host-pathogen interactions are complex relationships, and a central challenge is to reveal the interactions between pathogens and their hosts. Bacillus bombysepticus (Bb) which can produces spores and parasporal crystals was firstly separated from the corpses of the infected silkworms (Bombyx mori). Bb naturally infects the silkworm can cause an acute fuliginosa septicaemia and kill the silkworm larvae generally within one
Lulin Huang; Tingcai Cheng; Pingzhen Xu; Daojun Cheng; Ting Fang; Qingyou Xia; Georg H�cker
Proteolytic enzymes as markers of productivity and heterosis of silkworm
A positive correlation between the activity level of cysteine proteinases in developing eggs of common silkworm moth (Bombyx mori L.), on the one hand, and a set of commercial characteristics, on the other, was found. This allows the determination of cysteine proteinase activities (pH optima of 3.0, 3.6, and 8.6) to be recommended as a biochemical test for an early
I. D. Krylova; D. V. Yarygin; Yu. B. Filippovich
Trehazolin, a slow, tight-binding inhibitor of silkworm trehalase
Mechanisms of enzyme inhibition by trehazolin, a new inhibitor of trehalase (Ando et al. (1991) J. Antibiot. 44, 1165), were investigated using purified soluble silkworm trehalase and other glycosidases. Trehazolin inhibited trehalase with an IC50 value of 27 nM, whereas some other exo-?-glucosidases were inhibited only weakly, with IC50 values ranging from 7 to 370 ?M. Other glycosidases tested were
Osamu Ando; Mutsuo Nakajima; Mika Kifune; Hui Fang; Kazuhiko Tanzawa
Expression profile of cuticular genes of silkworm, Bombyx mori
BACKGROUND: Insect cuticle plays essential roles in many physiological functions. During molting and metamorphosis tremendous changes occur in silkworm cuticle where multiple proteins exist and genes encoding them constitute about 1.5% of all Bombyx mori genes. RESULTS: In an effort to determine their expression profiles, a microarray-based investigation was carried out using mRNA collected from larvae to pupae. The results
Jiubo Liang; Liang Zhang; Zhonghuai Xiang; Ningjia He
Vertical transmission of nucleopolyhedrovirus in the silkworm, Bombyx mori L
Nucleopolyhedrovirus (NPV) was tested for vertical transmission in the silkworm, Bombyx mori. Fifth instar larvae were exposed to four different dosages of BmNPV (830, 1300, 1800, and 2000OBs\\/larva) and a dosage of about 2000OBs\\/larva was found suitable for obtaining infected adults. Histopathological studies revealed the infection in susceptible tissues and organs initially, and at later stages of infection cycles the
A. M. Khurad; A. Mahulikar; M. K. Rathod; M. M. Rai; S. Kanginakudru; J. Nagaraju
Transgenic silkworms produce recombinant human type III procollagen in cocoons
We describe the generation of transgenic silkworms that produce cocoons containing recombinant human collagen. A fusion cDNA was constructed encoding a protein that incorporated a human type III procollagen mini-chain with C-propeptide deleted, a fibroin light chain (L-chain), and an enhanced green fluorescent protein (EGFP). This cDNA was ligated downstream of the fibroin L-chain promoter and inserted into a piggyBac
Masahiro Tomita; Hiroto Munetsuna; Tsutomu Sato; Takahiro Adachi; Rika Hino; Masahiro Hayashi; Katsuhiko Shimizu; Namiko Nakamura; Toshiki Tamura; Katsutoshi Yoshizato
Animal welfare and use of silkworm as a model animal.
Sacrificing model animals is required for developing effective drugs before being used in human beings. In Japan today, at least 4,210,000 mice and other mammals are sacrificed to a total of 6,140,000 per year for the purpose of medical studies. All the animals treated in Japan, including test animals, are managed under control of "Act on Welfare and Management of Animals". Under the principle of this Act, no person shall kill, injure, or inflict cruelty on animals without due cause. "Animal" addressed in the Act can be defined as a "vertebrate animal". If we can make use of invertebrate animals in testing instead of vertebrate ones, that would be a remarkable solution for the issue of animal welfare. Furthermore, there are numerous advantages of using invertebrate animal models: less space and small equipment are enough for taking care of a large number of animals and thus are cost-effective, they can be easily handled, and many biological processes and genes are conserved between mammals and invertebrates. Today, many invertebrates have been used as animal models, but silkworms have many beneficial traits compared to mammals as well as other insects. In a Genome Pharmaceutical Institute's study, we were able to achieve a lot making use of silkworms as model animals. We would like to suggest that pharmaceutical companies and institutes consider the use of the silkworm as a model animal which is efficacious both for financial value by cost cutting and ethical aspects in animals' welfare. PMID:23006994
Sekimizu, N; Paudel, A; Hamamoto, H
Identification and Characterization of an Arginine Kinase as a Major Allergen from Silkworm (Bombyx mori) Larvae
Background: The silkworm, Bombyx mori, is an important insect in the textile industry and its pupa are used in Chinese cuisine and traditional Chinese medicine. The silk, urine and dander of silkworms is often the cause of allergies in sericulture workers and the pupa has been found to be a food allergen in China. Recent studies have focused on reporting
Zhigang Liu; Lixin Xia; Yulan Wu; Qingyou Xia; Jiajie Chen; Kenneth H. Roux
Existence of an Enzyme catalysing the Hydrogenation of Oximes in Silkworms
RECENTLY1, I found that, in silkworms, nitrites, nitrates and ammonium salts could be converted into oximes, and suggested that this insect might utilize inorganic nitrogenous salts as nutritive substances. I have now been able to demonstrate that in the silkworm body there exists an enzyme which catalyses the transformation of oximes to corresponding amino compounds.
Kazuo Yamafuji
Correlation between yield and biochemical parameters in the mulberry silkworm, Bombyx mori L
A detailed study was carried out on six biochemical parameters and four yield attributes using multiple regression analysis to investigate their relationship in the mulberry silkworm,Bombyx mori. The study generated new information on the importance of digestive amylase activity for the survival of the silkworm and revealed the inability of other enzymes to affect this relationship. Data also substantiate the
S. N. Chatterjee; C. G. P. Rao; G. K. Chatterjee; S. K. Ashwath; A. K. Patnaik
Fine mapping of Ekp-1, a locus associated with silkworm (Bombyx mori) proleg development
The silkworm homeotic mutant Ekp has a pair of rudimentary abdominal legs, called prolegs, in its A2 segment. This phenotype is caused by a single dominant mutation at the Ekp-1 locus, which was previously mapped to chromosome 6. To explore the possible association of Hox genes with proleg development in the silkworm, a map-based cloning strategy was used to isolate
H Xiang; M Li; F Yang; Q Guo; S Zhan; H Lin; X Miao; Y Huang
Efficacy of silkworm ( Bombyx mori L.) chrysalis oil as a lipid source in adult Wistar rats
The effects of silkworm chrysalis oil, rich in n-3 ?-linolenic acid (ALA), on lipid metabolism in Wistar rats were investigated. The rats were fed diets containing 7% soybean oil (control), silkworm chrysalis oil (SWO), or fish oil (FO) for 8weeks. Plasma triglyceride and glucose levels were significantly lower in the SWO group after 8weeks compared to the control and FO
Feny Mentang; Masashi Maita; Hideki Ushio; Toshiaki Ohshima
A Draft Sequence for the Genome of the Domesticated Silkworm (Bombyx mori)
We report a draft sequence for the genome of the domesticated silkworm (Bombyx mori), covering 90.9% of all known silkworm genes. Our estimated gene count is 18,510, which exceeds the 13,379 genes reported for Drosophila melanogaster. Comparative analyses to fruitfly, mosquito, spider, and butterfly reveal both similarities and differences in gene content.
Qingyou Xia; Zeyang Zhou; Cheng Lu; Daojun Cheng; Fangyin Dai; Bin Li; Ping Zhao; Xingfu Zha; Tingcai Cheng; Chunli Chai; Guoqing Pan; Jinshan Xu; Chun Liu; Ying Lin; Jifeng Qian; Yong Hou; Zhengli Wu; Guanrong Li; Minhui Pan; Chunfeng Li; Yihong Shen; Xiqian Lan; Lianwei Yuan; Tian Li; Hanfu Xu; Guangwei Yang; Yongji Wan; Yong Zhu; Maode Yu; Weide Shen; Dayang Wu; Zhonghuai Xiang; Jun Yu; Jun Wang; Ruiqiang Li; Jianping Shi; Heng Li; Guangyuan Li; Jianning Su; Xiaoling Wang; Guoqing Li; Zengjin Zhang; Qingfa Wu; Jun Li; Qingpeng Zhang; Ning Wei; Jianzhe Xu; Haibo Sun; Le Dong; Dongyuan Liu; Shengli Zhao; Xiaolan Zhao; Qingshun Meng; Fengdi Lan; Xiangang Huang; Yuanzhe Li; Lin Fang; Changfeng Li; Dawei Li; Yongqiao Sun; Zhenpeng Zhang; Zheng Yang; Yanqing Huang; Yan Xi; Qiuhui Qi; Dandan He; Haiyan Huang; Xiaowei Zhang; Zhiqiang Wang; Wenjie Li; Yuzhu Cao; Yingpu Yu; Hong Yu; Jinhong Li; Jiehua Ye; Huan Chen; Yan Zhou; Bin Liu; Jing Wang; Jia Ye; Hai Ji; Shengting Li; Peixiang Ni; Jianguo Zhang; Yong Zhang; Hongkun Zheng; Bingyu Mao; Wen Wang; Chen Ye; Songgang Li; Jian Wang; Gane Ka-Shu Wong; Huanming Yang
Microsatellites in the silkworm, Bomby� mori : Abundance, polymorphism, and strain characterization
We have isolated and characterized microsatellites (simple sequence repeat (SSR) loci) from the silkworm genome. The screening of a partial genomic library by the conventional hybridization method led to the isolation of 28 microsatellites harbouring clones. The abundance of (CA)n repeats in the silkworm genome was akin to those reported in the other organisms such as honey bee, pig, and
K. Damodar Reddy; E. G. Abraham; J. Nagaraju
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.
Ishii, Kenichi; Hamamoto, Hiroshi; Imamura, Katsutoshi; Adachi, Tatsuo; Shoji, Mikio; Nakayama, Koji; Sekimizu, Kazuhisa
Transcriptome Analysis of the Silkworm (Bombyx mori) by High-Throughput RNA Sequencing
The domestic silkworm, Bombyx mori, is a model insect with important economic value for silk production that also acts as a bioreactor for biomaterial production. The functional complexity of the silkworm transcriptome has not yet been fully elucidated, although genomic sequencing and other tools have been widely used in its study. We explored the transcriptome of silkworm at different developmental stages using high-throughput paired-end RNA sequencing. A total of about 3.3 gigabases (Gb) of sequence was obtained, representing about a 7-fold coverage of the B. mori genome. From the reads that were mapped to the genome sequence; 23,461 transcripts were obtained, 5,428 of them were novel. Of the 14,623 predicted protein-coding genes in the silkworm genome database, 11,884 of them were found to be expressed in the silkworm transcriptome, giving a coverage of 81.3%. A total of 13,195 new exons were detected, of which, 5,911 were found in the annotated genes in the Silkworm Genome Database (SilkDB). An analysis of alternative splicing in the transcriptome revealed that 3,247 genes had undergone alternative splicing. To help with the data analysis, a transcriptome database that integrates our transcriptome data with the silkworm genome data was constructed and is publicly available at http://220.127.116.11/gbrowse2/. To our knowledge, this is the first study to elucidate the silkworm transcriptome using high-throughput RNA sequencing technology. Our data indicate that the transcriptome of silkworm is much more complex than previously anticipated. This work provides tools and resources for the identification of new functional elements and paves the way for future functional genomics studies.
Tian, Jian; Liu, Huifen; Yang, Huipeng; Yi, Yongzhu; Wang, Jinhui; Shi, Xiaofeng; Jiang, Feng; Yao, Bin; Zhang, Zhifang
Silkworm cocoons inspire models for random fiber and particulate composites.
The bioengineering design principles evolved in silkworm cocoons make them ideal natural prototypes and models for structural composites. Cocoons depend for their stiffness and strength on the connectivity of bonding between their constituent materials of silk fibers and sericin binder. Strain-activated mechanisms for loss of bonding connectivity in cocoons can be translated directly into a surprisingly simple yet universal set of physically realistic as well as predictive quantitative structure-property relations for a wide range of technologically important fiber and particulate composite materials. PMID:21230317
Chen, Fujia; Porter, David; Vollrath, Fritz
Silkworm cocoons inspire models for random fiber and particulate composites
The bioengineering design principles evolved in silkworm cocoons make them ideal natural prototypes and models for structural composites. Cocoons depend for their stiffness and strength on the connectivity of bonding between their constituent materials of silk fibers and sericin binder. Strain-activated mechanisms for loss of bonding connectivity in cocoons can be translated directly into a surprisingly simple yet universal set of physically realistic as well as predictive quantitative structure-property relations for a wide range of technologically important fiber and particulate composite materials.
Chen Fujia; Porter, David; Vollrath, Fritz [Department of Zoology, University of Oxford, Oxford OX1 3PS (United Kingdom)
[Silkworm excrement organic fertilizer: its nutrient properties and application effect].
In this paper, silkworm excrement was harmless-treated via controlled fermentation to prepare silkworm excrement organic fertilizer (SEOF). The nutrient properties of the SEOF were determined, and a pot experiment was conducted to examine the application effect of the fertilizer. After fermentation, the total N, P, and K contents in the SEOF had a significant increase, being 58.0%, 84.4% , and 29.7% higher than those in the raw material, respectively. The addition of microbial inoculants shortened the fermentation period, and decreased the carbon and nitrogen losses during fermentation. With the application of SEOF, the seed germination index of cabbage and tomato was higher than 80% , suggesting that the fertilizer had no inhibitory effect on the seed germination. The application of SEOF not only increased the Chinese cabbage yield and its nutrients and Vc contents, decreased the plant nitrate content, but also improved the soil pH value, and increased the soil available nutrients and organic matter contents and soil enzyme activities, with better effect than applying composted goat feces. PMID:22007458
Chen, Xiao-ping; Xie, Ya-jun; Luo, Guang-en; Shi, Wei-yong
Purification and characterization of a cysteine proteinase from silkworm eggs.
Eggs of the silkworm, Bombyx mori, contain a high level of a proteinase which is most active in acidic pH region. The proteinase was purified from an extract of eggs by a six-step procedure which included conventional chromatographic fractionations. The molecular mass of the proteinase was estimated to be 350 kDa by gel filtration and 47 kDa by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels, suggesting an octameric structure. The amino acid composition was found to resemble that of mammalian lysosomal cysteine proteinases, in particular cathepsin L. The NH2-terminal 10-residue sequence is Val-Gln-Phe-Phe-Asp-Leu-Val-Lys-Glu-Glu-. The enzyme appears to be a member of the class of cysteine proteinases since it was strongly inhibited by sulfhydryl-reactive compounds and N-[N-(1,3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). The enzyme hydrolyzed various protein substrates, such as hemoglobin, vitellogenin, vitellin, and lipophorin, with maximal activity around pH 3-3.5. The specificity of the cleavage sites in the oxidized B chain of insulin was rather well defined and there was high affinity for hydrophobic residues at the P2 and P3 positions. The cysteine proteinase is thought to be involved in protein degradation during embryonic development of silkworm eggs. PMID:2226439
Kageyama, T; Takahashi, S Y
Background Insect cuticle plays essential roles in many physiological functions. During molting and metamorphosis tremendous changes occur in silkworm cuticle where multiple proteins exist and genes encoding them constitute about 1.5% of all Bombyx mori genes. Results In an effort to determine their expression profiles, a microarray-based investigation was carried out using mRNA collected from larvae to pupae. The results showed that a total of 6676 genes involved in various functions and physiological pathways were activated. The vast majority (93%) of cuticular protein genes were expressed in selected stages with varying expression patterns. There was no correlation between expression patterns and the presence of conserved motifs. Twenty-six RR genes distributed in chromosome 22 were co-expressed at the larval and wandering stages. The 2 kb upstream regions of these genes were further analyzed and three putative elements were identified. Conclusions Data from the present study provide, for the first time, a comprehensive expression profile of genes in silkworm epidermal tissues and evidence that putative elements exist to allow massive production of mRNAs from specific cuticular protein genes.
Characterization of hemocytes proliferation in larval silkworm, Bombyx mori.
Hemocytes play multiple important roles during insect growth and development. Five types of hemocytes have been identified in the silkworm, Bombyx mori: prohemocyte, plasmatocyte, granulocyte, spherulocyte, and oenocytoid. We used the S-phase marker bromodeoxyuridine (BrdU) antibody along with the mitosis marker phosphohistone H3 (PHH3) antibody to monitor proliferation of hemocytes in vivo. The results indicate that silkworm hematopoiesis not only occurs in the circulatory system but also in hematopoietic organs (HPOs). During the 5th instar, the hemocyte proliferation in the circulatory system reaches a peak at the pre-wandering stage. Following infection by Escherichia coli, circulating hemocytes increase their cell divisions as demanded by the cellular immune response. All hemocytes, except spherulocytes, have the capacity to multiply in vivo. The BrdU label-retaining assay shows that a small portion of cells from the circulatory system and the HPOs are continuously labelled up to 9days and 4days respectively. A small number of long-term label retaining cells (LRCs) quiescently locate in circulatory system. All results indicate that there are a few quiescent stem cells or some progenitors in the larval circulatory system and HPO that produce new hemocytes and continuously release them into the circulating system. PMID:23557681
Tan, Juan; Xu, Man; Zhang, Kui; Wang, Xue; Chen, Siyuan; Li, Tai; Xiang, Zhonghuai; Cui, Hongjuan
Characterization and Comparison of Serratia Marcescens Isolated from Edible Cactus and from Silkworm for Virulence Potential and Chitosan Susceptibility
Representative strains of Serratia marcescens from an edible cactus plant and silkworms were characterized and a comparison based on their cellular fatty acid composition, 16S rRNA and groE gene sequence analysis as well as silkworm virulence and chitosan susceptibility was carried out. Results from this study indicate that there are no significant differences between the phenotypic and molecular characterization, virulence and chitosan susceptibility of the S. marcescens strains from the cactus plant and silkworms. Silkworms inoculated with S. marcescens from either plant or silkworm resulted in nearly 100% mortality. Chitosan solution exhibited strong antibacterial activity against S. marcescens. This activity increased with the increase of chitosan concentration and incubation time regardless of the strain source. Also, the results indicate that the plant associated S. marcescens maybe plays a possible role in the contamination of humans and animals, in particular silkworms, while chitosan showed a potential to control the contamination caused by S. marcescens.
Li, Bin; Yu, Rongrong; Liu, Baoping; Tang, Qiaomei; Zhang, Guoqing; Wang, Yanli; Xie, Guanlin; Sun, Guochang
Ontogeny of mouthpart sensilla of Muga silkworm: a scanning electron microscopic study.
Scanning electron microscopy on the postembryonic development of the mouthpart sensory structures of the Muga silk moth, Antheraea assamensis revealed the presence of a variety of sensilla. The types, distribution pattern of the sensilla remained almost the same in the different larval stages except for the number and dimension in some cases. The findings of the study assumes significance because the silk moth, Antheraea assamensis is a species endemic to the North Eastern part of India and very few reports are available on it. In fact, the present study provides the first report on the ontogeny of the mouth part sensilla of the silk moth species. Since mouth part sensilla and their surface micro structural characteristics are extremely important in understanding their roles in searching behavior during feeding, the present study will certainly help in developing strategies for proper rearing of this economically important insect species. PMID:24446353
Goldsmith, Anita; Dey, Sudip; Kalita, Jatin; Choudhury, Rina
Occurrence of Two Different Enzymes in the Silkworm, Bombyx mori, to reduce Folate and Sepiapterin
THE mutant lemon of the silkworm is characteristically yellow in colour, accumulating a fluorescent pigment, xanthopterin-B1, in the skin. Extract of the fat body of the wild-type silkworm converts this pigment into a non fluorescent product1. There is some evidence to support the view that xanthopterin-B1 is identical with sepiapterin2, which is known to be reduced, in the presence of
Masako Matsubara; Motoo Tsusue; Miki Akino
Occurrence of a Malic Enzyme free of Oxalacetic Decarboxylase in Silkworm H�molymph
H�MOLYMPH of the larval silkworm (Bombyx mori L.) contains a triphosphopyridine nucleotide-linked `malic' enzyme that has been purified by ammonium sulphate fractionation followed by adsorption and elution from calcium phosphate gel1,2. It was of interest to measure the oxalacetic decarboxylase activity of the `malic' enzyme from silkworm, since those of pigeon liver3,4 and wheat germ5,6 exhibit a constant ratio (approximately
P. Faulkner
Melatonin and arylalkylamine N-acetyltransferase activity in the silkworm, Bombyx mori
Melatonin (N-acetyl-5-methoxytryptamine) was identified in the head and hemolymph of the silkworm, Bombyx mori, using reversed-phase high-performance liquid chromatography coupled with fluorometric detection and radioimmunoassay. In addition, evidence of arylalkylamine (serotonin) N-acetyltransferase (NAT) a key enzyme controlling the synthesis of melatonin in vertebrates, was found in the head of the silkworm. Melatonin levels in the head and hemolymph and the
Masanori T. Itoh; Atsuhiko Hattori; Tsuyoshi Nomura; Yawara Sumi; Takuro Suzuki
Utilization of silkworm cocoon waste as a sorbent for the removal of oil from water
The aim of this study is to investigate the utilization of silkworm cocoon waste, such as pierced or stained cocoons, as a sorbent material for the removal of motor and vegetable oils from water. The oil-sorption capacity, rate and reusability of the material were evaluated. The results show the high sorption capacity of the silkworm cocoon waste sorbent (42�52goil\\/gsorbent for
Hiroshi Moriwaki; Shiori Kitajima; Masahiro Kurashima; Ayaka Hagiwara; Kazuma Haraguchi; Koji Shirai; Rensuke Kanekatsu; Kenji Kiguchi
Germ line transformation of the silkworm, Bombyx mori , using the transposable element Minos
We investigated the use of Minos as a vector for transgenesis in the silkworm, Bombyx mori. We first constructed a vector plasmid with the green fluorescent protein (GFP) gene fused with the silkworm cytoplasmic\\u000a actin gene (A3) promoter, and a helper plasmid with the Minos transposase gene controlled by the same A3 promoter. Injection of the vector and helper plasmid
K. Uchino; M. Imamura; K. Shimizu; T. Kanda; T. Tamura
Transcriptome analysis of silkworm, Bombyx mori, during early response to Beauveria bassiana challenges.
Host-pathogen interactions are complex processes and it is a central challenge to reveal these interactions. Fungal infection of silkworm, Bombyx mori, may induce a variety of responsive reaction. However, little is known about the molecular mechanism of silkworm immune response against the fungal infection. To obtain an overview of the interaction between silkworm and an entomopathogenic fungus Beauveria bassiana, Digital Gene Expression profiling, a tag based high-throughput transcriptome sequencing method, was employed to screen and identify differentially expressed genes (DEGs, FDR ? 0.001, ?log2ratio? ? 1) of silkworm larvae during early response against B. bassiana infection. Total 1430 DEGs including 960 up-regulated and 470 down-regulated ones were identified, of which 627 DEGs can be classified into GO categories by Gene Ontology (GO) analysis. KEGG pathways analysis of these DEGs suggested that many biological processes, such as defense and response, signal transduction, phagocytosis, regulation of gene expression, RNA splicing, biosynthesis and metabolism, protein transport etc. were involved in the interaction between the silkworm and B. bassiana. A number of differentially expressed fungal genes were also identified by mapping the sequencing tags to B. bassiana genome. These results provided new insights to the molecular mechanism of silkworm immune response to B. bassiana infection. PMID:24618587
Hou, Chengxiang; Qin, Guangxing; Liu, Ting; Geng, Tao; Gao, Kun; Pan, Zhonghua; Qian, Heying; Guo, Xijie
Specificity Determinants of the Silkworm Moth Sex Pheromone
The insect olfactory system, particularly the peripheral sensory system for sex pheromone reception in male moths, is highly selective, but specificity determinants at the receptor level are hitherto unknown. Using the Xenopus oocyte recording system, we conducted a thorough structure-activity relationship study with the sex pheromone receptor of the silkworm moth, Bombyx mori, BmorOR1. When co-expressed with the obligatory odorant receptor co-receptor (BmorOrco), BmorOR1 responded in a dose-dependent fashion to both bombykol and its related aldehyde, bombykal, but the threshold of the latter was about one order of magnitude higher. Solubilizing these ligands with a pheromone-binding protein (BmorPBP1) did not enhance selectivity. By contrast, both ligands were trapped by BmorPBP1 leading to dramatically reduced responses. The silkworm moth pheromone receptor was highly selective towards the stereochemistry of the conjugated diene, with robust response to the natural (10E,12Z)-isomer and very little or no response to the other three isomers. Shifting the conjugated diene towards the functional group or elongating the carbon chain rendered these molecules completely inactive. In contrast, an analogue shortened by two omega carbons elicited the same or slightly higher responses than bombykol. Flexibility of the saturated C1�C9 moiety is important for function as addition of a double or triple bond in position 4 led to reduced responses. The ligand is hypothesized to be accommodated by a large hydrophobic cavity within the helical bundle of transmembrane domains.
Xu, Pingxi; Hooper, Antony M.; Pickett, John A.; Leal, Walter S.
Evaluation of general toxicity and genotoxicity of the silkworm extract powder.
The silkworm extract powder contain 1-deoxynojirimycin (DNJ), a potent ?-glycosidase inhibitor, has therapeutic potency against diabetes mellitus. Therefore, natural products containing DNJ from mulberry leaves and silkworm are consumed as health functional food. The present study was performed to evaluate the safety of the silkworm extract powder, a health food which containing the DNJ. The repeated toxicity studies and gentic toxicity studies of the silkworm extract powder were performed to obtain the data for new functional food approval in MFDS. The safety was evaluated by a single-dose oral toxicity study and a 90 day repeated-dose oral toxicity study in Sprague-Dawley rats. The silkworm extract powder was also evaluated for its mutagenic potential in a battery of genetic toxicity test: in vitro bacterial reverse mutation assay, in vitro chromosomal aberration test, and in vivo mouse bone marrow micronucleus assay. The results of the genetic toxicology assays were negative in all of the assays. The approximate lethal dose in single oral dose toxicity study was considered to be higher than 5000 mg/kg in rats. In the 90 day study, the dose levels were wet at 0, 500, 1000, 2000 mg/kg/day, and 10 animals/sex/dose were treated with oral gavage. The parameters that were monitored were clinical signs, body weights, food and water consumptions, ophthalmic examination, urinalysis, hematology, serum biochemistry, necropsy findings, organ weights, and histopathological examination. No adverse effects were observed after the 90 day administration of the silkworm extract powder. The No-Observed-Adverse-Effect-Level (NOAEL) of silkworm extract powder in the 90 day study was 2000 mg/kg/day in both sexes, and no target organ was identified. PMID:24578797
Heo, Hyun-Suk; Choi, Jae-Hun; Oh, Jung-Ja; Lee, Woo-Joo; Kim, Seong-Sook; Lee, Do-Hoon; Lee, Hyun-Kul; Song, Si-Whan; Kim, Kap-Ho; Choi, Yang-Kyu; Ryu, Kang-Sun; Kang, Boo-Hyon
Expression system for production of bioactive compounds, recombinant human adiponectin, in the silk glands of transgenic silkworms.
Adiponectin is an adipocyte hormone involved in glucose and lipid metabolism. The aim of this study was to develop a human adiponectin expression system in transgenic silkworm using a human adiponectin expression vector. The silk gland of the silkworm is a highly specialized organ that has the wonderful ability to synthesize and secrete silk protein. To express human adiponectin in the silk gland of transgenic silkworm, targeting vectors pB-A3-adiponectin-IRES-RFP and pB-Ser1-adiponectin-IRES-RFP were constructed and then introduced into the silkworm pupa. The transgenic silkworms were verified by PCR and then generated. The level of adiponectin in the transgenic silkworm was 6-10 ng/50 mg of freeze-dried powder, and western blotting using an antibody against human adiponectin demonstrated a specific band with a molecular weight of 30 kDa in the silkworm. These results showed that human adiponectin introduced into the silkworm genome was expressed successfully on a large-scale. PMID:24272890
Shin, Seulmee; Kim, Bong-Yoon; Jeon, Hyung-Yook; Lee, Aeri; Lee, Sungwon; Sung, Seung-Hyun; Park, Chan-Su; Lee, Chong-Kil; Kong, Hyunseok; Song, Youngcheon; Kim, Kyungjae
Molecular Markers (RAPD) Associated with Growth, Yield, and Origin of the Silkworm, Bombyx mori L. in India
To identify the molecular markers associated with growth and yield parameters in silkworm, Bombyx mori, RAPD profiles generated with seven UBC primers for fourteen silkworm stocks, originated from China, Japan, India, and Russia, were statistically analyzed. Stepwise multiple regression analysis establishes significant association of 45 markers with larval span, growth indices and four cocoon yield parameters relevant for silk production
S. N. Chatterjee; A. R. Pradeep
The progress and future of enhancing antiviral capacity by transgenic technology in the silkworm Bombyx mori.
Bombyx mori is a common lepidopteran model and an important economic insect for silk production. B. mori nucleopolyhedrovirus (BmNPV) is a typical pathogenic baculovirus that causes serious economic losses in sericulture. B. mori and BmNPV are a model of insect host and pathogen interaction including invasion of the host by the pathogen, host response, and enhancement of host resistance. The antiviral capacity of silkworms can be improved by transgenic technology such as overexpression of an endogenous or exogenous antiviral gene, RNA interference of the BmNPV gene, or regulation of the immune pathway to inhibit BmNPV at different stages of infection. Antiviral capacity could be further increased by combining different methods. We discuss the future of an antiviral strategy in silkworm, including possible improvement of anti-BmNPV, the feasibility of constructing transgenic silkworms with resistance to multiple viruses, and the safety of transgenic silkworms. The silkworm model could provide a reference for disease control in other organisms. PMID:24561307
Jiang, Liang; Xia, Qingyou
Comparative proteomic analysis reveals that caspase-1 and serine protease may be involved in silkworm resistance to Bombyx mori nuclear polyhedrosis virus.
The silkworm Bombyx mori is of great economic value. The B. mori nuclear polyhedrosis virus (BmNPV) is one of the most common and severe pathogens for silkworm. Although certain immune mechanisms exist in silkworms, most silkworms are still susceptible to BmNPV infection. Interestingly, BmNPV infection resistance in some silkworm strains is varied and naturally existing. We have previously established a silkworm strain NB by genetic cross, which is highly resistant to BmNPV invasion. To investigate the molecular mechanism of silkworm resistance to BmNPV infection, we employed proteomic approach and genetic cross to globally identify proteins differentially expressed in parental silkworms NB and 306, a BmNPV-susceptible strain, and their F(1) hybrids. In all, 53 different proteins were found in direct cross group (NB?, 306?, F(1) hybrid) and 21 in reciprocal cross group (306?, NB?, F(1) hybrid). Gene ontology and KEGG pathway analyses showed that most of these different proteins are located in cytoplasm and are involved in many important metabolisms. Caspase-1 and serine protease expressed only in BmNPV-resistant silkworms, but not in BmNPV-susceptible silkworms, which was further confirmed by Western blot. Taken together, our data suggests that both caspase-1 and serine protease play a critical role in silkworm resistance against BmNPV infection. PMID:22546490
Qin, Lvgao; Xia, Hengchuan; Shi, Haifeng; Zhou, Yajing; Chen, Liang; Yao, Qin; Liu, Xiaoyong; Feng, Fan; Yuan, Yi; Chen, Keping
N-acetyltransferase (nat) is a critical conjunct of photoperiodism between the circadian system and endocrine axis in Antheraea pernyi.
Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16:8 (LD) and LD12:12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4 �C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNA(NAT) caused dysfunction of photoperiodism. dsRNA(PER) upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNA(NAT) decreased melatonin while dsRNA(PER) increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNA(NAT), to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism. PMID:24667367
Mohamed, Ahmed A M; Wang, Qiushi; Bembenek, Jadwiga; Ichihara, Naoyuki; Hiragaki, Susumu; Suzuki, Takeshi; Takeda, Makio
Silkworms culture as a source of protein for humans in space
NASA Astrophysics Data System (ADS)
This paper focuses on the problem about a configuration with complete nutrition for humans in a Controlled Ecological Life Support System (CELSS) applied in the spacebases. The possibility of feeding silkworms to provide edible animal protein with high quality for taikonauts during long-term spaceflights and lunar-based missions was investigated from several aspects, including the nutrition structure of silkworms, feeding method, processing methods, feeding equipment, growing conditions and the influences on the space environmental condition changes caused by the silkworms. The originally inedible silk is also regarded as a protein source. A possible process of edible silk protein was brought forward in this paper. After being processed, the silk can be converted to edible protein for humans. The conclusion provides a promising approach to solving the protein supply problem for the taikonauts living in space during an extended exploration period.
Yang, Yunan; Tang, Liman; Tong, Ling; Liu, Hong
Comparative analysis of proteome maps of silkworm hemolymph during different developmental stages
Background The silkworm Bombyx mori is a lepidopteran insect with four developmental stages: egg, larva (caterpillar), pupa, and adult. The hemolymph of the silkworm is in an open system that circulates among all organs, and functions in nutrient and hormone transport, injury, and immunity. To understand the intricate developmental mechanisms of metamorphosis, silkworm hemolymph from different developmental stages, including the 3rd day of fifth instar, the 6th day of fifth instar, the 3rd day of pupation, the 8th day of pupal stage and the first day of the moth stage, was investigated by two-dimensional electrophoresis and mass spectrometry. Results Two-dimensional polyacrylamide gel electrophoresis showed that from the larval to moth stages, silkworm hemolymph proteins changed markedly. Not only did major proteins such as SP1, SP2, and the 30 K lipoprotein change, but other proteins varied greatly at different stages. To understand the functions of these proteins in silkworm development, 56 spots were excised from gels for analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 34 proteins involved in metamorphosis, programmed cell death, food digestion, metabolism, and nutrient storage and transport. Most proteins showed different expression at different stages, suggesting functions in development and metamorphosis. An abundance of proteins related to immunity were found, including hemolin, prophenoloxidase, serine proteinase-like protein, paralytic peptide-binding protein, and protease inhibitor. Conclusions Proteomics research not only provides the opportunity for direct investigation of protein expression patterns, but also identifies many attractive candidates for further study. Two-dimensional maps of hemolymph proteins expressed during the growth and metamorphosis of the silkworm offer important insights into hemolymph function and insect metamorphosis.
Insect cytokine paralytic peptide activates innate immunity via nitric oxide production in the silkworm Bombyx mori.
Insect cytokine paralytic peptide (PP) upregulates the expression of immune-related genes and contributes to host defense in the silkworm Bombyx mori. The present findings demonstrated that PP promotes nitric oxide (NO) production and induces the expression of NO synthase. A pharmacologic NO synthase inhibitor suppressed the PP-dependent (i) induction of immune-related genes, (ii) activation of p38 mitogen-activated protein kinase, and (iii) killing delay of silkworm larvae by Staphylococcus aureus. The upstream mechanism of NO synthesis in insect immunity has been unknown, and the present results suggest for the first time that an insect cytokine induces NO and contributes to self-defense. PMID:23178406
Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Oonishi, Tadahiro; Kamimura, Manabu; Imamura, Katsutoshi; Sekimizu, Kazuhisa
Heterologous Expression Characteristics of Trichoderma viride Endoglucanase V in the Silkworm, Bombyx mori L
Efficient degradation of cellulose needs a synergistic reaction of the cellulolytic enzymes, which include exoglucanases,\\u000a endoglucanases, and ?-1,4-glucosidase. In this study, we used an improved Bac-to-Bac\\/BmNPV baculovirus expression system,\\u000a which lacks the virus-encoded chitinase cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV), to express the endoglucanase V (EG V) gene from Trichoderma viride in silkworm BmN cells and silkworm larvae,
Xing-hua Li; Mei-xian Wang; Peng Zhang; Jia-biao Hu; Chun-guang Sun; Xin-ju Liu; Fang Zhou; Yan-shan Niu; Firdose Ahmad Malik; Roy Bhaskar; Hua-jun Yang; Yun-gen Miao
[Cloning of silkworm 3-hydroxyisobutyrate dehydrogenase gene and its expression patterns analysis in simulated weightless environment].
The full length cDNA of silkworm hibadh gene was cloned by RT-PCR and RACE (Rapid amplification of cDNA ends) technique. The hibadh gene and its deduced amino acid sequences were analyzed. The tissue distribution of hibadh gene in 5th instar silkworm larvae was tested by RT-PCR. The expression patterns of hibadh gene in simulated weightless environment were analyzed by real time RT-PCR. The results showed that the full length hibadh cDNA sequence was 1074 bp in lenth, including an open read frame of 969 bp encoding the entire coding region of Hibadh (GenBank accession No. EU719652). The deduced amino acid sequence similarities of hibadh between silkworm and Burkholderia ambifaria, Drosophila melanogaster, Apis mellifera, Xenopus tropicalis, Mus musculus, Homo sapiens were 46%, 43%, 48%, 44%, 45%, 45%, respectively. Signal peptide analysis showed that Hibadh was a secretory protein. There wasn't glycosyl-phosphatidyl inositol anchor site in Hibadh amino acid sequence. Molecular weight and isoelectric point of Hibadh were 34.1 kD and 9.14 respectively. The RT-PCR tests indicated that the hibadh gene expressed in head, silk gland, midgut, cuticle, blood, fat body, tuba malpighii of the 5th instar silkworm larvae. There were different expression patterns of hibadh gene during different silkworm embryo period in simulated weightless environment. Simulated weightlessness resulted in the expression of silkworm hibadh gene up regulated 2.3-fold (P < 0.05), up regulated 4.6-fold (P<0.01), down regulated 7.6-fold (P < 0.01), down regulated 2.6-fold (P < 0.05) during apophysis formation period, inverse period, trachea formation period, and whole embryo period, respectively. There was no significant change of hibadh gene expression during other period of silkworm embryo between simulated weightless and control groups. There were different response patterns to simulated weightless environment between hibadh gene and whole body of silkworm. Gene showed much higher sensitivity compared to whole body in response to environment. This study is useful for the further research on the gravity biological mechanism of hibadh gene. PMID:19306573
Tian, Zongcheng; Zhou, Bo; Qian, Airong; Xu, Huiyun; Di, Shengmeng; Zhao, Yunpo; Zhang, Yuping; Liu, Jia; Huang, Yongping; Shang, Peng
Characterization of Argonaute family members in the silkworm, Bombyx mori.
The Argonaute protein family is a highly conserved group of proteins, which have been implicated in RNA silencing in both plants and animals. Here, four members of the Argonaute family were systemically identified based on the genome sequence of Bombyx mori. Based on their sequence similarity, BmAgo1 and BmAgo2 belong to the Ago subfamily, while BmAgo3 and BmPiwi are in the Piwi subfamily. Phylogenetic analysis reveals that silkworm Argonaute family members are conserved in insects. Conserved amino acid residues involved in recognition of the 5' end of the small RNA guide strand and of the conserved (aspartate, aspartate and histidine [DDH]) motif present in their PIWI domains suggest that these four Argonaute family members may have conserved slicer activities. The results of microarray expression analysis show that there is a low expression level for B. mori Argonaute family members in different tissues and different developmental stages, except for BmPiwi. All four B. mori Argonaute family members are upregulated upon infection with B. mori nucleopolyhedrovirus. The complete coding sequence of BmPiwi, the homolog of Drosophila piwi, was cloned and its expression occurred mainly in the area where spermatogonia and spermatocytes appear. Our results provide an overview of the B. mori Argonaute family members and suggest that they may have multiple roles. In addition, this is also the first report, to our knowledge, of the response of RNA silencing machinery to DNA virus infection in insects. PMID:23955828
Wang, Gen-Hong; Jiang, Liang; Zhu, Li; Cheng, Ting-Cai; Niu, Wei-Huan; Yan, Ya-Fei; Xia, Qing-You
Thermally induced increase in energy transport capacity of silkworm silks.
This work reports on the first study of thermally induced effect on energy transport in single filaments of silkworm (Bombyx mori) fibroin degummed mild (type 1), moderate (type 2), to strong (type 3). After heat treatment from 140 to 220�C, the thermal diffusivity of silk fibroin type 1, 2, and 3 increases up to 37.9, 20.9, and 21.5%, respectively. Our detailed scanning electron microscopy study confirms that the sample diameter change is almost negligible before and after heat treatment. Raman analysis is performed on the original and heat-treated (at 147�C) samples. After heat treatment at 147�C, the Raman peaks at 1081, 1230, and 1665�cm(-1) become stronger and narrower, indicating structural transformation from amorphous to crystalline. A structure model composed of amorphous, crystalline, and laterally ordered regions is proposed to explain the structural change by heat treatment. Owing to the close packing of more adjacent laterally ordered regions, the number and size of the crystalline regions of Bombyx mori silk fibroin increase by heat treatment. This structure change gives the observed significant thermal diffusivity increase by heat treatment. � 2014 Wiley Periodicals, Inc. Biopolymers 101: 1029-1037, 2014. PMID:24723331
Liu, Guoqing; Xu, Shen; Cao, Ting-Ting; Lin, Huan; Tang, Xiaoduan; Zhang, Yu-Qing; Wang, Xinwei
Vertical transmission of nucleopolyhedrovirus in the silkworm, Bombyx mori L.
Nucleopolyhedrovirus (NPV) was tested for vertical transmission in the silkworm, Bombyx mori. Fifth instar larvae were exposed to four different dosages of BmNPV (830, 1300, 1800, and 2000OBs/larva) and a dosage of about 2000OBs/larva was found suitable for obtaining infected adults. Histopathological studies revealed the infection in susceptible tissues and organs initially, and at later stages of infection cycles the spermatocytes and nurse cells in the young oocytes were infected in the larval rudiments of testis and ovary, respectively. The mating of infected females with uninfected males resulted in significant reduction in fecundity (P < 0.01) and hatching of eggs (P < 0.001) due to transovarial transmission of BmNPV. Mating tests of uninfected females and infected males also confirmed venereal transmission as there was a significant reduction in hatching of eggs (P < 0.01). Further, among the F1 hybrid offspring (infected female x uninfected male) that were infected transovarially, larval progeny died at first and second instar stages, whereas those infected venereally developed acute lethal infection late and died by the end of third and fourth instar stage. PCR amplification and sequencing of 473bp of immediate early-1 (ie-1) gene of BmNPV isolated from the viral-infected parent and the F1 offspring confirmed that the viral infection is vertically transmitted to the progeny. PMID:15491594
Khurad, A M; Mahulikar, A; Rathod, M K; Rai, M M; Kanginakudru, S; Nagaraju, J
Evaluation of therapeutic effects and pharmacokinetics of antibacterial chromogenic agents in a silkworm model of Staphylococcus aureus infection.
The therapeutic effect of dye compounds with antibacterial activity was evaluated in a silkworm model of Staphylococcus aureus infection. Among 13 chromogenic agents that show antibacterial activity against S. aureus (MIC = 0.02 to 19 ?g/mL), rifampicin had a therapeutic effect. The ED(50) value in the silkworm model was consistent with that in a murine model. Other 12 dyes did not increase survival of the infected silkworms. We examined the reason for the lack of therapeutic efficacy. Amidol, pyronin G, and safranin were toxic to silkworms, which explained the lack of therapeutic effects. Fuchsin basic and methyl green disappeared quickly from the hemolymph after injection, suggesting that they are not stable in the hemolymph. Although coomassie brilliant blue R250/G250, cresyl blue, and nigrosin showed no toxic effects or instability in the hemolymph, they also did not have a therapeutic effect. The in vitro antibacterial actions of these dyes were inhibited by silkworm plasma or bovine serum albumin and filtration experiments demonstrated that cresyl blue bound to plasma proteins in the silkworm, suggesting that plasma protein binding inhibited the therapeutic efficacy of these four dyes. These findings indicate that drug screening using the silkworm infection model is useful for evaluating toxicity and pharmacokinetics of potential antibiotics. PMID:22491238
Fujiyuki, T; Imamura, K; Hamamoto, H; Sekimizu, K
Oak Savanna Ecology
NSDL National Science Digital Library
Students will study the oak savannah ecosystems in Oregon to determine the environmental conditions that support this specific ecosystem. This lesson will be an inquiry based lesson that will create the foundation of a larger class project in which students explore the ecological changes to an area over time, and, using this data, develop an ecosystem determination and restoration plan for a site east of Cottage Grove. This resource includes both a teaching guide and student worksheets.
Aumack, Stefan
Co-Sensitization to Silkworm Moth (Bombyx mori) and 9 Inhalant Allergens among Allergic Patients in Guangzhou, Southern China
Objectives This study aimed to investigate the profile of sensitization to silkworm moth (Bombyx mori) and other 9 common inhalant allergens among patients with allergic diseases in southern China. Methods A total of 175 patients were tested for serum sIgE against silkworm moth in addition to combinations of other allergens: Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Blattella germanica, Periplaneta americana, cat dander, dog dander, Aspergillus fumigatus and Artemisia vulgaris by using the ImmunoCAP system. Correlation between sensitization to silkworm moth and to the other allergens was analyzed. Results Of the 175 serum samples tested, 86 (49.14%) were positive for silkworm moth sIgE. With high concordance rates, these silkworm moth sensitized patients were concomitantly sensitized to Dermatophagoides pteronyssinus (94.34%), Dermatophagoides farinae (86.57%), Blomia tropicalis (93.33%), Blattella germanica (96.08%), and Periplaneta americana (79.41%). Moreover, there was a correlation in serum sIgE level between silkworm moth and Dermatophagoides pteronyssinus (r?=?0.518), Dermatophagoides farinae (r?=?0.702), Blomia tropicalis (r?=?0.701), Blattella germanica (r?=?0.878), and Periplaneta americana (r?=?0.531) among patients co-sensitized to silkworm moth and each of these five allergens. Conclusion In southern Chinese patients with allergic diseases, we showed a high prevalence of sensitization to silkworm moth, and a co-sensitization between silkworm moth and other five common inhalant allergens. Further serum inhibition studies are warranted to verify whether cross-reactivity exists among these allergens.
Wei, Nili; Huang, Huimin; Zeng, Guangqiao
The effect of W chromosome origin on sex-chromosome pairing in ZZWW tetraploid females of the domesticated silkworm, Bombyx mori, and the congenic wild silkworm, Bombyx mandarina.
To analyze the degree of pairing of the Z and W chromosomes in ZZWW tetraploid female silkworms that have the W chromosomes of the domesticated silkworm, Bombyx mori, and those of the wild silkworm, Bobyx mandarina, we induced two types of ZZWW tetraploid female silkworms (Cr4n, Wr4n) through cold treatment of the eggs. The Wr4n female is congenic to the Cr4n female for W chromosomes; namely, the W chromosomes of the Wr4n female are derived from those of B. mandarina. Each of the sex ratios (female/male) in filial triploids from the Cr4n females was shown to be in the range of 3.9-5.3 (4.6 as an average of six cases). On the other hand, each of the sex ratios (female/male) in filial triploids from the Wr4n females was shown to be in the range of 6.2-9.0 (6.9 as an average of nine cases). The results of a t-test indicated that the difference in sex ratios in the two groups is highly significant (at the 0.1% level). These results suggest that, in the meiosis of the ZZWW tetraploid female, the frequency of pairing of the W chromosome of B. mandarina and the Z chromosome of B. mori is lower than that of the pairing of the W and Z chromosomes of B. mori. Furthermore, the t-test results are evidence that the W chromosomes have undergone significant evolutional change. PMID:11990764
Tanaka, N; Yokoyama, T; Abe, H; Ninagi, O; Oshiki, T
A BAC-based integrated linkage map of the silkworm Bombyx mori
BACKGROUND: In 2004, draft sequences of the model lepidopteran Bombyx mori were reported using whole-genome shotgun sequencing. Because of relatively shallow genome coverage, the silkworm genome remains fragmented, hampering annotation and comparative genome studies. For a more complete genome analysis, we developed extended scaffolds combining physical maps with improved genetic maps. RESULTS: We mapped 1,755 single nucleotide polymorphism (SNP) markers
Kimiko Yamamoto; Junko Nohata; Keiko Kadono-Okuda; Junko Narukawa; Motoe Sasanuma; Shun-ichi Sasanuma; Hiroshi Minami; Michihiko Shimomura; Yoshitaka Suetsugu; Yutaka Banno; Kazutoyo Osoegawa; Pieter J de Jong; Marian R Goldsmith; Kazuei Mita
Phenol oxidase is a necessary enzyme for the silkworm molting which is regulated by molting hormone.
Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. The molting process includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis excoriation etc. Polyphenol oxidases (PPOs), dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the phenol oxidase was considered as an enzyme for epidermal layer's tanning and melanization. This work suggested that polyphenol oxidases are one set of the key enzymes in molting, which closely related with the role of ecdysone in regulation of molting processes. The data showed that the expression peak of phenol oxidase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the phenol oxidase expression was significantly elevated compared to the control. PPO1 RNAi induced phenol oxidase expression obviously declined in the silkworm larvae, and caused the pupae incomplete pupation. Overall, the results described that the phenol oxidase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting. PMID:23275200
Wang, Mei-xian; Lu, Yan; Cai, Zi-zheng; Liang, Shuang; Niu, Yan-shan; Miao, Yun-gen
Expression and functions of dopa decarboxylase in the silkworm, Bombyx mori was regulated by molting hormone.
Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. Molting includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis shed and other series of continuous processes. Polyphenol oxidases, dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the dopa decarboxylase (BmDdc) was considered as an enzyme for epidermal layer's tanning and melanization. This work suggested that dopa decarboxylase is one set of the key enzymes in molting, which closely related with the regulation of ecdysone at the time of biological molting processes. The data showed that the expression peak of dopa decarboxylase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was also observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the dopa decarboxylase expression was significantly elevated compared to the control. BmDdc RNAi induced dopa decarboxylase expression obviously declined in the silkworm larvae, and caused the pupae appeared no pupation or incomplete pupation. BmDdc was mainly expressed and stored in the peripheral plasma area near the nucleus in BmN cells. In larval, BmDdc was mainly located in the brain and epidermis, which is consisted with its function in sclerotization and melanization. Overall, the results described that the dopa decarboxylase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting. PMID:23640098
Wang, Mei-xian; Cai, Zi-zheng; Lu, Yan; Xin, Hu-Hu; Chen, Rui-ting; Liang, Shuang; Singh, Chabungbam Orville; Kim, Jong-Nam; Niu, Yan-shan; Miao, Yun-gen
Host plant urease in the hemolymph of the silkworm, Bombyx mori
Urease activity was detected in the hemolymph of the silkworm, Bombyx mori from the beginning of spinning to the pharate adult stage if the larvae were reared on mulberry leaves throughout the 5th-instar (the last larval instar). In contrast, no urease activity was detected in the hemolymph of insects fed artificial diets, resulting in accumulation of urea during the spinning
Chikara Hirayama; Masahiro Sugimura; Hitoshi Saito; Masatoshi Nakamura
Induction of dauer pupae by fenoxycarb in the silkworm, Bombyx mori
Topical application of fenoxycarb (1 ?g per animal) at 129 or 132 h of the fifth instar larvae of the silkworm, Bombyx mori, did not induce morphological abnormalities in the pupal stage, but these animals became dauer (permanent) pupae. This condition of B. mori and the endocrine events leading to permanent pupae are discussed in this work. Application of fenoxycarb
S. G. Dedos; F. Szurdoki; A. Sz�k�cs; A. Mizoguchi; H. Fugo
Purification and Partial Characterization of an Induced Antibacterial Protein in the Silkworm, Bombyx mori
Injection of live Escherichia coli into larvae of the silkworm, Bombyx mori, induces antibacterial activity in the hemolymph. The major induced antibacterial activity was purified in two steps by CM-Sephadex C-50 and Sephadex G-100 column chromatography. After trypsin treatment, the purified antibacterial protein lost its activity and the antibacterial activity was found to be partially heat labile. The purified protein
E. G Abraham; J Nagaraju; D Salunke; H. M Gupta; R. K Datta
Amino-Terminal Amino Acid Sequence of the Silkworm Prothoracicotropic Hormone: Homology with Insulin
Three molecular forms of prothoracicotropic hormone were isolated from the head of the adult silkworm, Bombyx mori, and the amino acid sequence of 19 amino acid residues in the amino terminus of these prothoracicotropic hormones was determined. These residues exhibit significant homology with insulin and insulin-like growth factors.
Hiromichi Nagasawa; Hiroshi Kataoka; Akira Isogai; Saburo Tamura; Akinori Suzuki; Hironori Ishizaki; Akira Mizoguchi; Yuko Fujiwara; Atsushi Suzuki
Diapause hormone of the silkworm, Bombyx mori: Structure, gene expression and function
Diapause hormone (DH) is a neuropeptide hormone which is secreted from the suboesophageal ganglion (SG) and is responsible for induction of embryonic diapause of the silkworm, Bombyx mori. DH is isolated from SGs and determined to be a 24 amino acid peptide amide. The cDNA encodes the polyprotein precursor from which DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other
Okitsugu Yamashita
Eicosanoids mediate induction of immune genes in the fat body of the silkworm, Bombyx mori
The expression of cecropin and lysozyme genes is induced in response to bacterial peptidoglycan in the fat body of the silkworm, Bombyx mori. Specific inhibitors of either phospholipase A2, cyclooxygenase or lipoxygenase significantly inhibit the induction of the immune genes both in vivo and in cultured fat body as detected by means of Northern hybridization. Arachidonic acid injected into the
Isao Morishima; Yoshiaki Yamano; Kenji Inoue; Noriyuki Matsuo
Amino Acid Sequence of a Prothoracicotropic Hormone of the Silkworm Bombyx mori
We have determined the complete amino acid sequence of 4K-PTTH-II, one of three forms of the Mr 4400 prothoracicotropic hormone of the silkworm Bombyx mori, active to brainless pupae of Samia cynthia ricini. Like vertebrate insulin, it consists of two nonidentical peptide chains (A and B chains). The A chain consists of 20 amino acid residues. The B chain is
Hiromichi Nagasawa; Hiroshi Kataoka; Akira Isogai; Saburo Tamura; Akinori Suzuki; Akira Mizoguchi; Yuko Fujiwara; Atsushi Suzuki; Susumu Y. Takahashi; Hironori Ishizaki
The DNA content of sperm and hemocyte nuclei of the silkworm, Bombyx mori L
To estimate the size of the haploid genome of the silkworm, Bombyx mori (Lepidoptera), amounts of Feulgen-DNA staining in individual nuclei of primary spermatocytes, spermatids, maturing sperm, and larval or pupal hemocytes were determined with an integrating microdensitometer and compared with the Feulgen-DNA levels found for chicken erythrocyte nuclei, or the sperm and erythrocyte nuclei of Xenopus laevis that were
Ellen M. Rasch
Glucose Stimulates the Release of Bombyxin, an Insulin-Related Peptide of the Silkworm Bombyx mori
The effects of starvation and feeding on the release of bombyxin, a peptide of insulin superfamily in insects, from the larval brain of the silkworm Bombyx mori were investigated. Following starvation, the bombyxin titer in the hemolymph of larvae decreased, whereas its content in the brain increased. On the other hand, refeeding of the starved larvae resulted in an increase
Makoto Masumura; Shin'Ichiro Satake; Hironao Saegusa; Akira Mizoguchi
Synthesis of Blood Protein by the Fat Body in the Silkworm, Bombyx mori L
THE concentration of protein in the blood of insects changes remarkably in the course of metamorphosis1. I have shown that the concentration of blood protein increases after the middle period of the last larval instar2 in the silkworm. It is not yet clear, however, what organ is concerned in the synthesis of blood protein in larval stage, although there are
Hajime Shigematsu
Conversion of Glycogen to Sorbitol and Glycerol in the Diapause Egg of the Bombyx Silkworm
IN the course of biochemical studies1 on the embryonic diapause of the Bombyx silkworm, it has been shown that the glycogen content of the egg decreases markedly at the onset of diapause and reaches the lowest level at about thirty days after oviposition. When diapause is broken by cold treatment, glycogen increases progressively even at low temperature and regains the
Haruo Chino
Silkworm powder containing manganese superoxide dismutase regulated the immunity and inhibited the growth of Hepatoma 22 cell in mice
The effects of SOD contained silkworm powder on immune regulation and inhibition against Hepatoma 22 tumor cells in�vivo were\\u000a investigated. The activity of natural killer cell (NK) and the ConA-stimulated spleen proliferation were measured. The results\\u000a found that the SOD-contained silkworm powder caused an enhancement on NK cell activity, which implied this material modulated\\u000a the immune system in mice in�vivo.
Wan-Fu Yue; Wen Deng; Xing-Hua Li; Bhaskar Roy; Guang-Li Li; Jian-Mei Liu; Xiao-Feng Wu; Hong-Xiang Sun; Min-Li Yao; Wan Chi Cheong David; Yun-Gen Miao
Isolation of a novel peptide from silkworm pupae protein components and interaction characteristics to angiotensin I-converting enzyme
The hydrolysate of silkworm protein produced by acid protease (Asperqiius usamii NO. 537) contains angiotensin I-converting enzyme inhibitory activity. Four kinds of protein components were identified in\\u000a silkworm pupae protein, albumin, globulin, prolamin and glutelin, and the albumin was in the highest quantity. The four component\\u000a proteins were then further hydrolyzed by acid protease under the same conditions, and the
Wei WangNan; Nan Wang; Yu Zhou; Yu Zhang; Lihong Xu; Junfeng Xu; Fengqin Feng; Guoqing He
[Physiologically active substances in the oral excreta produced by honey bee--effects of royal jelly on silkworm].
The effect of royal jelly on the growth, development, weight of a cocoon and the number of eggs of the silkworm, Bombyx mori, was tested by the use of artificial diets. The growth and developmental speed of the silkworm were quickened by addition of royal jelly. The number of eggs increased markedly by the feeding of raw royal jelly but not of boiled royal jelly. PMID:2640797
Saikatsu, S; Ikeno, K; Hanada, Y; Ikeno, T
Purification and Molecular Cloning of an Inducible Gram-Negative Bacteria-Binding Protein from the Silkworm, Bombyx mori
A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori. The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced. Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region
Won-Jae Lee; Jiing-Dwan Lee; Vladimir V. Kravchenko; Richard J. Ulevitch; Paul T. Brey
Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system
Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori
Tomoko Motohashi; Tsukasa Shimojima; Tatsuo Fukagawa; Katsumi Maenaka; Enoch Y. Park
Identification and Expression Profiling of the BTB Domain-Containing Protein Gene Family in the Silkworm, Bombyx mori
The BTB domain is a conserved protein-protein interaction motif. In this study, we identified 56 BTB domain-containing protein genes in the silkworm, in addition to 46 in the honey bee, 55 in the red flour beetle, and 53 in the monarch butterfly. Silkworm BTB protein genes were classified into nine subfamilies according to their domain architecture, and most of them could be mapped on the different chromosomes. Phylogenetic analysis suggests that silkworm BTB protein genes may have undergone a duplication event in three subfamilies: BTB-BACK-Kelch, BTB-BACK-PHR, and BTB-FLYWCH. Comparative analysis demonstrated that the orthologs of each of 13 BTB protein genes present a rigorous orthologous relationship in the silkworm and other surveyed insects, indicating conserved functions of these genes during insect evolution. Furthermore, several silkworm BTB protein genes exhibited sex-specific expression in larval tissues or at different stages during metamorphosis. These findings not only contribute to a better understanding of the evolution of insect BTB protein gene families but also provide a basis for further investigation of the functions of BTB protein genes in the silkworm.
Cheng, Daojun; Qian, Wenliang; Meng, Meng; Wang, Yonghu; Peng, Jian; Xia, Qingyou
Utilization of silkworm cocoon waste as a sorbent for the removal of oil from water.
The aim of this study is to investigate the utilization of silkworm cocoon waste, such as pierced or stained cocoons, as a sorbent material for the removal of motor and vegetable oils from water. The oil-sorption capacity, rate and reusability of the material were evaluated. The results show the high sorption capacity of the silkworm cocoon waste sorbent (42-52 g(oil)/g(sorbent) for motor oil and 37-60 g(oil)/g(sorbent) for vegetable oil). The oil sorbed onto the material could be recovered by squeezing the sorbent, and the squeezed material showed an oil-sorption capacity over 15 g(oil)/g(sorbent). We concluded that the material shows a high performance as a low cost and environmental friendly sorbent for the removal of oil from water. PMID:19008047
Moriwaki, Hiroshi; Kitajima, Shiori; Kurashima, Masahiro; Hagiwara, Ayaka; Haraguchi, Kazuma; Shirai, Koji; Kanekatsu, Rensuke; Kiguchi, Kenji
Microarray Analysis of the Juvenile Hormone Response in Larval Integument of the Silkworm, Bombyx mori
Juvenile hormone (JH) coordinates with 20-hydroxyecdysone (20E) to regulate larval growth and molting in insects. However, little is known about how this cooperative control is achieved during larval stages. Here, we induced silkworm superlarvae by applying the JH analogue (JHA) methoprene and used a microarray approach to survey the mRNA expression changes in response to JHA in the silkworm integument. We found that JHA application significantly increased the expression levels of most genes involved in basic metabolic processes and protein processing and decreased the expression of genes associated with oxidative phosphorylation in the integument. Several key genes involved in the pathways of insulin/insulin-like growth factor signaling (IIS) and 20E signaling were also upregulated after JHA application. Taken together, we suggest that JH may mediate the nutrient-dependent IIS pathway by regulating various metabolic pathways and further modulate 20E signaling.
Cheng, Daojun; Peng, Jian; Meng, Meng; Wei, Ling; Kang, Lixia; Qian, Wenliang; Xia, Qingyou
Directly obtaining high strength silk fiber from silkworm by feeding carbon nanotubes.
By feeding silkworm with the carbon nanotube, CNT, we directly obtained high strength silk fiber, SF, from silkworm. The CNT-based SF, SF/CNT, has a stress at 1.69GPa and a strain at about 24% both higher than those of the SF and are capable to compare with the super SF and even the spider fiber. Morphology comparison showed that the presence of CNT in SF caused the cross-section changed from triangle to ellipse. X-ray diffraction and infrared analysis indicated that the embedded CNT in SF caused an increase in silk-I structure. Specifically the amide-II structure reduced by about 5% and the amide-III structure increased by about 10%. Thermogravimetric analyses indicated that the presence of CNT in SF enhanced the thermal stability. Additionally, the presence of CNT in SF also enhanced the electrical property. PMID:24268277
Wang, Jun-Ting; Li, Lu-Lu; Zhang, Meng-Yuan; Liu, Si-Lu; Jiang, Lin-Hai; Shen, Qing
Toyama Kametaro and Vernon Kellogg: silkworm inheritance experiments in Japan, Siam, and the United States, 1900-1912.
Japanese agricultural scientist Toyama Kametaro's report about the Mendelian inheritance of silkworm cocoon color in Studies on the Hybridology of Insects (1906) spurred changes in Japanese silk production and thrust Toyama and his work into a scholarly exchange with American entomologist Vernon Kellogg. Toyama's work, based on research conducted in Japan and Siam, came under international scrutiny at a time when analyses of inheritance flourished after the "rediscovery" of Mendel's laws of heredity in 1900. The hybrid silkworm studies in Asia attracted the attention of Kellogg, who was concerned with how experimental biology would be used to study the causes of natural selection. He challenged Toyama's conclusions that Mendelism alone could explain the inheritance patterns of silkworm characters such as cocoon color because they had been subject to hundreds of years of artificial selection, or breeding. This examination of the intersection of Japanese sericulture and American entomology probes how practical differences in scientific interests, societal responsibilities, and silkworm materiality were negotiated throughout the processes of legitimating Mendelian genetics on opposite sides of the Pacific. The ways in which Toyama and Kellogg assigned importance to certain silkworm properties show how conflicting intellectual orientations arose in studies of the same organism. Contestation about Mendelism took place not just on a theoretical level, but the debate was fashioned through each scientist's rationale about the categorization of silkworm breeds and races and what counted as "natural". This further mediated the acceptability of the silkworm not as an experimental organism, but as an appropriately "natural" insect with which to demonstrate laws of inheritance. All these shed light on the challenges that came along with the use of agricultural animals to convincingly articulate new biological principles. PMID:20665229
Onaga, Lisa
Molecular cloning, sequence analysis and expression of genome segment 7 (S7) of Antheraea mylitta cypovirus (AmCPV) that encodes a viral structural protein.
The Genome segment 7 (S7) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus (AmCPV) was converted to cDNA, cloned and sequenced. The nucleotide sequence showed that segment 7 consisted of 1789 nucleotides with an ORF of 530 amino acids and could encode a protein of approximately 61 kDa, termed P61. The 5' terminal sequence, AGTAAT and the 3' terminal sequence, AGAGC of the plus strand was found to be the same as genome segment 10 of AmCPV encoding polyhedrin. No sequence similarity was found by searching nucleic acid and protein sequence databases using BLAST. The secondary structure prediction showed the presence of 17 alpha-helices, 18 extended beta-sheets along the entire length of P61. The ORF of segment 7 was expressed in E. coli as His-tagged fusion protein, purified through Ni-NTA chromatography, and polyclonal antibody was raised in rabbit indicating that P61 is immunogenic. Immunoblot analysis using this antibody on viral infected cells as well as purified polyhedra showed that P61 is a viral structural protein. Motif scan search showed some similarity of P61 with Inosine monophosphate dehydrogenase (IMPDH) cystathionine-beta-synthase (CBS) domain at the C-terminus and it was hypothesized that by binding to single stranded viral RNA through its CBS domain P61 may help in virus replication or transcription. PMID:17253125
Chavali, Venkata Ramana Murthy; Ghosh, Ananta K
Purification and characterization of a 1,3-?-D-glucan recognition protein from Antheraea pernyi larve that is regulated after a specific immune challenge.
Pattern recognition receptors are known to participate in the activation of Prophenoloxidase system. In this study, a 1,3-?-D-glucan recognition protein was detected for the first time in Antheraea pernyi larvae (Ap-?GRP). Ap-?GRP was purified to 99.9% homogeneity from the hemolymph using traditional chromatographic methods. Ap-?GRP specifically bind 1,3-?-D-glucan and yeast, but not E. coli or M. luteus. The 1,3-?-D-glucan dependent phenoloxidase (PO) activity of the hemolymph inhibited by anti-Ap-?GRP antibody could be recovered by addition of purified Ap-?GRP. These results demonstrate that Ap-?GRP acts as a biosensor of 1,3-?-Dglucan to trigger the Prophenoloxidase system. A trace mount of 1,3-?-D-glucan or Ap-?GRP alone was unable to trigger the proPO system, but they both did. Ap-?GRP was specifically degraded following the activation of proPO with 1,3-?-Dglucan. These results indicate the variation in the amount of Ap-?GRP after specific immune challenge in A. pernyi hemolymph is an important regulation mechanism to immune response. PMID:23710637
Youlei, Ma; Jinghai, Zhang; Yuntao, Zhang; Jiaoshu, Lin; Tianyi, Wang; Chunfu, Wu; Rong, Zhang
Xanthurenic acid is an endogenous substrate for the silkworm cytosolic sulfotransferase, bmST1
Sulfotransferase enzymes are known to regulate physiologically active substances such as steroids and catecholamines in mammals. Although invertebrates also express sulfotransferases, their biological function is mostly unclear. In a previous study, we reported that 4-nitrocatechol and the gallete ester are substrates for the silkworm sulfotransferase bmST1. The Km of bmST1 for these substrates is high. However, endogenous substrates of bmST1
Akira Kushida; Ryo Horie; Kenji Hattori; Hiroshi Hamamoto; Kazuhisa Sekimizu; Hiroomi Tamura
Purification and characterization of NADH-dependent glutamate synthase from the silkworm fat body ( Bombyx mori)
NADH-dependent glutamate synthase (NADH-GOGAT; EC 18.104.22.168) 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 procedure including ammonium sulfate fraction, Q-Sepharose HP ion exchange column chromatography, Blue Sepharose FF affinity column chromatography and Superdex 200HR gel filtration. The purified enzyme yielded a single band
Chikara Hirayama; Hitoshi Saito; Kotaro Konno; Hiroshi Shinbo
Molecular cloning and characterization of hatching enzyme-like gene in the silkworm, Bombyx mori
Hatching is the important process for the life of the metazoan, in which hatching enzyme (HE) plays a key role. In this paper,\\u000a we cloned the full-length sequence of hatching enzyme-like cDNA from bluish-silkworm-eggs of Bombyx mori (BmHEL) by the method of in silico cloning, SMART cDNA synthesis and RACE-PCR technique. The BmHEL is 974�bp in length, and contains an
Fu-hao Lu; Shun-ming Tang; Xing-jia Shen; Na Wang; Qiao-ling Zhao; Guo-zheng Zhang; Xi-jie Guo
Expression of open reading frames in silkworm pupal cDNA library
A cDNA library containing 2409 singletons was constructed from whole silkworm pupae (Bombyx mori) In addition, the types of genes overexpressed in pupa were analyzed. These genes contained 79 types of proteins with the\\u000a exception of enzyme, mitochondrial DNA, andribosomal protein. Also analyzed were the expression and nonexpression of open\\u000a reading frame (ORF) sequences in Escherichia coli. cDNA sequences were
Yao-Zhou Zhang; Jian Chen; Zuo-Ming Nie; Zheng-Bing L�; Dan Wang; Cai-Ying Jiang; Ping-An He; Li-Li Liu; Yu-Lan Lou; Li Song; Xiang-Fu Wu
Genetics of insect hemolymph ?- N -acetylglucosaminidase in the silkworm, Bombyx mori
The distribution of insect hemolymph �-N-acetylglucosaminidase was investigated in the silkworm, Bombyx mori. Activity in 115 varieties was 6.92�3.22 units\\/ml, ranging from 1.4 to 17.0 units\\/ml. No enzyme-deficient individuals were observed. By selecting individuals showing either high or low enzyme activities, homozygotes were separated with activities varying 10-fold between isolates. No differences in activity of a-mannosidase and �-galactosidase were observed.
Shigeru Kimura
Structure of insect chitin isolated from beetle larva cuticle and silkworm ( Bombyx mori) pupa exuvia
Chitin samples in a ?-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 NaBH4. 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
M. Zhang; A. Haga; H. Sekiguchi; S. Hirano
Occurrence of Pyridoxal 5?-Phosphate-Dependent Serine Racemase in Silkworm, Bombyx mori
D-Serine is known to occur in the silkwormBombyx morias well as in the mammalian central nervous systems. We found that serine racemase occurs in the insect, catalyzing the conversion of L-serine to its antipode. The enzyme was partially purified from pupae of the insect, and was inactivated by treatment with hydroxylamine and reactivated with pyridoxal 5?-phosphate (PLP). L-Alanine was racemized
Takuma Uo; Tohru Yoshimura; Susumu Shimizu; Nobuyoshi Esaki
Alkaline protease in the midgut of the silkworm Bombyx mori L: changes during metamorphosis
The protease activity in the midgut of bivoltine and muitivoltine races of the silkworm,Bombyx mori, was studied. The enzyme activity increased during fifth instar reached a peak, and decreased significantly through pre-pupal\\u000a stage to the lowest level at the pupal stagTouring pharate adult period, the protease activity increased to reach another\\u000a peak just before emergence of the moth, and decreased
S K Sarangi
Characterization of a silkworm thioredoxin peroxidase that is induced by external temperature stimulus and viral infection
A thioredoxin peroxidase (TPx) that reduces H2O2 was firstly characterized in the lepidopteran insect, silkworm Bombyx mori. The B. mori TPx (BmTPx) cDNA contains an open reading frame of 585bp encoding 195 amino acid residues and possesses two cysteine residues that are characteristic of 2-Cys subgroup of peroxiredoxin family. The deduced amino acid sequence of the BmTPx cDNA showed 78%
Kwang Sik Lee; Seong Ryul Kim; Nam Sook Park; Iksoo Kim; Pil Dong Kang; Bong Hee Sohn; Kwang Ho Choi; Seok Woo Kang; Yeon Ho Je; Sang Mong Lee; Hung Dae Sohn; Byung Rae Jin
Functional analysis of the larval serum protein gene promoter from silkworm Bombyx mori
The regulation region of larval serum protein gene, Bombyx mori. (BmLSP), consisting of the first intron, the first exon,\\u000a the central promoter region and 5�-upstream region, is cloned from genomic DNA from the silkworm variety of Suju � Minghu.\\u000a Using PCR and restriction endonuclease methods, a series of luciferase reporter plasmids, driven by different length of BmLSP\\u000a promoters, are constructed.
Shimming Tang; Yongzhu Yi; Xingjia Shen; Zhifang Zhang; Yiren Li; Jialu He
Shotgun proteomic analysis of the fat body during metamorphosis of domesticated silkworm ( Bombyx mori )
Protein expression profiles in the fat bodies of larval, pupal, and moth stages of silkworm were determined using shotgun\\u000a proteomics and MS sequencing. We identified 138, 217, and 86 proteins from the larval, pupal and moth stages, respectively,\\u000a of which 12 were shared by the 3 stages. There were 92, 150, and 45 specific proteins identified in the larval, pupal
Huijuan Yang; Zhonghua Zhou; Huarong Zhang; Ming Chen; Jianying Li; Yingying Ma; Boxiong Zhong
Modified fiber qualities of the transgenic cotton expressing a silkworm fibroin gene
A silkworm gene for fibroin was introduced into the upland cotton WC line by Agrobacterium-mediated transformation. PCR detection for fibroin, nptII and gus genes, Kanamycin (Km)-resistance analysis and GUS-histochemical assay were conducted on 30 regenerated plants from\\u000a 9 callus lines, and 17 positive plants were obtained by these 5 screening methods. By Km-resistance analysis and PCR for fibroin, 6 homozygous
FeiFei Li; ShenJie Wu; FenNi L�; TianZi Chen; Ming Ju; HaiHai Wang; YanJie Jiang; Jie Zhang; WangZhen Guo; TianZhen Zhang
Genetic characterization of the silkworm Bombyx mori by simple sequence repeat (SSR)-anchored PCR
Thirteen diverse strains of the silkworm Bombyx mori were analysed using the simple sequence repeat anchored polymerase chain reaction (SSR-anchored PCR) or Inter-SSR-PCR (ISSR-PCR). A set of four 5�-anchored and two 3�-anchored repeat primers amplified a total of 239 bands out of which 184 (77%) were polymorphic. The 5�-anchored primers revealed more distinct polymorphic markers than the 3�-anchored primers and
Sexual attraction in the silkworm moth: structure of the pheromone-binding-protein�bombykol complex
Background: Insects use volatile organic molecules to communicate messages with remarkable sensitivity and specificity. In one of the most studied systems, female silkworm moths (Bombyx mori) attract male mates with the pheromone bombykol, a volatile 16-carbon alcohol. In the male moth�s antennae, a pheromone-binding protein conveys bombykol to a membrane-bound receptor on a nerve cell. The structure of the pheromone-binding
Benjamin H Sandler; Larisa Nikonova; Walter S Leal; Jon Clardy
Detection of Peptidoglycan and Endotoxin in Dialysate, Using Silkworm Larvae Plasma and Limulus Amebocyte Lysate Methods
Silkworm larvae plasma (SLP) reagent is activated by peptidoglycan (PG), a fragment of both the gram-positive and gram-negative bacterial cell wall, as well as ?-glucan (BG), a component of fungi. It is possible to measure contamination of gram-positive bacteria quantitatively by combining the conventional limulus amebocyte lysate (LAL) and PG measurement methods. Therefore, a more highly accurate analysis of dialysate
K. Tsuchida; Y. Takemoto; S. Yamagami; H. Edney; M. Niwa; M. Tsuchiya; T. Kishimoto; S. Shaldon
Expression pattern of immunoglobulin superfamily members in the silkworm, Bombyx mori.
Immunoglobulin superfamily (IgSF) proteins are involved in cell adhesion, cell communication and immune functions. In this study, 152 IgSF genes containing at least one immunoglobulin (Ig) domain were predicted in the Bombyx mori silkworm genome. Of these, 145 were distributed on 25 chromosomes with no genes on chromosomes 16, 18 and 26. Multiple sequence alignments and phylogenetic evolution analysis indicated that IgSFs evolved rapidly. Gene ontology (GO) annotation indicated that IgSF members functioned as cellular components and in molecular functions and biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that IgSF proteins were involved in signal transduction, signaling molecules and interaction, and cell communication. Microarray-based expression data showed tissue expression for 136 genes in anterior silkgland, middle silkgland, posterior silkgland, testis, ovary, fat body, midgut, integument, hemocyte, malpighian tubule and head. Expression pattern of IgSF genes in the silkworm ovary and midgut was analyzed by RNA-Seq. Expression of 105 genes was detected in the ovary in strain Dazao. Expression in the midgut was detected for 74 genes in strain Lan5 and 75 genes in strain Ou17. Expression of 34 IgSF genes in the midgut relative to the actin A3 gene was significantly different between strains Lan5 and Ou17. Furthermore, 1 IgSF gene was upregulated and 1 IgSF gene was downregulated in strain Lan5, and 4 IgSF genes were upregulated and 2 IgSF genes were downregulated in strain Ou17 after silkworms were challenged with B. mori cypovirus (BmCPV), indicating potential involvement in the response to BmCPV-infection. These results provide an overview of IgSF family members in silkworms, and lay the foundation for further functional studies. PMID:25020261
He, Lei; Cao, Guangli; Huang, Moli; Xue, Renyu; Hu, Xiaolong; Gong, Chengliang
M�ssbauer studies of subfossil oak
Subfossil oak wood found in a dried-up bog in Bavaria, Germany, was studied by M�ssbauer spectroscopy. The bog oaks contain substantial amounts of iron taken up from the bog waters and presumably forming complexes with the tanning agents in the oak wood. The iron is mainly Fe3 + and much of this exhibits an uncommonly large quadrupole splitting of up to 1.6 mm/s that can tentatively be explained by the formation of oxo-bridged iron dimers. Only rarely, mainly in the dense wood of the roots of bog oaks, was divalent iron found. When the wood was ground to a powder the divalent iron oxidized to Fe3 + within hours. This suggests that iron is taken up from the bog water as Fe2 + and oxidizes only when the wood emerges from the water and comes into contact with air.
van B�rck, Uwe; Wagner, Friedrich E.; Lerf, Anton
Molecular cloning and characterization of lactate dehydrogenase gene 1 in the silkworm, Bombyx mori.
Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate into lactate and constitutes a major checkpoint of anaerobic glycolysis. Recently, LDH draws a great deal of attention for its potential to be used as a novel diagnostic and therapeutic target for various diseases, including cancer and malaria. Insect LDHs have been mainly identified from fruit fly and mosquitoes, but not from silkworm. In this study, a novel LDH homologue, designated as BmLDH1, was firstly identified and characterized from the silkworm, Bombyx mori. The BmLDH1 cDNA contains an open reading frame of 996�bp, and encodes a protein of 331 amino acid residues with calculated molecular mass of 36�kDa. Sequence comparison showed BmLDH1 is a highly conserved protein. RT-PCR revealed BmLDH1 is transcripted in all tissues and in all developmental stages tested, indicating its essential roles for silkworm physiology and development. The BmLDH1 gene was subcloned and expressed in E. coli, and was further characterized by Western blot and Mass Spectrometry. The expressed protein contained the LDH activity, and could be inhibited by reduced glutathione in vitro. Immunofluoresence showed that the BmLDH1 was located in the cytoplasm. The cloned BmLDH1 sequence was deposited in the GenBank (accession number EU334850). PMID:20852941
Xia, Hengchuan; Wu, Chao; Xu, Qinggang; Shi, Jing; Feng, Fan; Chen, Keping; Yao, Qin; Wang, Yong; Wang, Lin
[Trials using drugs in diseases of the silkworm (Bombyx mori L.)].
Studied was the pharmacokinetic of chloramphenicol, tylosine, ampicillin, and penicillin in silkworm moths. The antibiotics were applied only once in the form of water solutions in concentrations of 0.062 to 1 per cent for chloramphenicol, 50 to 1500 IU/cm3 for tylosine, 0.03 to 0.5 per cent for ampicillin, and 300 to 2500 IU/cm3 for benzilpenicillin at the rate of 25 cm3/cm2 of a mulberry tree leaf. It was found that chloramphenicol in conc. 0.25 to 0.5 per cent was absorbed in the alimentary canal of the silkworm and was found in the haemolymph in bacteriostatic levels up to the 96th hour following treatment. Tylosine was also absorbed, and in concentrations of 500 to 1500 IU/cm3 was shown to maintain bacteriostatic levels in the haemolymph of the moths for a period of 24-32 hours. Ampicillin and penicillin were absorbed to a slight extent, and were eliminated from the haemolymph up to the eighth hour. It is suggested to use chloramphenicol and tylosine in the concentrations referred to for the control of the spontaneous nuclear polyhedria and flasheria in silkworm moths. PMID:6740927
Manchev, M; Doneva, M; Donev, B
Silkworm Hemolymph Down-Regulates the Expression of Endoplasmic Reticulum Chaperones under Radiation-Irradiation
We demonstrated that up-regulation of gene expression of endoplasmic reticulum (ER) chaperones (BiP, calnexin, calreticulin, ERp29) and ER membrane kinases (IRE1, PERK, ATF6) was induced by radiation in neuronal PC12 cells. However, addition of silkworm, Bombyx mori, hemolymph to irradiated cells resulted in an obvious decrease in expression of these genes, compared with a single radiation treatment. In contrast, one of the ER chaperones, �ischemia-responsive protein 94 kDa� (irp94), was up-regulated by radiation. However, addition of silkworm hemolymph resulted in no change in the expression of irp94, with an expression pattern that differed from that of ER chaperones. Based on these results, we propose that silkworm hemolymph contains factors that regulate a decrease in the expression of ER chaperones under radiation-irradiation conditions, with the exception of irp94, which is not down-regulated. We suggest that this difference in the molecular character of irp94 may provide a clue to the biological functions associated with ER stress pathways, particularly the effects of radiation.
Lee, Kyeong Ryong; Kim, Seung-Whan; Kim, Young Kook; Kwon, Kisang; Choi, Jong-Soon; Yu, Kweon; Kwon, O-Yu
Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system
Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G{sub i}{alpha}) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [{sup 35}S]GTP{gamma}S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.
Kajikawa, Mizuho; Sasaki, Kaori [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)] [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Wakimoto, Yoshitaro; Toyooka, Masaru [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan)] [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Motohashi, Tomoko; Shimojima, Tsukasa [National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540 (Japan)] [National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540 (Japan); Takeda, Shigeki [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan)] [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Park, Enoch Y. [Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka, Shizuoka 422-8529 (Japan)] [Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka, Shizuoka 422-8529 (Japan); Maenaka, Katsumi, E-mail: firstname.lastname@example.com [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)] [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)
Cloning, expression and enzymatic properties analysis of dihydrofolate reductase gene from the silkworm, Bombyx mori.
Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases, which control the levels of monoamine neurotransmitters. BH4 deficiency has been associated with many neuropsychological disorders. Dihydrofolate reductase (DHFR) can catalyze 7,8-dihydrobiopterin to 5,6,7,8-tetrahydrobiopterin (BH4) in the salvage pathway of BH4 synthesis from sepiapterin (SP), a major pigment component contained in the integument of silkworm Bombyx mori mutant lemon (lem) in high concentration. In this study, we report the cloning of DHFR gene from the silkworm B. mori (BmDhfr) and identification of enzymatic properties of BmDHFR. BmDhfr is located on scaffold Bm_199 with a predicted gene model BGIBMGA013340, which encodes a 185-aa polypeptide with a predicted molecular mass of about 21 kDa. Biochemical analyses showed that the recombinant BmDHFR protein exhibited high enzymatic activity and suitable parameters to substrate. Together with our previous studies on SP reductase of B. mori (BmSPR) and the lem mutant, it may be an effective way to industrially extract SP from the lem silkworms in large scale to produce BH4 in vitro by co-expressing BmSPR and BmDHFR and using the extracted SP as a substrate in the future. PMID:23065260
Wang, Wenjing; Gao, Junshan; Wang, Jing; Liu, Chaoliang; Meng, Yan
Molecular cloning and expression characterization of translationally controlled tumor protein in silkworm pupae.
A Bombyx mori (B. mori) cDNA was isolated from silkworm pupae cDNA library encoding a homologue of translationally controlled tumor protein (BmTCTPk). BmTCTPk was expressed in E. coli; SDS-PAGE and Western blot showed the molecular weight of recombinant and native BmTCTPk is approximately 28 and 25 kDa, respectively; they are larger than the theoretical molecular weight. Immunohistochemical studies showed that BmTCTPk is uniformly distributed throughout the cytoplasm of BmN cells. In silkworm pupae, BmTCTPk is expressed in the midgut wall, the midgut cavity, and some fat body tissues lying between the midgut wall and body wall. Western blot and ELISAs performed on total protein extracts isolated from silkworm pupae at different development stages showed that, although BmTCTPk is expressed during all pupae stages, its expression level increases dramatically during late pupae stages, suggesting that BmTCTPk may play an important role during the developmental transition from pupa to imago. PMID:19757184
Nie, Zuoming; Lv, Zhengbing; Qian, Jiawei; Chen, Jian; Li, Shanshan; Sheng, Qing; Wang, Dan; Shen, Hongdan; Yu, Wei; Wu, Xiangfu; Zhang, Yaozhou
The promoter of Bmlp3 gene can direct fat body-specific expression in the transgenic silkworm, Bombyx mori.
The fat body plays multiple, crucial roles in the life of silkworms. Targeted expression of transgenes in the fat body of the silkworm, Bombyx mori, is important not only for clarifying the function of endogenous genes expressed in this tissue, but also for producing valuable recombinant proteins. However, fat body-specific gene expression remains difficult due to a lack of suitable tissue-specific promoters. Here we report the isolation of the fat body-specific promoter of Bmlp3, a member of the 30K protein family of silkworms. The 1.1�kb fragment from -374 to +738 of Bmlp3 displayed strong promoter activity in the cell lines BmE and Spli-221. In transgenic silkworms, a DsRed reporter gene controlled by the 1.1�kb Bmlp3 promoter fragment was expressed specifically in the fat body in a stage-specific pattern that was nearly identical to the endogenous Bmlp3 gene. We conclude that the 1.1�kb Bmlp3 promoter fragment is sufficient to direct tissue- and stage-specific expression of transgenes in the fat body of silkworms, highlighting the potential use of this promoter for both functional genomics research and biotechnology applications. PMID:23543408
Deng, Dangjun; Xu, Hanfu; Wang, Feng; Duan, Xiaoli; Ma, Sanyuan; Xiang, Zhonghuai; Xia, Qingyou
In vivo site-specific integration of transgene in silkworm via PhiC31 integrase-mediated cassette exchange.
Current techniques for genetic engineering of the silkworm Bombyx mori genome utilize transposable elements, which result in positional effects and insertional mutagenesis through random insertion of exogenous DNA. New methods for introducing transgenes at specific positions are therefore needed to overcome the limitations of transposon-based strategies. Although site-specific recombination systems have proven powerful tools for genome manipulation in many organisms, their use has not yet been well established for the integration of transgenes in the silkworm. We describe a method for integrating target genes at pre-defined chromosomal sites in the silkworm via phiC31/att site-specific recombination system-mediated cassette exchange. Successful recombinase-mediated cassette exchange (RMCE) was observed in the two transgenic target strains with an estimated transformation efficiency of 3.84-7.01%. Our results suggest that RMCE events between chromosomal attP/attP target sites and incoming attB/attB sites were more frequent than those in the reciprocal direction. This is the first report of in vivo RMCE via phiC31 integrase in the silkworm, and thus represents a key step toward establishing genome manipulation technologies in silkworms and other lepidopteran species. PMID:23974010
Long, Dingpei; Zhao, Aichun; Xu, Longxia; Lu, Weijian; Guo, Qing; Zhang, Yang; Xiang, Zhonghuai
Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori.
A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori. The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced. Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region displaying significant homology to the putative catalytic region of a group of bacterial beta-1,3 glucanases and beta-1,3-1,4 glucanases. Silkworm GNBP was also shown to have amino acid sequence similarity to the vertebrate lipopolysaccharide receptor CD14 and was recognized specifically by a polygonal anti-CD14 antibody. Northern blot analysis showed that GNBP was constitutively expressed in fat body, as well as in cuticular epithelial cells of naive silkworms. Intense transcription was, however, rapidly induced following a cuticular or hemoceolien bacterial challenge. An mRNA that hybridized with GNBP cDNA was also found in the l(2)mbn immunocompetent Drosophila cell line. These observations suggest that GNBP is an inducible acute phase protein implicated in the immune response of the silkworm and perhaps other insects. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 Fig. 6
Lee, W J; Lee, J D; Kravchenko, V V; Ulevitch, R J; Brey, P T
Characterization and expression patterns of let-7 microRNA in the silkworm (Bombyx mori)
Background lin-4 and let-7, the two founding members of heterochronic microRNA genes, are firstly confirmed in Caenorhabditis elegans to control the proper timing of developmental programs in a heterochronic pathway. let-7 has been thought to trigger the onset of adulthood across animal phyla. Ecdysone and Broad-Complex are required for the temporal expression of let-7 in Drosophila melanogaster. For a better understanding of the conservation and functions of let-7, we seek to explore how it is expressed in the silkworm (Bombyx mori). Results One member of let-7 family has been identified in silkworm computationally and experimentally. All known members of this family share the same nucleotides at ten positions within the mature sequences. Sequence logo and phylogenetic tree show that they are not only conserved but diversify to some extent among some species. The bmo-let-7 was very lowly expressed in ova harvested from newborn unmated female adult and in individuals from the first molt to the early third instar, highly expressed after the third molt, and the most abundant expression was observed after mounting, particularly after pupation. The expression levels were higher at the end of each instar and at the beginning of each molt than at other periods, coinciding with the pulse of ecdysone and BR-C as a whole. Using cultured ovary cell line, BmN-SWU1, we examined the effect of altered ecdysone levels on bmo-let-7 expression. The expression was also detected in various tissues of day 3 of the fifth instar and of from day 7 of the fifth to pupa, suggesting a wide distributing pattern with various signal intensities. Conclusion bmo-let-7 is stage- and tissue-specifically expressed in the silkworm. Although no signals were detected during embryonic development and first larval instar stages, the expression of bmo-let-7 was observed from the first molt, suggesting that it might also function at early larval stage of the silkworm. The detailed expression profiles in the whole life cycle and cultured cell line of silkworm showed a clear association with ecdysone pulse and a variety of biological processes.
Liu, Shiping; Xia, Qingyou; Zhao, Ping; Cheng, Tingcai; Hong, Kaili; Xiang, Zhonghuai
Differentially Expressed Genes in the Cuticle and Hemolymph of the Silkworm, Bombyx mori, Injected with the Fungus Beauveria bassiana
The most important pathogenic fungus of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), is Beauveria bassiana (Balsamo-Crivelli ) Vuillemin (Hypocreales: Clavicipitaceae), which causes significant damage to sericulture production. Therefore, diagnosing fungal disease and developing new control measures are crucial to silk production. To better understand the responsive and interactive mechanisms between the host silkworm and this fungus, variations in silkworm gene expression were investigated using the suppression subtractive hybridization method following the injection of B. bassiana conidia. Two cDNA libraries were constructed, and 140 cDNA clones were isolated. Of the 50 differentially expressed genes identified, 45 (112 clones) were identified in the forward library, and 5 (28 clones) were identified in the reverse library. Expression profiling of six of these genes by quantitative polymerase chain reaction (qPCR) verified that they were induced by the fungal challenge. The present study provides insight into the interaction between lepidopteran insects and pathogenic fungi.
Hou, Cheng-Xiang; Qin, Guang-Xing; Liu, Ting; Mei, Xing-Lin; Li, Bing; Shen, Zhong-Yuan; Guo, Xi-Jie
Differentially Expressed Genes in the Cuticle and Hemolymph of the Silkworm, Bombyx mori, Injected with the Fungus Beauveria bassiana.
Abstract The most important pathogenic fungus of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), is Beauveria bassiana (Balsamo-Crivelli ) Vuillemin (Hypocreales: Clavicipitaceae), which causes significant damage to sericulture production. Therefore, diagnosing fungal disease and developing new control measures are crucial to silk production. To better understand the responsive and interactive mechanisms between the host silkworm and this fungus, variations in silkworm gene expression were investigated using the suppression subtractive hybridization method following the injection of B. bassiana conidia. Two cDNA libraries were constructed, and 140 cDNA clones were isolated. Of the 50 differentially expressed genes identified, 45 (112 clones) were identified in the forward library, and 5 (28 clones) were identified in the reverse library. Expression profiling of six of these genes by quantitative polymerase chain reaction (qPCR) verified that they were induced by the fungal challenge. The present study provides insight into the interaction between lepidopteran insects and pathogenic fungi. PMID:24794288
Transcriptome Analysis of Integument Differentially Expressed Genes in the Pigment Mutant (quail) during Molting of Silkworm, Bombyx mori.
In the silkworm Bombyx mori, pigment mutants with diverse body colors have been maintained throughout domestication for about 5000 years. The silkworm larval body color is formed through the mutual interaction of melanin, ommochromes, pteridines and uric acid. These pigments/compounds are synthesized by the cooperative action of various genes and enzymes. Previous reports showed that melanin, ommochrome and pteridine are increased in silkworm quail (q) mutants. To understand the pigment increase and alterations in pigment synthesis in q mutant, transcriptome profiles of the silkworm integument were investigated at 16 h after head capsule slippage in the fourth molt in q mutants and wild-type (Dazao). Compared to the wild-type, 1161 genes were differentially expressed in the q mutant. Of these modulated genes, 62.4% (725 genes) were upregulated and 37.6% (436 genes) were downregulated in the q mutant. The molecular function of differently expressed genes was analyzed by Blast2GO. The results showed that upregulated genes were mainly involved in protein binding, small molecule binding, transferase activity, nucleic acid binding, specific DNA-binding transcription factor activity and chromatin binding, while exclusively down-expressed genes functioned in oxidoreductase activity, cofactor binding, tetrapyrrole binding, peroxidase activity and pigment binding. We focused on genes related to melanin, pteridine and ommochrome biosynthesis; transport of uric acid; and juvenile hormone metabolism because of their importance in integument coloration during molting. This study identified differently expressed genes implicated in silkworm integument formation and pigmentation using silkworm q mutant. The results estimated the number and types of genes that drive new integument formation. PMID:24718369
Nie, Hongyi; Liu, Chun; Cheng, Tingcai; Li, Qiongyan; Wu, Yuqian; Zhou, Mengting; Zhang, Yinxia; Xia, Qingyou
Antheraea pernyi Silk Fibroin-Coated PEI/DNA Complexes for Targeted Gene Delivery in HEK 293 and HCT 116 Cells
Polyethylenimine (PEI) has attracted much attention as a DNA condenser, but its toxicity and non-specific targeting limit its potential. To overcome these limitations, Antheraea pernyi silk fibroin (ASF), a natural protein rich in arginyl-glycyl-aspartic acid (RGD) peptides that contains negative surface charges in a neutral aqueous solution, was used to coat PEI/DNA complexes to form ASF/PEI/DNA ternary complexes. Coating these complexes with ASF caused fewer surface charges and greater size compared with the PEI/DNA complexes alone. In vitro transfection studies revealed that incorporation of ASF led to greater transfection efficiencies in both HEK (human embryonic kidney) 293 and HCT (human colorectal carcinoma) 116 cells, albeit with less electrostatic binding affinity for the cells. Moreover, the transfection efficiency in the HCT 116 cells was higher than that in the HEK 293 cells under the same conditions, which may be due to the target bonding affinity of the RGD peptides in ASF for integrins on the HCT 116 cell surface. This result indicated that the RGD binding affinity in ASF for integrins can enhance the specific targeting affinity to compensate for the reduction in electrostatic binding between ASF-coated PEI carriers and cells. Cell viability measurements showed higher cell viability after transfection of ASF/PEI/DNA ternary complexes than after transfection of PEI/DNA binary complexes alone. Lactate dehydrogenase (LDH) release studies further confirmed the improvement in the targeting effect of ASF/PEI/DNA ternary complexes to cells. These results suggest that ASF-coated PEI is a preferred transfection reagent and useful for improving both the transfection efficiency and cell viability of PEI-based nonviral vectors.
Liu, Yu; You, Renchuan; Liu, Guiyang; Li, Xiufang; Sheng, Weihua; Yang, Jicheng; Li, Mingzhong
Sampling and Analysis Plan for White Oak Creek Watershed Remedial Investigation supplemental sampling, Oak Ridge National Laboratory, Oak Ridge, Tennessee.
This Sampling and Analysis (SAP) presents the project requirements for proposed soil sampling to support the White Oak Creek Remedial Investigation/Feasibility Study at Oak Ridge National Laboratory. During the Data Quality Objectives process for the proj...
Diversity analysis of Beauveria bassiana isolated from infected silkworm in southwest China based on molecular data and morphological features of colony.
Beauveria bassiana is an important entomopathogenic fungus that not only often causes infection and epidemics of wild insects but some strains also show pathogenicity to the silkworm, Bombyx mori. The present study is about diversity of B. bassiana isolated from the silkworm in southwest China. Five strains of B. bassiana were isolated from infected silkworm. Two isolates, GXtr1009 and GXtr1010, were isolated from infected silkworms treated with two kinds of biological pesticides applied in Guangxi province, and three isolates, SCsk1006, YNsk1106 and GXsk1011, were collected from naturally infected silkworms from different geographical locations in Yunnan and Sichuan. All of the isolates showed highly similar conidia and conidial fructification, but the colony characteristics demonstrated great differences among the isolates. The ITS and 18S rDNA sequence analysis was sufficient to identify all five isolates as B. bassiana. However, the dendrogram, based on the ISSR data, produced two large genetic groups. GXtr1009 and GXtr1010 comprised one group, and SCsk1006, YNsk1106 and GXsk1011 converged in a different large group. The results suggested that, although all of these five B. bassiana strains were pathogenic to silkworms, strains of biological pesticides could be differentiated from strains of naturally infected silkworm via ISSR analysis. PMID:23417263
Wang, Jing-jie; Yang, Li; Qiu, Xin; Liu, Yong-gui; Zhou, Wei; Wan, Yong-Ji
77 FR 23506 - Notice of Inventory Completion: The Region of Three Oaks Museum, Three Oaks, MI
Federal Register 2010, 2011, 2012, 2013
...Completion: The Region of Three Oaks Museum, Three Oaks, MI AGENCY: National Park...SUMMARY: The Region of Three Oaks Museum has completed an inventory of human remains...remains may contact The Region of Three Oaks Museum. Repatriation of the human remains...
Production of scFv-displaying BmNPV in silkworm larvae and its efficient purification.
Baculovirus-display technology utilizing the gp64 envelope protein has been developed. A simple and efficient process to separate the virus from the majority of the protein contaminants may be needed for the future demand of pure and functional baculovirus vectors ideal for vaccine- and gene-delivery applications. In the present study, using Bombyx mori (silkworm) larvae as a host, scFv (single-chain variable fragment)-surface displaying recombinant baculovirus production and its purification from silkworm larval haemolymph by SEC (size-exclusion chromatography) were demonstrated. The amounts of scFv were 4-8 ?g/ml in the haemolymph. The scFv-gp64 fusion protein was confirmed to be incorporated into the cell membrane and the BmNPV (B. mori nucleopolyhedrovirus) surface by immunofluorescence microscopy and Western blotting. rBmNPV (recombinant BmNPV) was purified to higher purity by SEC using Sephacryl S-1000 column chromatography than by sucrose-density-gradient centrifugation. The recovery of purified rBmNPV was 22.2%, and the virus purity in the SEC fraction was increased 269-fold compared with its purity in haemolymph. Judging from the results of ELISA, approx. 0.9% of the total baculovirus-particle proteins were occupied by scFv on their surface. A BmNPV-based silkworm-larval system is suitable for large-scale production of baculovirus-surface-displayed proteins or peptides in comparison with a cell-culture system. The present study will be useful for future BmNPV-application studies for gene delivery and vaccine trials. PMID:20923414
Dojima, Takashi; Nishina, Takuya; Kato, Tatsuya; Ueda, Hiroshi; Park, Enoch Y
Conceptual design of a bioregenerative life support system containing crops and silkworms
This article summarizes a conceptual design of a bioregenerative life support system for permanent lunar base or planetary exploration. The system consists of seven compartments - higher plants cultivation, animal rearing, human habitation, water recovery, waste treatment, atmosphere management, and storages. Fifteen kinds of crops, such as wheat, rice, soybean, lettuce, and mulberry, were selected as main life support contributors to provide the crew with air, water, and vegetable food. Silkworms fed by crop leaves were designated to produce partial animal nutrition for the crew. Various physical-chemical and biological methods were combined to reclaim wastewater and solid waste. Condensate collected from atmosphere was recycled into potable water through granular activated carbon adsorption, iodine sterilization, and trace element supplementation. All grey water was also purified though multifiltration and ultraviolet sterilization. Plant residue, human excrement, silkworm feces, etc. were decomposed into inorganic substances which were finally absorbed by higher plants. Some meat, ingredients, as well as nitrogen fertilizer were prestored and resupplied periodically. Meanwhile, the same amount and chemical composition of organic waste was dumped to maintain the steady state of the system. A nutritional balanced diet was developed by means of the linear programming method. It could provide 2721 kcal of energy, 375.5 g of carbohydrate, 99.47 g of protein, and 91.19 g of fat per capita per day. Silkworm powder covered 12.54% of total animal protein intakes. The balance of material flows between compartments was described by the system of stoichiometric equations. Basic life support requirements for crews including oxygen, food, potable and hygiene water summed up to 29.68 kg per capita per day. The coefficient of system material closure reached 99.40%.
Hu, Enzhu; Bartsev, Sergey I.; Liu, Hong
Fire Effects in Northeastern Forests: Oak.
Effects of fire on the oak timber type are reviewed. Many oak stands of today originated under severe fire regimes. Fire can kill or injure oak trees. Factors determining direct injury or mortality from fire include: season of year; bark characteristics; ...
C. Rouse
Herbivore insect guild of oaks in Hungary
Oaks play outstanding role both from economical and ecological point of view in Hungary. 32.5% (ca. 568 thousand hectares) of the Hungarian forested is covered by oak forests. 658 species of herbivore insects have been recorded so far to feed on oaks. This number is far higher than the species richness recorded from any other tree genus in Europe. 46.7%
Gy�rgy Cs�ka
Structure of autophagy-related protein Atg8 from the silkworm Bombyx mori
Autophagy-related protein Atg8 is ubiquitous in all eukaryotes. It is involved in�the Atg8�PE ubiquitin-like conjugation system, which is essential for autophagosome formation. The structures of Atg8 from different species are very similar and share a ubiquitin-fold domain at the C-terminus. In the 2.40?� crystal structure of Atg8 from the silkworm Bombyx mori reported here, the ubiquitin fold at the C-terminus is preceded by two additional helices at the N-�terminus.
Hu, Chen; Zhang, Xuan; Teng, Yan-Bin; Hu, Hai-Xi; Li, Wei-Fang
The nicotinic acetylcholine receptor gene family of the silkworm, Bombyx mori
Background Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic cholinergic transmission in the insect central nervous system. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Like mammalian nAChRs, insect nAChRs are considered to be made up of five subunits, coded by homologous genes belonging to the same family. The nAChR subunit genes of Drosophila melanogaster, Apis mellifera and Anopheles gambiae have been cloned previously based on their genome sequences. The silkworm Bombyx mori is a model insect of Lepidoptera, among which are many agricultural pests. Identification and characterization of B. mori nAChR genes could provide valuable basic information for this important family of receptor genes and for the study of the molecular mechanisms of neonicotinoid action and resistance. Results We searched the genome sequence database of B. mori with the fruit fly and honeybee nAChRs by tBlastn and cloned all putative silkworm nAChR cDNAs by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. B. mori appears to have the largest known insect nAChR gene family to date, including nine ?-type subunits and three ?-type subunits. The silkworm possesses three genes having low identity with others, including one ? and two ? subunits, ?9, ?2 and ?3. Like the fruit fly and honeybee counterparts, silkworm nAChR gene ?6 has RNA-editing sites, and ?4, ?6 and ?8 undergo alternative splicing. In particular, alternative exon 7 of Bm?8 may have arisen from a recent duplication event. Truncated transcripts were found for Bm?4 and Bm?5. Conclusion B. mori possesses a largest known insect nAChR gene family characterized to date, including nine ?-type subunits and three ?-type subunits. RNA-editing, alternative splicing and truncated transcripts were found in several subunit genes, which might enhance the diversity of the gene family.
Shao, Ya-Ming; Dong, Ke; Zhang, Chuan-Xi
Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system
We constructed the fibroin H-chain expression system to produce recombinant proteins in the cocoon of transgenic silkworms. Feline interferon (FeIFN) was used for production and to assess the quality of the product. Two types of FeIFN fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, were designed to be secreted into the lumen of the posterior silk glands. The expression of the FeIFN/H-chain fusion gene was regulated by the fibroin H-chain promoter domain. The transgenic silkworms introduced these constructs with the piggyBac transposon-derived vector, which produced the normal sized cocoons containing each FeIFN/H-chain fusion protein. Although the native-protein produced by transgenic silkworms have almost no antiviral activity, the proteins after the treatment with PreScission protease to eliminate fibroin H-chain derived N- and C-terminal sequences from the products, had very high antiviral activity. This H-chain expression system, using transgenic silkworms, could be an alternative method to produce an active recombinant protein and silk-based biomaterials.
Kurihara, H. [Toray Industries, Inc., New Frontiers Research Laboratories, 1111 Tebiro, Kamakura, Kanagawa 248-8555 (Japan)]. E-mail: email@example.com; Sezutsu, H. [Transgenic Silkworm Research Center, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634 (Japan); Tamura, T. [Transgenic Silkworm Research Center, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634 (Japan); Yamada, K. [Toray Industries, Inc., New Frontiers Research Laboratories, 1111 Tebiro, Kamakura, Kanagawa 248-8555 (Japan)
Changes in Carbohydrate Metabolism in Hemolymph and Fat Body of the Silkworm, Bombyx mori L., Exposed to Organophosphorus Insecticides
Effects of lethal and sublethal doses of widely used commercial-grade organophosphorus insecticides, namely fenitrothion and ethion, as a function of carbohydrate metabolism of hemolymph and fat body on Days 1, 3, 5, and 7 of the fifth-instar silkworm, Bombyx mori, were investigated. The results showed that pyruvate level and lactate dehydrogenase activity decreased with elevated lactate levels, indicating reduced mobilization
B. Surendra Nath
A Study of the Complex of Proteolytic Enzymes and Their Protein Inhibitors in the Embryogenesis of the Silkworm
We studied changes in the activities of serin, thiol, and aspartyl proteinases and their protein inhibitors during embryogenesis of the silkworm. The dynamics of the activities of the protein inhibitors and specific proteinases were interrelated, thus providing for the coordination and fine regulation of the functioning of the proteolytic complex of enzymes during embryogenesis. Possible functions of peptidohydrolases and their
D. V. Yarygin; S. M. Klunova; Yu. B. Filippovich
The Cytochrome System of the Cecropia Silkworm. II. Spectrophotometric Studies of Oxidative Enzyme Systems in the Wing Epithelium
As shown by spectroscopic studies, the wing epithelium of the Cecropia silkworm is typical of the insect's non-muscular tissues in undergoing a virtual breakdown of the cytochrome system at the outset of pupal diapause, and a prominent resynthesis of this system when diapause is terminated. Further information on these marked changes has now been provided by spectrophotometric determinations of DPNH
D. G. Shappirio; C. M. Williams
Differential sequestration of storage proteins by various fat body tissues during post-larval development in silkworm, Bombyx mori L
During the final larval stadium of the silkworm, Bombyx mori, storage proteins SP1 and SP2 are synthesized mainly by peripheral and dorsal perivisceral fat body tissues. No further synthesis occurs during the non-feeding pupal stage. The ventral perivisceral tissue has no role in this process, but is specialized for the selective storage of the proteins. Storage proteins are selectively sequestered
Genetic mapping of Z chromosome and identification of W chromosome-specific markers in the silkworm, Bombyx mori.
In the silkworm, Bombyx mori, the female is the heterogametic (ZW) sex and the male is homogametic (ZZ). The female heterogamety is a typical situation in the insect order Lepidoptera. Although the W chromosome in silkworm is strongly female determining, no W-linked gene for a morphological character has been found on it. The Z chromosome carries important traits of economic value as well as genes for various phenotypic traits, but only 2% of molecular information based on its relative size is known. Studies conducted so far indicate that the Z-linked genes are not dosage compensated. In the present study, we constructed a genetic map of randomly amplified polymorphic DNA fragments (RAPD), simple sequence repeats (SSR), and fluorescent intersimple sequence repeat PCR (FISSR) markers for the Z chromosome using a backcross mapping population. A total of 16 Z-linked markers were identified, characterized, and mapped using od, a recessive trait for translucent skin as an anchor marker yielding a total recombination map of 334.5 cM. The linkage distances obtained suggested that the markers were distributed throughout the Z chromosome. Four RAPD and four SSR markers that were linked to W chromosome were also identified. The proposed mapping approach should be useful to identify and map sex-linked traits in the silkworm. The economic and evolutionary significance of Z- and W-linked genes in silkworm, in particular, and lepidopterans, in general, is discussed. PMID:15931240
Nagaraja, G M; Mahesh, G; Satish, V; Madhu, M; Muthulakshmi, M; Nagaraju, J
Swallowing motor pattern triggered and modified by sucrose stimulation in the larvae of the silkworm, Bombyx mori
To determine the contribution of sucrose signals to swallowing motor patterns, a series of behavioral, morphological and electrophysiological experiments were carried out in the larvae of the silkworm, Bombyx mori. The larvae ingested a droplet of sucrose solution applied to the mouth. The rate of ingestion was increased for higher sucrose concentrations. The swallowing movements were produced by a cibarial
Ken Sasaki; Kiyoshi Asaoka
Applying fenoxycarb at the penultimate instar triggers an additional ecdysteroid surge and induces perfect extra larval molting in the silkworm
When the juvenile hormone analog fenoxycarb was topically applied to the silkworm Bombyx mori at the beginning of the 3rd or 4th (penultimate) instar, an extra larval molt was induced. The 5th instar period was shortened to about 5 days and the extra 6th instar ranged from 8 to more than 20 days, depending on the dose applied. Starvation before
Manabu Kamimura; Makoto Kiuchi
Effects of Fenoxycarb on the Secretory Activity of the Prothoracic Glands in the Fifth Instar of the Silkworm, Bombyx mori
Effects of fenoxycarb on the secretory activity of the prothoracic glands (PGs) of the silkworm,Bombyx mori,were investigated. Fenoxycarb inhibited the secretory activity of PGsin vitrothroughout the fifth instar. Incubation of PGs in the presence of 1 ?g of fenoxycarb resulted in a ?50% inhibition of their secretory activity. This inhibition of fenoxycarb was dose dependent. Stimulation of the PGs by
Skarlatos G. Dedos; Hajime Fugo
In the previous papers of this series (Williams, 1946b, 1947, 1948a) an endo crine basis was described for the production and termination of pupal diapause in the Cecropia silkworm. The onset of diapause was correlated with a temporary failure of the brain in secreting a hormone required for the initiation of adult development. The ultimate release of this "�?brain hormone"
Morphological and physiological properties of pheromone-triggered flipflopping descending interneurons of the male silkworm moth, Bombyx mori
1.The morphology of descending interneurons (DNs) which have arborizations in the lateral accessory lobe (LAL) of the protocerebrum, the higher order olfactory center, and have an axon in the ventral nerve cord (VNC), were characterized in the male silkworm moth, Bombyx mori.2.Two clusters (group I, group II) of DNs which have arborizations mainly in the LALs were morphologically characterized. The
R. Kanzaki; A. Ikeda; T. Shibuya
A novel peptide inhibitor of platelet aggregation from stiff silkworm, Bombyx batryticatus.
A novel platelet aggregation inhibitory peptide, named BB octapeptide, was isolated from stiff silkworm (Bombyx batryticatus) by gel filtration, anion-exchange, and reverse-phase high performance liquid chromatography. The molecular mass of the peptide was determined to be 885 Da using electrospray ionization mass spectrometry, and the sequence was identified as Asp-Pro-Asp-Ala-Asp-IIe-Leu-Gln using the Edman degradation method. To test its biological activity, the peptide was chemically synthesized using Fmoc solid-phase synthesis method. BB octapeptide inhibited rabbit platelet aggregation that was induced by collagen and epinephrine, with the IC50 values of 91.14 ?M and 104.50 ?M, respectively. After intravenous administrated in mice (30 mg/kg, 4 days), BB octapeptide showed similar ex vivo efficacy of inhibiting platelet aggregation as aspirin (10 mg/kg). In addition, this peptide prevented paralysis and death in pulmonary thromboembolism model and significantly reduced ferric chloride-induced thrombus formation in rats. Moreover, it exhibited low cytotoxicity in a cellular model. In conclusion, this is the first report that a novel platelet aggregation inhibitory peptide was isolated from stiff silkworm (B. batryticatus). Due to the excellent efficacy in reducing platelet aggregation and low toxicity, it can be a valuable lead compound for new drug design and development. PMID:24361453
Kong, Yi; Xu, Cheng; He, Zhi-Long; Zhou, Qiu-Mei; Wang, Jin-Bin; Li, Zhi-Yu; Ming, Xin
Xanthurenic acid is an endogenous substrate for the silkworm cytosolic sulfotransferase, bmST1.
Sulfotransferase enzymes are known to regulate physiologically active substances such as steroids and catecholamines in mammals. Although invertebrates also express sulfotransferases, their biological function is mostly unclear. In a previous study, we reported that 4-nitrocatechol and the gallete ester are substrates for the silkworm sulfotransferase bmST1. The K(m) of bmST1 for these substrates is high. However, endogenous substrates of bmST1 have not yet been determined. We therefore investigated endogenous bmST1 substrates and carried out a detailed expression profile analysis of bmST1. We found that xanthurenic acid, a tryptophan metabolite, is a possible endogenous substrate of bmST1. The K(m) of bmST1 for xanthurenic acid is low, in the ?M range, which is lower than that for previously reported substrates. Additionally, xanthurenic acid is a tryptophan metabolite that characteristically shows toxicity in vivo. High dose administration of xanthurenic acid resulted in inhibition of cuticular biosynthesis. The expression of the bmST1 gene reached a maximal level in the Malpighian tubule at the 4th molting stage, when amino acid metabolism might be activated. Our results suggest that bmST1 plays a role in detoxification of xanthurenic acid in the silkworm. PMID:22036934
Kushida, Akira; Horie, Ryo; Hattori, Kenji; Hamamoto, Hiroshi; Sekimizu, Kazuhisa; Tamura, Hiroomi
Shotgun proteomic analysis of wing discs from the domesticated silkworm (Bombyx mori) during metamorphosis.
Proteomic profiles from the wing discs of silkworms at the larval, pupal, and adult moth stages were determined using shotgun proteomics and MS sequencing. We identified 241, 218, and 223 proteins from the larval, pupal, and adult moth stages, respectively, of which 139 were shared by all three stages. In addition, there were 55, 37, and 43 specific proteins identified at the larval, pupal, and adult moth stages, respectively. More metabolic enzymes were identified among the specific proteins expressed in the wing disc of larvae compared with pupae and moths. The identification of FKBP45 and the chitinase-like protein EN03 as two proteins solely expressed at the larval stage indicate these two proteins may be involved in the immunological functions of larvae. The myosin heavy chain was identified in the pupal wing disc, suggesting its involvement in the formation of wing muscle. Some proteins, such as proteasome alpha 3 subunits and ribosomal proteins, specifically identified from the moth stage may be involved in the degradation of old cuticle proteins and new cuticle protein synthesis. Gene ontology analysis of proteins specific to each of these three stages enabled their association with cellular component, molecular function, and biological process categories. The analysis of similarities and differences in these identified proteins will greatly further our understanding of wing disc development in silkworm and other insects. PMID:24005483
Zhang, Yi-ling; Xue, Ren-yu; Cao, Guang-li; Zhu, Yue-xiong; Pan, Zhong-hua; Gong, Cheng-liang
The effect of calorie restriction on growth and development in silkworm, Bombyx mori.
Caloric restriction (CR) is known to extend the life span in different species from yeast to mammals. In this report, a simple organism silkworm (Bombyx mori) was used to study the effect of moderate CR on the growth and development processes of insects. Here we show that an extension of life span upon moderate CR was observed in the silkworm. The total protein level in the 5th instar larvae hemolymph appeared to decline significantly under CR. SDS-PAGE analysis showed that the influence of CR was sex-dependent. The CR effects on female animals were much more significant than on the males. The MALDI-TOF MS study identified 16 proteins that expressed differentially among six groups of the male or female larvae fed at different time frequencies. Four of them, storage protein 1 (SP1), arylphorin (SP2), imaginal disk growth factor (IDGF), and 30-kDa lipoprotein, showed significant differences. It was demonstrated that these four proteins were up-regulated when the larvae were over-fed and down-regulated when the larvae were less-fed. PMID:19479736
Li, Yijia; Chen, Keping; Yao, Qin; Li, Jun; Wang, Yong; Liu, Haijun; Zhang, Chiyu; Huang, Guoping
Nested retrotransposons on the W chromosome of the wild silkworm Bombyx mandarina.
The W chromosome of the silkworms Bombyx mori or B. mandarina is recombinationally isolated from the Z chromosome and the autosomes. We previously characterized a female-specific randomly amplified polymorphic DNA (RAPD), designated W-Yamato, derived from the W chromosome of the wild silkworm Bombyx mandarina. To further analyse the W chromosome of B. mandarina, we obtained a lambda phage clone that contains the W-Yamato RAPD sequence and sequenced the 16.7 kb DNA insert. We found that this DNA comprises a nested structure of at least seven elements: six retrotransposons and one transposable element-like sequence. The transposable element-like sequence is inserted into a micropia-like retrotransposon (Karate). The Karate and the non-long terminal repeat (non-LTR) retrotransposon BMC1 are inserted into a 412-like retrotransposon (Judo). Furthermore, this Judo, and two non-LTR retrotransposons (Kurosawa and Kendo) are inserted into a Pao-like retrotransposon (Yamato). These results indicate that the retrotransposons inserted into the W chromosome are not efficiently removed but accumulate gradually as strata without recombination. PMID:12144695
Abe, H; Sugasaki, T; Terada, T; Kanehara, M; Ohbayashi, F; Shimada, T; Kawai, S; Mita, K; Oshiki, T
Existence of Prophenoloxidase in Wing Discs: A Source of Plasma Prophenoloxidase in the Silkworm, Bombyx mori
In insects, hemocytes are considered as the only source of plasma prophenoloxidase (PPO). PPO also exists in the hemocytes of the hematopoietic organ that is connected to the wing disc of Bombyx mori. It is unknown whether there are other cells or tissues that can produce PPO and release it into the hemolymph besides circulating hemocytes. In this study, we use the silkworm as a model to explore this possibility. Through tissue staining and biochemical assays, we found that wing discs contain PPO that can be released into the culture medium in vitro. An in situ assay showed that some cells in the cavity of wing discs have PPO1 and PPO2 mRNA. We conclude that the hematopoietic organ may wrongly release hemocytes into wing discs since they are connected through many tubes as repost in previous paper. In wing discs, the infiltrating hemocytes produce and release PPO probably through cell lysis and the PPO is later transported into hemolymph. Therefore, this might be another source of plasma PPO in the silkworm: some infiltrated hemocytes sourced from the hematopoietic organ release PPO via wing discs.
Diao, Yupu; Lu, Anrui; Yang, Bing; Hu, Wenli; Peng, Qing; Ling, Qing-Zhi; Beerntsen, Brenda T.; Soderhall, Kenneth; Ling, Erjun
Heritable Genome Editing with CRISPR/Cas9 in the Silkworm, Bombyx mori
We report the establishment of an efficient and heritable gene mutagenesis method in the silkworm Bombyx mori using modified type II clustered regularly interspaced short palindromic repeats (CRISPR) with an associated protein (Cas9) system. Using four loci Bm-ok, BmKMO, BmTH, and Bmtan as candidates, we proved that genome alterations at specific sites could be induced by direct microinjection of specific guide RNA and Cas9-mRNA into silkworm embryos. Mutation frequencies of 16.7�35.0% were observed in the injected generation, and DNA fragments deletions were also noted. Bm-ok mosaic mutants were used to test for mutant heritability due to the easily determined translucent epidermal phenotype of Bm-ok-disrupted cells. Two crossing strategies were used. In the first, injected Bm-ok moths were crossed with wild-type moths, and a 28.6% frequency of germline mutation transmission was observed. In the second strategy, two Bm-ok mosaic mutant moths were crossed with each other, and 93.6% of the offsprings appeared mutations in both alleles of Bm-ok gene (compound heterozygous). In summary, the CRISPR/Cas9 system can act as a highly specific and heritable gene-editing tool in Bombyx mori.
Roy, Bhaskar; Dai, Junbiao; Miao, Yungen; Gao, Guanjun
Overexpression and functional characterization of an Aspergillus niger phytase in the fat body of transgenic silkworm, Bombyx mori.
In a previous study, we isolated 1,119�bp of upstream promoter sequence from Bmlp3, a gene encoding a member of the silkworm 30�K storage protein family, and demonstrated that it was sufficient to direct fat body-specific expression of a reporter gene in a transgenic silkworm, thus highlighting the potential use of this promoter for both functional genomics research and biotechnology applications. To test whether the Bmlp3 promoter can be used to produce recombinant proteins in the fat body of silkworm pupae, we generated a transgenic line of Bombyx mori which harbors a codon-optimized Aspergillus niger phytase gene (phyA) under the control of the Bmlp3 promoter. Here we show that the Bmlp3 promoter drives high levels of phyA expression in the fat body, and that the recombinant phyA protein is highly active (99.05 and 54.80 U/g in fat body extracts and fresh pupa, respectively). We also show that the recombinant phyA has two optimum pH ranges (1.5-2.0 and 5.5-6.0), and two optimum temperatures (55 and 37��C). The activity of recombinant phyA was lost after high-temperature drying, but treating with boiling water was less harmful, its residual activity was approximately 84�% of the level observed in untreated samples. These results offer an opportunity not only for better utilization of large amounts of silkworm pupae generated during silk production, but also provide a novel method for mass production of low-cost recombinant phytase using transgenic silkworms. PMID:24719047
Xu, Hanfu; Liu, Yaowen; Wang, Feng; Yuan, Lin; Wang, Yuancheng; Ma, Sanyuan; Bene�, Helen; Xia, QingYou
Oak Ridge National Laboratory Review
This report presents brief descriptions of the following programs at Oak Ridge National Laboratory: The effects of pollution and climate change on forests; automation to improve the safety and efficiency of rearming battle tanks; new technologies for DNA sequencing; ORNL probes the human genome; ORNL as a supercomputer research center; paving the way to superconcrete made with polystyrene; a new look at supercritical water used in waste treatment; and small mammals as environmental monitors.
Krause, C.; Pearce, J.; Zucker, A. (eds.)
High level expression of a frog ?-amidating enzyme, AE-II, in cultured cells and silkworm larvae using a Bombyx mori nuclear polyhedrosis virus expression vector
AXenopus laevis peptidyl C-terminal ?-amidating enzyme (AE-II) gene, modified by deletion of a region encoding the putative membrane-spanning\\u000a domain and the putative C-terminal cytosolic tail, was expressed in BoMo-15 AIIc insect cells and silkworm larvae using aBombyx mori baculovirus expression vector system. The expressed enzyme was identified predominantly in the culture medium and the hemolymph\\u000a of silkworm larvae, indicating successful
Jun Kobayashi; Shigeo Imanishi; Hajime Inoue; Kazuhiro Ohsuye; Kozo Yamaichi; Nobuo Tsuruoka; Shoji Tanaka
Expression of Trichoderma viride endoglucanase III in the larvae of silkworm, Bombyx mori L. and characteristic analysis of the recombinant protein
Endoglucanase is a part of cellulase which hydrolyzes cellulose into glucose. In this study, we cloned endoglucanase III (EG\\u000a III) gene from Trichoderma viride strain AS 3.3711 using a PCR-based exon splicing method, and expressed EG III recombinant protein in both silkworm BmN cell\\u000a line and silkworm larvae with an improved Bac-to-Bac\\/BmNPV mutant baculovirus expression system, which lacks the chiA
Xing-hua Li; Peng Zhang; Mei-xian Wang; Fang Zhou; Firdose Ahmad Malik; Hua-jun Yang; Roy Bhaskar; Jia-biao Hu; Chun-guang Sun; Yun-gen Miao
Manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L enhances the NK activity and splenocyte proliferation against Sarcoma 180 tumor cells in vivo.
Natural killer cell (NK) is known as a major immune system in body through mediating cell death via several possible pathways, and one of three subpopulations of lymphocytes functioning as scavenger of tumor, virus infected cells etc. Our present results found that the SOD-contained silkworm larvae powder caused an enhancement of the effect on NK cell cytotoxicity, which implied this material modulated the immune system in mice in vivo. The NK cell activities of S180 tumor modeled mice treated with silkworm powder including SOD were enhanced significantly ranging from 30% to 48%, respectively, compare to a distilled water feeding control and silkworm powder without SOD. Meanwhile, the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control both in T cells or B cells. The average tumor weight of S180 modeled mice treated with doses of SOD-contained silkworm powder was lighter than that of water control showing the tumor inhibition rates (IR) reached to 22.51% to 37%, respectively. In conclusion, these findings demonstrate that administration of silkworm larvae powder containing SOD results in activation of NK cells and immune T-cell and B-cell, suggesting the silkworm larvae powder containing SOD play a positive role in tumor inhibition. PMID:17934870
Yue, Wan-Fu; Yao, Min-Li; Liu, Jian-Mei; Li, Guang-Li; Li, Xing-Hua; Wu, Xiao-Feng; Deng, Wen; Sun, Hong-Xiang; Zhou, Ji-Yong; Zhang, Chuan-Xi; Miao, Yun-Gen
Molecular Markers for Biomass Traits: Association, Interaction and Genetic Divergence in Silkworm Bombyx mori
Improvement of high yielding, disease resistant silkworm strains became imminent to increase production of silk, which is a major revenue earner for sericulturists. Since environment interacts with phenotype, conventional breeding did not result in commendable yield improvement in synthetic strains of silkworm, Bombyx mori. Identification of DNA markers associated with different economically important biomass traits and its introgression could assist molecular breeding and expression of stabilized high yielding characters, but genetic basis of most quantitative traits in silkworm is poorly understood due to its polygenic control. Correlation analysis (R = 0.9) revealed significant interrelation among biomass traits viz., larval duration (TLD), larval weight (LWT), cocoon weight (CWT), shell weight (SWT), shell ratio (SR) and floss content. PCR using inter simple sequence repeat (ISSR) primers revealed 92% polymorphism among 14 tropical and temperate strains of B. mori, with average diversity index of 0.747. Stepwise multiple regression analysis (MRA) selected 35 ISSR markers positively or negatively correlated with different biomass traits, illustrated polygenic control. ISSR marker 830.81050bp was significantly associated with LWT, CWT, SWT, SR and floss content, indicated its pleiotropic role. Two ISSR markers, 835.51950bp and 825.9710bp showed significant association with floss content and TLD. These markers were segregated in F2 generation and Chi-square test confirmed (?2 = ~45; P < 0.05) its genetic contribution to the associated biomass traits. Strains, with both positively and negatively correlated markers, had intermediate mean value for biomass traits (eg. SWT = 0.17 � 0.014 g in GNM and Moria) indicated interaction of loci in natural populations. Low yielding Indian strains grouped together by Hierarchical clustering. Chinese and Japanese strains were distributed in the periphery of ALSCAL matrix indicated convergence of genetic characters in Indian strains. Average genetic distance between Chinese strains and Indian strains (0.193) significantly (P < 0.01) varied from that between Chinese and Japanese strains. Interaction of loci and allelic substitutions induced phenotypic plasticity in temperate B. mori populations on tropic adaptation in India. These outcomes show possibility to combine favorable alleles at different QTL to increase larval, cocoon and shell weight.
Pradeep, Appukuttannair R; Jingade, Anuradha H; Urs, Raje S
Molecular Markers for Biomass Traits: Association, Interaction and Genetic Divergence in Silkworm Bombyx mori.
Improvement of high yielding, disease resistant silkworm strains became imminent to increase production of silk, which is a major revenue earner for sericulturists. Since environment interacts with phenotype, conventional breeding did not result in commendable yield improvement in synthetic strains of silkworm, Bombyx mori. Identification of DNA markers associated with different economically important biomass traits and its introgression could assist molecular breeding and expression of stabilized high yielding characters, but genetic basis of most quantitative traits in silkworm is poorly understood due to its polygenic control. Correlation analysis (R = 0.9) revealed significant interrelation among biomass traits viz., larval duration (TLD), larval weight (LWT), cocoon weight (CWT), shell weight (SWT), shell ratio (SR) and floss content. PCR using inter simple sequence repeat (ISSR) primers revealed 92% polymorphism among 14 tropical and temperate strains of B. mori, with average diversity index of 0.747. Stepwise multiple regression analysis (MRA) selected 35 ISSR markers positively or negatively correlated with different biomass traits, illustrated polygenic control. ISSR marker 830.8(1050bp) was significantly associated with LWT, CWT, SWT, SR and floss content, indicated its pleiotropic role. Two ISSR markers, 835.5(1950bp) and 825.9(710bp) showed significant association with floss content and TLD. These markers were segregated in F(2) generation and Chi-square test confirmed (chi(2) = ~45; P < 0.05) its genetic contribution to the associated biomass traits. Strains, with both positively and negatively correlated markers, had intermediate mean value for biomass traits (eg. SWT = 0.17 +/- 0.014 g in GNM and Moria) indicated interaction of loci in natural populations. Low yielding Indian strains grouped together by Hierarchical clustering. Chinese and Japanese strains were distributed in the periphery of ALSCAL matrix indicated convergence of genetic characters in Indian strains. Average genetic distance between Chinese strains and Indian strains (0.193) significantly (P < 0.01) varied from that between Chinese and Japanese strains. Interaction of loci and allelic substitutions induced phenotypic plasticity in temperate B. mori populations on tropic adaptation in India. These outcomes show possibility to combine favorable alleles at different QTL to increase larval, cocoon and shell weight. PMID:19662204
Comprehensive integrated planning: A process for the Oak Ridge Reservation, Oak Ridge, Tennessee.
The Oak Ridge Comprehensive Integrated Plan is intended to assist the US Department of Energy (DOE) and contractor personnel in implementing a comprehensive integrated planning process consistent with DOE Order 430.1, Life Cycle Asset Management and Oak R...
The OakMapper WebGIS: Improved Access to Sudden Oak Death Spatial Data1
Access to timely and accurate sudden oak death (SOD) location data is critical for SOD monitoring, management and research. Several websites (hereafter called the OakMapper sites) associated with sudden oak death monitoring efforts have been maintained with up-to- date SOD location information for over five years, providing information and maps of the most current spatial and attribute data on Phytophthora
K. Tuxen; M. Kelly
The horned oak gall wasp (Callirhytis cornigera) induces large, woody galls on twigs of oak; these galls can disfigure trees and result in extensive branch dieback. This paper reviews our recent research on the pest's biology and management on cultivated pin oaks (Quercus palustris) in Lexington, Kentucky, U.S., especially aspects that are of greatest relevance to tree care professionals. The
Eileen A. Eliason; Daniel A. Potter
Identification of novel random amplified polymorphic DNAs (RAPDs) on the W chromosome of the domesticated silkworm, Bombyx mori, and the wild silkworm, B. mandarina, and their retrotransposable element-related nucleotide sequences.
Genomic DNAs were compared between males and females of the domesticated silkworm, Bombyx mori, strains C108, C137, J137, p50, and WILD-W (constructed by crossing a wild silkworm, B. mandarina, female with a male of strain C108) by polymerase chain reaction (PCR) with 700 arbitrary 10-mer primers. Four female-specific RAPDs (W-Kabuki, W-Samurai, W-Kamikaze, and W-Yamato) were found. The sex chromosome formulas of B. mori and B. mandarina are ZW (XY) for the female and ZZ (XX) for the male. The four female-specific RAPDs are assumed to be derived from the W chromosome because the other chromosomes are shared by both sexes. A computer search for deduced amino acid sequences of these four RAPDs revealed that all of them showed homology to previously reported amino acid sequences encoded in known retrotransposable elements from various organisms. PMID:9880922
Abe, H; Kanehara, M; Terada, T; Ohbayashi, F; Shimada, T; Kawai, S; Suzuki, M; Sugasaki, T; Oshiki, T
Embryonic thermosensitive TRPA1 determines transgenerational diapause phenotype of the silkworm, Bombyx mori.
In the bivoltine strain of the silkworm, Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother; however, its molecular mechanisms are largely unknown. Here, we show that the Bombyx TRPA1 ortholog (BmTrpA1) acts as a thermosensitive transient receptor potential (TRP) channel that is activated at temperatures above ? 21 �C and affects the induction of diapause in progeny. In addition, we show that embryonic RNAi of BmTrpA1 affects diapause hormone release during pupal-adult development. This study identifying a thermosensitive TRP channel that acts as a molecular switch for a relatively long-term predictive adaptive response by inducing an alternative phenotype to seasonal polyphenism is unique. PMID:24639527
Sato, Azusa; Sokabe, Takaaki; Kashio, Makiko; Yasukochi, Yuji; Tominaga, Makoto; Shiomi, Kunihiro
Catalytic function of an ?-class glutathione S-transferase of the silkworm.
The glutathione S-transferase (GST) superfamily is involved in the detoxification of various xenobiotics. A silkworm GST, belonging to a previously reported Epsilon-class GST family, was identified, named bmGSTE, cloned, and produced in Escherichia coli. Investigation of this enzyme's properties showed that it was able to catalyse glutathione (GSH) with 1-chloro-2,4-dinitrobenzene and ethacrynic acid, and also that it possessed GSH-dependent peroxidase activity. The enzyme's highly conserved amino acid residues, including Ser11, His53, Val55, Ser68 and Arg112, were of interest regarding their possible involvement in its catalytic activity. These residues were replaced with alanine by site-directed mutagenesis and subsequent kinetic analysis of bmGSTE mutants indicated that His53, Val55, and Ser68 were important for enzyme function. PMID:23803169
Yamamoto, K; Aso, Y; Yamada, N
Cre-mediated targeted gene activation in the middle silk glands of transgenic silkworms (Bombyx mori).
Cre-mediated recombination is widely used to manipulate defined genes spatiotemporally in vivo. The present study evaluated the Cre/loxP system in Bombyx mori by establishing two transgenic lines. One line contained a Cre recombinase gene controlled by a sericin-1 gene (Ser1) promoter. The other line contained a loxP-Stop-loxP-DsRed cassette driven by the same Ser1 promoter. The precise deletion of the Stop fragment was found to be triggered by Cre-mediated site-specific excision, and led to the expression of DsRed fluorescence protein in the middle silk glands of all double-transgenic hybrids. This result was also confirmed by phenotypical analysis. Hence, the current study demonstrated the feasibility of Cre-mediated site-specific recombination in B. mori, and opened a new window for further refining genetic tools in silkworms. PMID:23264031
Duan, Jianping; Xu, Hanfu; Ma, Sanyuan; Guo, Huizhen; Wang, Feng; Zhao, Ping; Xia, Qingyou
Transgene-based, female-specific lethality system for genetic sexing of the silkworm, Bombyx mori.
Transgene-based genetic sexing methods are being developed for insects of agricultural and public health importance. Male-only rearing has long been sought in sericulture because males show superior economic characteristics, such as better fitness, lower food consumption, and higher silk yield. Here we report the establishment of a transgene-based genetic sexing system for the silkworm, Bombyx mori. We developed a construct in which a positive feedback loop regulated by sex-specific alternative splicing leads to high-level expression of the tetracycline-repressible transactivator in females only. Transgenic animals show female-specific lethality during embryonic and early larval stages, leading to male-only cocoons. This transgene-based female-specific lethal system not only has wide application in sericulture, but also has great potential in lepidopteran pest control. PMID:23569267
Tan, Anjiang; Fu, Guoliang; Jin, Li; Guo, Qiuhong; Li, Zhiqian; Niu, Baolong; Meng, Zhiqi; Morrison, Neil I; Alphey, Luke; Huang, Yongping
Biological Activity of Recombinant Bovine Interferon ? Produced by a Silkworm-Baculovirus Gene Expression System
ABSTRACT Bovine interferon (bIFN) ? plays a crucial role in maternal-fetal recognition and was expressed using a Bombyx mori (Bm) nuclear polyhedrosis virus (silkworm baculovirus) gene expression system. The biological effects of Bm-recombinant bIFN? (rbIFN?) on prostaglandin (PG) F2? synthesis were investigated in cultured bovine endometrial epithelial cells with oxytocin (OT, 100 nM) and on the in vitro development of bovine embryos. Bm-rbIFN? and OT were shown to suppress PGF2? production in a dose-dependent manner. When in vitro produced morula stage embryos were cultured for 72 hr in modified CR1aa medium supplemented with or without rbIFN?, Bm-rbIFN? (10 ng/ml) significantly promoted development to the expanded blastocyst stage. In conclusion, Bm-rbIFN? was suggested to have the same bioactivity as native IFN?.
TAKAHASHI, Hitomi; TSUNAZAKI, Makoto; HAMANO, Takashi; TAKAHASHI, Masashi; OKUDA, Kiyoshi; INUMARU, Shigeki; OKANO, Akira; GESHI, Masaya; HIRAKO, Makoto
Embryonic thermosensitive TRPA1 determines transgenerational diapause phenotype of the silkworm, Bombyx mori
In the bivoltine strain of the silkworm, Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother; however, its molecular mechanisms are largely unknown. Here, we show that the Bombyx TRPA1 ortholog (BmTrpA1) acts as a thermosensitive transient receptor potential (TRP) channel that is activated at temperatures above ?21 �C and affects the induction of diapause in progeny. In addition, we show that embryonic RNAi of BmTrpA1 affects diapause hormone release during pupal-adult development. This study identifying a thermosensitive TRP channel that acts as a molecular switch for a relatively long-term predictive adaptive response by inducing an alternative phenotype to seasonal polyphenism is unique.
An emerging functional natural silk biomaterial from the only domesticated non-mulberry silkworm Samia ricini.
Mulberry silk fibroin is a widely used biomaterial and recent work on non-mulberry silk fibroin also suggests it may have similar uses. We expect silk fibroin from the only domesticated non-mulberry eri silkworm, Samia ricini, to possess useful properties as a biomaterial. Eri silk gland fibroin is a heterodimeric protein of approximately 450?kDa. Cytocompatibility evaluation with fibroblasts and osteoblast-like cells shows good cell attachment, viability and proliferation. The matrices, which have high thermal stability and good swellability, are also haemocompatible. Eri silk production is cost effective as no agronomic practices are required for their host plant cultivation. This fibroin provide new opportunities as an alternative natural functional biomaterial in various biomedical applications. PMID:23733347
Pal, Shilpa; Kundu, Joydip; Talukdar, Sarmistha; Thomas, Tintu; Kundu, Subhas C
A novel sodium N-fatty acyl amino acid surfactant using silkworm pupae as stock material
A novel sodium N-fatty acyl amino acid (SFAAA) surfactant was synthesized using pupa oil and pupa protein hydrolysates (PPH) from a waste product of the silk industry. The aliphatic acids from pupa oil were modified into N-fatty acyl chlorides by thionyl chloride (SOCl2). SFAAA was synthesized using acyl chlorides and PPH. GC-MS analysis showed fatty acids from pupa oil consist mainly of unsaturated linolenic and linoleic acids and saturated palmitic and stearic acids. SFAAA had a low critical micelle concentration, great efficiency in lowering surface tension and strong adsorption at an air/water interface. SFAAA had a high emulsifying power, as well as a high foaming power. The emulsifying power of PPH and SFAAA in an oil/water emulsion was better with ethyl acetate as the oil phase compared to n-hexane. The environment-friendly surfactant made entirely from silkworm pupae could promote sustainable development of the silk industry.
Wu, Min-Hui; Wan, Liang-Ze; Zhang, Yu-Qing
A novel sodium N-fatty acyl amino acid surfactant using silkworm pupae as stock material.
A novel sodium N-fatty acyl amino acid (SFAAA) surfactant was synthesized using pupa oil and pupa protein hydrolysates (PPH) from a waste product of the silk industry. The aliphatic acids from pupa oil were modified into N-fatty acyl chlorides by thionyl chloride (SOCl2). SFAAA was synthesized using acyl chlorides and PPH. GC-MS analysis showed fatty acids from pupa oil consist mainly of unsaturated linolenic and linoleic acids and saturated palmitic and stearic acids. SFAAA had a low critical micelle concentration, great efficiency in lowering surface tension and strong adsorption at an air/water interface. SFAAA had a high emulsifying power, as well as a high foaming power. The emulsifying power of PPH and SFAAA in an oil/water emulsion was better with ethyl acetate as the oil phase compared to n-hexane. The environment-friendly surfactant made entirely from silkworm pupae could promote sustainable development of the silk industry. PMID:24651079
Transgene-based, female-specific lethality system for genetic sexing of the silkworm, Bombyx mori
Transgene-based genetic sexing methods are being developed for insects of agricultural and public health importance. Male-only rearing has long been sought in sericulture because males show superior economic characteristics, such as better fitness, lower food consumption, and higher silk yield. Here we report the establishment of a transgene-based genetic sexing system for the silkworm, Bombyx mori. We developed a construct in which a positive feedback loop regulated by sex-specific alternative splicing leads to high-level expression of the tetracycline-repressible transactivator in females only. Transgenic animals show female-specific lethality during embryonic and early larval stages, leading to male-only cocoons. This transgene-based female-specific lethal system not only has wide application in sericulture, but also has great potential in lepidopteran pest control.
Tan, Anjiang; Fu, Guoliang; Jin, Li; Guo, Qiuhong; Li, Zhiqian; Niu, Baolong; Meng, Zhiqi; Morrison, Neil I.; Alphey, Luke; Huang, Yongping
Melanin and urate act to prevent ultraviolet damage in the integument of the silkworm, Bombyx mori.
The phenomenon that epidermal cells under the white stripes rather than black stripes contain many uric acid granules was found in larvae of several Lepidopteran species. However, the biological mechanism of this phenomenon is still unknown. In the present study, we take advantage of several silkworm (Bombyx mori) body color mutant strains to investigate the deposition patterns and biological mechanism of urate and melanin in the integuments of these mutant larvae. By imaging with transmission electron microscope, we found that there were some melanin granules in the larval cuticle in black body color mutant plain Black (p(B) ), but not in background strain plain (p) with white larval body color. In contrast, the larval epidermal cell of background strain had much more urate granules than that of black one. Furthermore, the uric acid content under the black stripes was significantly lower than that under the white stripes in a single individual of mottled stripe (p(S) ) with black and white stripes in each segment. Ultraviolet A (UVA) exposure experiments showed that the distinct oily (od) mutant individuals with translucent larval integument were more sensitive to the UVA damage than black body color mutant and background strain without any pigmentation in the larval cuticle. This is likely due to the absence of melanin granules and few urate granules in the integument of od mutant. Thus, both the deposited melanin granules in the cuticle and the abundant urate granules in the epidermis cells constitute effective barriers for the silkworm to resist UVA-induced damage. PMID:23575996
Hu, Yong-Gang; Shen, Yi-Hong; Zhang, Ze; Shi, Gui-Qin
Shotgun proteomic analysis on the diapause and non-diapause eggs of domesticated silkworm Bombyx mori.
To clarify the molecular mechanisms of silkworm diapause, it is necessary to investigate the molecular basis at protein level. Here, the spectra of peptides digested from silkworm diapause and non-diapause eggs were obtained from liquid chromatography tandem mass spectrometry (LC-MS/MS) and were analyzed by bioinformatics methods. A total of 501 and 562 proteins were identified from the diapause and non-diapause eggs respectively, of which 309 proteins were shared commonly. Among these common-expressed proteins, three main storage proteins (vitellogenin precursor, egg-specific protein and low molecular lipoprotein 30 K precursor), nine heat shock proteins (HSP19.9, 20.1, 20.4, 20.8, 21.4, 23.7, 70, 90-kDa heat shock protein and heat shock cognate protein), 37 metabolic enzymes, 22 ribosomal proteins were identified. There were 192 and 253 unique proteins identified in the diapause and non-diapause eggs respectively, of which 24 and 48 had functional annotations, these unique proteins indicated that the metabolism, translation of the mRNA and synthesis of proteins were potentially more highly represented in the non-dipause eggs than that in the diapause eggs. The relative mRNA levels of four identified proteins in the two kinds of eggs were also compared using quantitative reverse transcription PCR (qRT-PCR) and showed some inconsistencies with protein expression. GO signatures of 486 out of the 502 and 545 out of the 562 proteins identified in the diapause and non-diapause eggs respectively were available. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed the Metabolism, Translation and Transcription pathway were potentially more active in the non-dipause eggs at this stage. PMID:23580252
Fan, Lanfen; Lin, Jianrong; Zhong, Yangsheng; Liu, Jingyi
Shotgun Proteomic Analysis on the Diapause and Non-Diapause Eggs of Domesticated Silkworm Bombyx mori
To clarify the molecular mechanisms of silkworm diapause, it is necessary to investigate the molecular basis at protein level. Here, the spectra of peptides digested from silkworm diapause and non-diapause eggs were obtained from liquid chromatography tandem mass spectrometry (LC-MS/MS) and were analyzed by bioinformatics methods. A total of 501 and 562 proteins were identified from the diapause and non-diapause eggs respectively, of which 309 proteins were shared commonly. Among these common-expressed proteins, three main storage proteins (vitellogenin precursor, egg-specific protein and low molecular lipoprotein 30 K precursor), nine heat shock proteins (HSP19.9, 20.1, 20.4, 20.8, 21.4, 23.7, 70, 90-kDa heat shock protein and heat shock cognate protein), 37 metabolic enzymes, 22 ribosomal proteins were identified. There were 192 and 253 unique proteins identified in the diapause and non-diapause eggs respectively, of which 24 and 48 had functional annotations, these unique proteins indicated that the metabolism, translation of the mRNA and synthesis of proteins were potentially more highly represented in the non-dipause eggs than that in the diapause eggs. The relative mRNA levels of four identified proteins in the two kinds of eggs were also compared using quantitative reverse transcription PCR (qRT-PCR) and showed some inconsistencies with protein expression. GO signatures of 486 out of the 502 and 545 out of the 562 proteins identified in the diapause and non-diapause eggs respectively were available. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed the Metabolism, Translation and Transcription pathway were potentially more active in the non-dipause eggs at this stage.
Transgenesis approaches for functional analysis of peptidergic cells in the silkworm Bombyx mori
The domestic silkworm, Bombyx mori represents an insect model of great scientific and economic importance. Besides the establishment of a stable germline transformation using the PiggyBac vector, technically feasible methods for in vivo gene delivery and transient gene expression were developed using viral based vectors, especially Sindbis viruses and baculoviruses. The recombinant baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), commonly used for large-scale protein production in permissive cell lines or insects, has been used for foreign gene transfer into specific peptidergic cells of B. mori in vivo. Since targeted gene expression is essential for functional analysis of neuropeptide genes and their receptors, the baculovirus-mediated gene transfer can serve as a reliable approach in reverse genetic studies in the silkworm. We review various strategies employing the baculovirus vector system for transient expression of molecular markers and transcription factors in specific peptidergic cells to investigate their roles in B. mori. We also use this system for functional analysis of neuropeptide signaling in the ecdysis behavioral sequence. Our data indicate that the AcMNPV vector is suitable for efficient delivery of foreign genes and their expression directed into specific peptidergic neurons and endocrine cells of B. mori larvae and pupae. However, some modifications of the vector and steps for optimization are necessary to minimize negative effects of viral infection on the host development. The transient gene expression using the AcMNPV and other virus vectors are promising tools for analysis of molecular mechanisms underlying various neuroendocrine processes during development of B. mori.
Daubnerova, Ivana; Roller, Ladislav; Zitnan, Dusan
Insect food for astronauts: gas exchange in silkworms fed on mulberry and lettuce and the nutritional value of these insects for human consumption during deep space flights.
In this study, silkworm moth (Bombyx mori L.) larvae were regarded as an animal protein source for astronauts in the bioregenerative life support system during long-term deep space exploration in the future. They were fed with mulberry and stem lettuce leaves during the first three instars and the last two instars, respectively. In addition, this kind of environmental approach, which utilised inedible biomass of plants to produce animal protein of high quality, can likewise be applied terrestrially to provide food for people living in extreme environments and/or impoverished agro-ecosystems, such as in polar regions, isolated military bases, ships, submarines, etc. Respiration characteristics of the larvae during development under two main physiological conditions, namely eating and not-eating of leaves, were studied. Nutrient compositions of silkworm powder (SP), ground and freeze-dried silkworms on the 3rd day of the 5th instar larvae, including protein, fat, vitamins, minerals and fatty acids, were measured using international standard methods. Silkworms' respiration rates, measured when larvae were eating mulberry leaves, were higher than those of similar larvae that hadn't eaten such leaves. There was a significant difference between silkworms fed on mulberry leaves and those fed on stem lettuce in the 4th and 5th instars (P<0.01). Amounts of CO2 exhaled by the silkworms under the two physiological regimes differed from each other (P<0.01). There was also a significant difference between the amount of O2 inhaled when the insects were under the two physiological statuses (P<0.01). Moreover, silkworms' respiration quotient under the eating regime was larger than when under the not-eating regime. The SP was found to be rich in protein and amino acids in total; 12 essential vitamins, nine minerals and twelve fatty acids were detected. Moreover, 359?kcal could be generated per 100?gram of SP (dry weight). PMID:21554801
Tong, L; Yu, X; Liu, H
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 184.108.40.206), 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
Teruhiko Iino; Hiroshi Sawada; Motoo Tsusu�; Shin-Ichiro Takikawa
Amino Acid deprivation-induced expression of asparagine synthetase regulates the growth and survival of cultured silkworm cells.
Expression of Bombyx mori Asparagine synthetase (BmASNS), one gene that encodes an enzyme catalyzing asparagine biosynthesis, is transcriptionally induced following amino acid deprivation. Previous transcriptional analysis of the BmASNS gene showed the involvement of Polycomb proteins, epigenetic repressors, in suppressing BmASNS expression in a cell cycle-dependent manner. However, the role of BmAsns protein in these cellular processes remains unclear. The present study thus exploited the potential function of BmAsns protein in cultured silkworm cells. Our results showed that ectopic overexpression of BmASNS gene effectively inhibited cell growth in silkworm cells, whereas its overexpression could rescue cell growth upon amino acid deprivation treatment. We found that the cells expressing BmAsns protein were capable of influencing the formation of autophagic vacuoles stimulated by amino acid deprivation. We speculated that the recovery of cell growth by overexpressed BmAsns protein is due to the rapid turnover of autophagic vacuoles in the cells. To further assess the effects of BmAsns on cell development, we used RNA interference to silence BmASNS expression in silkworm cells in the presence or absence of amino acids. Our results revealed a significant change of cell proliferation as well as cell cycle distribution after knockdown of BmASNS. Importantly, silkworm cells lacking BmASNS under the condition of amino acid deprivation showed severely impaired proliferation. Altogether, we concluded that the up-regulated expression of BmASNS would be able to protect cells from impairment induced by amino acid deprivation, which in turn facilitates cell growth and survival. PMID:23633098
Li, Zhiqing; Zhu, Li; Xu, Jian; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro
The Enzyme That Synthesizes Tetrahydrobiopterin from 6-Pyruvoyl-tetrahydropterin in the lemon Mutant Silkworm Consists of Two Carbonyl Reductases
Tetrahydrobiopterin plays an important role in the biosynthesis of certain neurotransmitters. Using DEAE�Sepharose FF column chromatography, we separated the enzyme that synthesizes tetrahydrobiopterin from 6-pyruvoyl-tetrahydropterin [which is different from sepiapterin reductase (EC 220.127.116.11)] in the lemon mutant of the silkworm Bombyx mori into two fractions, which were named carbonyl reductase I (CR I) and carbonyl reductase II (CR II). The
Teruhiko Iino; Shin-Ichiro Takikawa; Toshio Yamamoto; Hiroshi Sawada
Molecular cloning of ecdysone 20-hydroxylase and expression pattern of the enzyme during embryonic development of silkworm Bombyx mori
In various insects, 20-hydroxyecdysone (20E) is indispensable for embryonic development. In eggs of the silkworm Bombyx mori, 20E has been demonstrated to be produced by two metabolic pathways: de novo synthesis from cholesterol and dephosphorylation of ovary-derived physiologically inactive ecdysteroid phosphates. In the former, ecdysone 20-hydroxylase (E20OHase) has been suggested to be a key enzyme. In the latter, it has
Sayaka Maeda; Asuka Nakashima; Ryouichi Yamada; Noriyuki Hara; Yoshinori Fujimoto; Yoichi Ito; Haruyuki Sonobe
A comparative study of rac- and S-metolachlor on some activities and metabolism of silkworm, Bombyx mori L
The racemic product of metolachlor contains two R-enantiomers and two S-enantiomers. S-Metolachlor is a more effective herbicidal agent than rac-metolachlor; currently, it is widely used instead of rac-metolachlor. The comparative toxicity of rac- and S-metolachlor was assessed on the basis of their effects on some enzyme activities of the fifth instar of the silkworm, Bombyx mori L. These results suggest
Xiu-ming Zhan; Hui-jun Liu; Yun-gen Miao; Wei-ping Liu
The Bmdsx transgene including trimmed introns is sex-specifically spliced in tissues of the silkworm, Bombyx mori
Bmdsx is an orthologue of the sex-determining gene doublesex (dsx) and known to be sex-specifically expressed in various tissues of the silkworm, Bombyx mori. Its pre-mRNA is sex-specifically spliced and encodes female-specific or male-specific polypeptides. The open reading frame of Bmdsx consists of 5 exons, of which exons 3 and 4 are female-specific and its pre-mRNA was known to undergo
Shunsuke Funaguma; Masataka G. Suzuki; Toshiki Tamura; Toru Shimada
In vitro transcription systems from cultured cells and fat-body tissue of the silkworm, Bombyx mori
This article describes the development of cell-free transcription systems from the cultured cells and fat body tissues of\\u000a a Lepidpteran insect, the silkworm,Bombyx mori. Detailed protocols are provided for the culture of aB. mori cell line, rearing larvae, preparation of whole cell as well as nuclear extract and conditions for in vitro transcription\\u000a of cloned plasma protein gene templates.
Eriko Mine; Hiroshi Sakurai; Susumu Izumi; Shiro Tomino
Peptidoglycan recognition protein (PGRP) from eri-silkworm, Samia cynthia ricini; protein purification and induction of the gene expression
Peptidoglycan recognition protein (PGRP) was isolated from immunized hemolymph of the wild silkworm, Samia cynthia ricini, detecting the biding activity with 125I-labeled peptidoglycan (PGN). The binding specificity of PGRP was tested by competitive inhibition of the binding to 125I-labeled-PGN by a large excess amount of non-labeled-PGN or other glucans. The binding to labeled uncross-linked Lys-type PGN from Micrococcus luteus was
Hiroko Onoe; Akiyoshi Matsumoto; Kazuhiko Hashimoto; Yoshiaki Yamano; Isao Morishima
cDNA cloning and characterization of LASP1 from silkworm, Bombyx mori, involved in cytoplasmic polyhedrosis virus infection.
Full-length cDNA of a LIM and SH3 contained protein 1 (named BmLASP1) was identified from the silkworm, Bombyx mori, for the first time by rapid amplification of cDNA ends. The full-length cDNA of BmLASP1 is 2094 bp, consisting of a 5'-terminal untranslated region (UTR) of 117 bp, and a 3'-UTR of 610 bp with two poly-adenylation signal sequence AATAAA and a poly (A) tail. The BmLASP1 cDNA encodes a polypeptide comprising 455 amino acids, including a LIM domain, two nebulin domains and an SH3 domain. The theoretical isoelectric point is 7.07 and the predicted molecular weight is 51.8 kDa. BmLASP1 has no signal peptide but three potential N-glycosylation sites. Sequence similarity and phylogenic analyses indicated that BmLASP1 belonged to the group of insect LASP1 with a longer linker region which is different from vertebrate LASP1. The LASP1 in silkworm contained eight exons in its coding regions, and the last exon-intron boundary was conserved the same as in mammalian and Ciona intestinalis LASP1 genes. By fluorescent quantitative real-time polymerase chain reaction, the mRNA transcripts of BmLASP1 were mainly detected in the gonad, head, and spiracle, and slightly in the silk gland, vasa mucosa, midgut, fat body, and hemocytes. After silkworm larvae were infected by B. mori cytoplasmic polyhedrosis virus (BmCPV), the relative expression level of BmLASP1 was down-regulated in the midgut. This result suggested that BmLASP1 may play an important role in the response of silkworm to BmCPV infection. PMID:23031809
Gao, Kun; Deng, Xiang-yuan; Qian, He-ying; Wu, Ping; Qin, Guang-xing; Liu, Ting; Guo, Xi-jie
Bombyxin, an Insulin-Related Peptide of Insects, Reduces the Major Storage Carbohydrates in the Silkworm Bombyx mori
The effects of an insect insulin-related peptide, bombyxin, on carbohydrate metabolism were investigated in the silkworm Bombyx mori. Bombyxin lowered the concentration of the major hemolymph sugar, trehalose, in a dose-dependent manner when injected into neck-ligated larvae. Bombyxin also caused elevated trehalase activity in the midgut and muscle, suggesting that bombyxin induces hypotrehalosemia by promoting the hydrolysis of hemolymph trehalose
Shin'Ichiro Satake; Makoto Masumura; Hironori Ishizaki; Koji Nagata; Hiroshi Kataoka; Akinori Suzuki; Akira Mizoguchi
A Dense Genetic Map of the Silkworm, Bombyx mori, Covering All Chromosomes Based on 1018 Molecular Markers
A dense linkage map was constructed for the silkworm, Bombyx mori, containing 1018 genetic markers on all 27 autosomes and the Z chromosome. Most of the markers, covering z2000 cM, were randomly amplified polymorphic DNAs amplified with primer-pairs in combinations of 140 commercially available decanucleotides. In addition, eight known genes and five visible mutations were mapped. Bombyx homo- logues of
Yuji Yasukochi
Pheromone-triggered flip-flopping interneurons in the ventral nerve cord of the silkworm moth, Bombyx mori
1.A group of extracellularly recorded descending interneurons in the ventral nerve cord of the male silkworm mothBombyx mori share a common flip-flopping input. In response to repeated stimuli these flip-flopping interneurons switch back and forth between long lasting high and low firing rates (Figs. 1, 2).2.Changes in the level of the female pheromone bombykol in an airstream directed at the
Robert M. Olberg
Bmmarl: a basal lineage of the mariner family of transposable elements in the silkworm moth, Bombyx mori
We describe a transposable element, called Bmmarl, from the genome of the silkworm moth, Bombyx mori. This element has features of the Tcl-mariner superfamily of transposable elements. Bmmarl was first detected as a fragment in the 5? region of the larval serum protein (BmLSP) gene. Six genomic clones characterized each differed from a consensus sequence by 3�5 insertions and deletions,
Hugh M. Robertson; Michele L. Asplund
Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector
We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac
Toshiki Tamura; Chantal Thibert; Corinne Royer; Toshio Kanda; Abraham Eappen; Mari Kamba; Natuo K�moto; Jean-Luc Thomas; Bernard Mauchamp; G�rard Chavancy; Paul Shirk; Malcolm Fraser; Jean-Claude Prudhomme; Pierre Couble
Nanoparticle-induced morphological transition of Bombyx mori nucleopolyhedrovirus: a novel method to treat silkworm grasserie disease.
Grasserie, a polyorganotrophic disease caused by Bombyx mori nucleopolyhedrovirus (BmNPV), accounts for lethal infection to fifth instar silkworm larvae. It was found that nanoparticle (NP)-induced morphological transformation of BmNPV polyhedra could reduce the infectivity of BmNPV both in cell line and in silkworm larvae. Initially, 11 NPs were screened for evaluation of their nature of interaction with polyhedra surface through scanning electron microscopy. Amongst these NPs, lipophilically coated silica nanoparticle (SNPL), alumina nanoparticles in the hexagonal close-packed ? structure and aspartate capped gold nanoparticle transformed polyhedra were tested for their infectivity in B. mori cell line using cytopathic effect and plaque reduction assay. SNPL was evaluated for its bio-efficacy in fifth instar silkworm larvae. The study of polyhedra morphology as a function of NP concentration showed severe 'roughening' of the polyhedra with replacement of the regular facets by a large number of irregular ones by SNPL, and this caused transition of highly infectious polyhedra into a nearly spherical, non-infectious structure. A moderate polyhedra roughening was observed for alumina NPs, and no roughening was noticed for gold NPs. The morphological changes could be correlated with reduction of virus-induced cytopathic effect and plaque formation, and increased survival rate of SNPL transformed polyhedra infected silkworm larvae to 70.09�6.61% after 96 h. In this group, 61.04�8.03% larvae formed normal cocoons from which moths eclosed, laid eggs and larvae emerged. This study could lead to open up newer pathways for designing nano pharmaceuticals to combat other viral diseases. PMID:23588933
Das, Sumistha; Bhattacharya, Ankita; Debnath, Nitai; Datta, Alokmay; Goswami, Arunava
Construction of a Single Nucleotide Polymorphism Linkage Map for the Silkworm, Bombyx mori, Based on Bacterial Artificial Chromosome End Sequences
We have developed a linkage map for the silkworm Bombyx mori based on single nucleotide poly- morphisms (SNPs) between strains p50T and C108T initially found on regions corresponding to the end sequences of bacterial artificial chromosome (BAC) clones. Using 190 segregants from a backcross of a p50T female 3 an F1 (p50T 3 C108T) male, we analyzed segregation patterns of
Kimiko Yamamoto; Junko Narukawa; Keiko Kadono-Okuda; Junko Nohata; Motoe Sasanuma; Yoshitaka Suetsugu; Yutaka Banno; Hiroshi Fujii; Marian R. Goldsmith; Kazuei Mita
White oaks phylogeography in the Iberian Peninsula
The geographic distribution of maternally inherited chloroplast DNA polymorphisms was studied to determine the phylogeographic structure of white oaks in the Iberian Peninsula. Almost 1000 mature trees from nearly 200 populations were sampled in the distribution areas of the six white oak species that are recognised in Iberia. The analysed trees roughly represent the species distribution and their respective abundance.
Marta Olalde; Ana Herr�n; Santiago Espinel; Pablo G. Goicoechea
Occupational poison ivy and oak dermatitis.
Among the growing and diverse groups of outdoor and environmental workers, poison ivy and poison oak continue to be the major cause of occupational contact dermatitis. This article reviews the practical and theoretic means to prevent poison ivy and poison oak dermatitis in workers occupationally exposed to these weeds. PMID:7923948
Epstein, W L
Insects That Damage Northern Red Oaks Acorns.
Insect damage to northern red oak acorns is extensive throughout the range of northern red oak, and greatly reduces the number of acorns available to produce seedlings and feed wildlife. Five species of Curculio weevils and two species of gall wasps cause...
L. P. Gibson
Emergency preparedness at Oak Ridge National Laboratory, Oak Ridge, Tennessee
Emergency preparedness for industry was commonly believed to be an essential responsibility on the part of management. Therefore, this study was conducted to research and accumulate information and data on emergency preparedness at Oak Ridge National Laboratory (ORNL). The objective of this study was to conduct a thorough evaluation of emergency preparedness knowledge among employees to determine if they were properly informed or if they needed more training. Also, this study was conducted to provide insight to management as to what their responsibility was concerning this training. To assess employee emergency preparedness knowledge, a questionnaire was developed and administered to 100 employees at ORNL. The data was analyzed using frequencies and percentages of response and was displayed through the use of graphs within the report. 22 refs., 22 figs.
Skipper, M.N.
Utilization of silkworm litter and pupal waste-an eco-friendly approach for mass production of Bacillus thuringiensis.
The objective of the present study was to investigate the utilization of pupal waste and silkworm litter separately as production media for the mass cultivation of the potential biopesticide, Bacillus thuringiensis (Bt). Bt is the most successful commercial biopesticide accounting for 90% of all biopesticides sold all over the world. Biochemical analysis of the dry pupal waste revealed to be consisting of 4% carbohydrates, 44.9% proteins and 40% lipids. Similarly the biochemical composition of dry silkworm litter was found to be 4% carbohydrates, 57.5% proteins and 30.5% lipids. B. thuringiensis NCIM No. 2159 was mass cultivated in a semi-solid-state fermentation at a pH 7.0 and temperature 32�C. Changes in the pH and biochemical composition of the substrates were evaluated during the course of the fermentation. The reliability of the two substrates as production media was evaluated by determination of growth at regular intervals. Maximum growth was recorded at 96h incubation showing a spore count in the order of 3.5�10(10) and 3.0�10(10)CFU/g in pupal waste and silkworm litter respectively. PMID:23403062
Patil, Sarvamangala R; Amena, S; Vikas, A; Rahul, P; Jagadeesh, K; Praveen, K
Hydroforming Applications at Oak Ridge
Hydroforming technology is a robust forming process that produces components with high precision and complexity. The goal of this paper is to present a brief description of the sheet hydroforming process with respect to the authors' experience and capabilities. Following the authors' discussion of the sheet-metal forming application, the tubular hydroforming process is described in the context of one of our technology development programs with an automotive industrial partner. After that is a summary of the tubular hydroforming advisor (expert system) development activity, which was a significant part of this overall program based on previous experience in developing a design and manufacturing support hydroforming advisor for the Oak Ridge Y-12 Plant's weapons-component manufacturing needs. Therefore, this paper is divided into three sections: (1) Hydroforming of Stainless Steel Parts, (2) Tubular Hydroforming, and (3) Components of a Tubular Hydroforming Advisor.
bird, e.l.; ludtka, g.m.
Localization of Na(+)/K(+)-ATPase in silkworm brain: a possible mechanism for protection of Na(+)/K(+)-ATPase from Ca(2+).
In mammalian blood, the Na(+) concentration is higher than the K(+) concentration, whereas in hemolymph of lepidopterous insects, the K(+) concentration is higher than the Na(+) concentration. Na(+)/K(+)-ATPase regulates Na(+) and K(+) concentrations in mammalian blood. Therefore, the absence of Na(+)/K(+)-ATPase in lepidopterous insects might be expected. However, we have observed that Na(+)/K(+)-ATPase is abundant in nerve tissues of larvae of silkworm, a lepidopterous insect. Furthermore, we found that silkworm Na(+)/K(+)-ATPase was completely inhibited by 3 mM Ca(2+)in vitro (Homareda, 2010), although the Ca(2+) concentration is very high (30-50 mM) in the hemolymph of silkworm larvae. To investigate the reason why silkworm Na(+)/K(+)-ATPase is not inhibited by Ca(2+)in vivo, we observed the localization of Na(+)/K(+)-ATPase in nerve tissues using immunohistochemical techniques. Na(+)/K(+)-ATPase was distributed in the cortex and neuropile but not in the perineurium of the silkworm brain, while plasma membrane Ca(2+)-ATPase appeared to distribute in the perineurium as well as in the cortex and neuropile. These results support a possibility that neuronal Na(+)/K(+)-ATPase is protected from a high Ca(2+) concentration by the blood-brain barrier consisting of perineurial glial cells with plasma membrane Ca(2+)-ATPase. PMID:23274012
Homareda, Haruo; Otsu, Masahiro
Partial deletions of the W chromosome due to reciprocal translocation in the silkworm Bombyx mori.
In the silkworm, Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which chromosomal fragments containing autosomal markers have been translocated on to W. In this study, we analysed the W chromosomal regions of the Zebra-W strain (T(W;3)Ze chromosome) and the Black-egg-W strain (T(W;10)+(w-2) chromosome) at the molecular level. Initially, we undertook a project to identify W-specific RAPD markers, in addition to the three already established W-specific RAPD markers (W-Kabuki, W-Samurai and W-Kamikaze). Following the screening of 3648 arbitrary 10-mer primers, we obtained nine W-specific RAPD marker sequences (W-Bonsai, W-Mikan, W-Musashi, W-Rikishi, W-Sakura, W-Sasuke, W-Yukemuri-L, W-Yukemuri-S and BMC1-Kabuki), almost all of which contained the border regions of retrotransposons, namely portions of nested retrotransposons. We confirmed the presence of eleven out of twelve W-specific RAPD markers in the normal W chromosomes of twenty-five silkworm strains maintained in Japan. These results indicate that the W chromosomes of the strains in Japan are almost identical in type. The Zebra-W strain (T(W;3)Ze chromosome) lacked the W-Samurai and W-Mikan RAPD markers and the Black-egg-W strain (T(W;10)+(w-2) chromosome) lacked the W-Mikan RAPD marker. These results strongly indicate that the regions containing the W-Samurai and W-Mikan RAPD markers or the W-Mikan RAPD marker were deleted in the T(W;3)Ze and T(W;10)+(w-2) chromosomes, respectively, due to reciprocal translocation between the W chromosome and the autosome. This deletion apparently does not affect the expression of Fem; therefore, this deleted region of the W chromosome does not contain the putative Fem gene. PMID:16033428
Abe, H; Seki, M; Ohbayashi, F; Tanaka, N; Yamashita, J; Fujii, T; Yokoyama, T; Takahashi, M; Banno, Y; Sahara, K; Yoshido, A; Ihara, J; Yasukochi, Y; Mita, K; Ajimura, M; Suzuki, M G; Oshiki, T; Shimada, T
Evaluation of Propiconazole Operational Treatments of Oaks for Wilt Control.
Oaks commercially treated with propiconazole on 29 sites in Minnesota in 1998 were evaluated for efficacy in controlling oak wilt. Root graft spread occurred in 39 percent of preventively treated red oaks over 5 years; spread in white oaks occurred only o...
J. Eggers J. Juzwik S. Bernick L. Mordaunt
Small mammal response to oak savanna restoration in northwest Indiana
The first objective of this thesis was to determine if differences existed in the composition of the small mammal community in oak savannas relative to the community found in adjacent oak woodland. Specifically, from June to August 2009, I estimated and compared abundance, density, and micro-habitat affiliations of small mammals in two oak savanna and four oak woodland sites at
Valerie J Clarkston
Oak Ridge Uranium Market Model (for Microcomputers).
The Oak Ridge Uranium Market Model (ORUMM) projects prices, production, imports, inventory, capital expenditures, and employment in the uranium mining and milling industry. The model considers every major production center and utility on a worldwide basis...
Identifying new volatile compounds in toasted oak.
Toasting wood to be used in barrels for aging wine produces a great number of volatile and odiferous compounds. Three new volatile odorous compounds in toasted oak were identified. Analysis by high-performance gas chromatography of toasted oak extracts, combined with olfactory detection, enabled various chromatographic peaks with these specific aromas to be isolated. These same odors were simultaneously studied by heating glucose both with and without proline and phenylalanine. Aromatic compounds of interest were identified thanks to a combination of gas chromatography and both mass and infrared spectrometry. An analysis RMN was also used. Hydroxymaltol, 2,5-furanedicarbaldehyde, and furylhydroxymethyl ketone have been detected in extract of toasted oak wood. These molecules may be formed by direct pyrolysis of sugar or Maillard reactions. The acetylformoine was not detected in extract of toasted oak wood, whereas it was detected in heated extracts of various sugars and sugars mixtures with amino acids. PMID:10564035
Cutzach, I; Chatonnet, P; Henry, R; Dubourdieu, D
Field Use of NMIS at Oak Ridge
The Nuclear Materials Identification System (NMIS), developed by the Oak Ridge National Laboratory and Oak Ridge Y-12 Plant (Y-12), has been successfully used at Y-12 for nuclear material control and accountability (NMC&A). It is particularly useful in the high gamma-ray background of storage arrays and for shielded HEU. With three systems in use at Y-12, NMIS has enhanced the NMC&A
L. G. Chiang; M. Conger; S. S. Hughes; J. K. Mattingly; J. A. McEvers; J. T. Mihalczo; J. A. Mullens; R. B. Perez; C. R. Turner; T. Uckan; T. E. Valentine
Chloroplast DNA variation in European white oaks
A consortium of 16 laboratories have studied chloroplast DNA (cpDNA) variation in European white oaks. A common strategy for molecular screening, based on restriction analysis of four PCR-amplified cpDNA fragments, was used to allow comparison among the different laboratories. A total of 2613 oak populations (12,214 individual trees from eight species) were sampled from 37 countries, and analysed with the
R�my J. Petit; Ulrike M. Csaikl; S�ndor Bord�cs; Kornel Burg; Els Coart; Joan Cottrell; Barbara van Dam; John D. Deans; Sylvie Dumolin-Lap�gue; Silvia Fineschi; Reiner Finkeldey; Amanda Gillies; Izabela Glaz; Pablo G. Goicoechea; Jan S. Jensen; Armin O. K�nig; Andrew J. Lowe; S�ren F. Madsen; Gabor M�ty�s; Robert C. Munro; Maria Olalde; Marie-H�l�ne Pemonge; Flaviu Popescu; Danko Slade; Helen Tabbener; Daniela Taurchini; Sven G. M. de Vries; Birgit Ziegenhagen; Antoine Kremer
Oak Ridge rf Test Facility
The rf Test Facility (RFTF) of Oak Ridge National Laboratory (ORNL) provides a national facility for the testing and evaluation of steady-state, high-power (approx.1.0-MW) ion cyclotron resonance heating (ICRH) systems and components. The facility consists of a vacuum vessel and two fully tested superconducting development magnets from the ELMO Bumpy Torus Proof-of-Principle (EBT-P) program. These are arranged as a simple mirror with a mirror ratio of 4.8. The axial centerline distance between magnet throat centers is 112 cm. The vacuum vessel cavity has a large port (74 by 163 cm) and a test volume adequate for testing prototypic launchers for Doublet III-D (DIII-D), Tore Supra, and the Tokamak Fusion Test Reactor (TFTR). Attached to the internal vessel walls are water-cooled panels for removing the injected rf power. The magnets are capable of generating a steady-state field of approx.3 T on axis in the magnet throats. Steady-state plasmas are generated in the facility by cyclotron resonance breakdown using a dedicated 200-kW, 28-GHz gyrotron. Available rf sources cover a frequency range of 2 to 200 MHz at 1.5 kW and 3 to 18 MHz at 200 kW, with several sources at intermediate parameters. Available in July 1986 will be a >1.0-MW, cw source spanning 40 to 80 MHz. 5 figs.
Gardner, W.L.; Hoffman, D.J.; McCurdy, H.C.; McManamy, T.J.; Moeller, J.A.; Ryan, P.M.
The silkworm Z chromosome is enriched in testis-specific genes.
The role of sex chromosomes in sex determination has been well studied in diverse groups of organisms. However, the role of the genes on the sex chromosomes in conferring sexual dimorphism is still being experimentally evaluated. An unequal complement of sex chromosomes between two sexes makes them amenable to sex-specific evolutionary forces. Sex-linked genes preferentially expressed in one sex over the other offer a potential means of addressing the role of sex chromosomes in sexual dimorphism. We examined the testis transcriptome of the silkworm, Bombyx mori, which has a ZW chromosome constitution in the female and ZZ in the male, and show that the Z chromosome harbors a significantly higher number of genes expressed preferentially in testis compared to the autosomes. We hypothesize that sexual antagonism and absence of dosage compensation have possibly led to the accumulation of many male-specific genes on the Z chromosome. Further, our analysis of testis-specific paralogous genes suggests that the accumulation on the Z chromosome of genes advantageous to males has occurred primarily by translocation or tandem duplication. PMID:19332883
Arunkumar, K P; Mita, Kazuei; Nagaraju, J
Positional cloning of a gene responsible for the cts mutation of the silkworm, Bombyx mori.
The larval head cuticle and anal plates of the silkworm mutant cheek and tail spot (cts) have chocolate-colored spots, unlike the entirely white appearance of the wild-type (WT) strain. We report the identification and characterization of the gene responsible for the cts mutation. Positional cloning revealed a cts candidate on chromosome 16, designated BmMFS, based on the high similarity of the deduced amino acid sequence between the candidate gene from the WT strain and the major facilitator superfamily (MFS) protein. BmMFS likely encodes a membrane protein with 11 putative transmembrane domains, while the putative structure deduced from the cts-type allele possesses only 10-pass transmembrane domains owing to a deletion in its coding region. Quantitative RT-PCR analysis showed that BmMFS mRNA was strongly expressed in the integument of the head and tail, where the cts phenotype is observed; expression markedly increased at the molting and newly ecdysed stages. These results indicate that the novel BmMFS gene is cts and the membrane structure of its protein accounts for the cts phenotype. These expression profiles and the cts phenotype are quite similar to those of melanin-related genes, such as Bmyellow-e and Bm-iAANAT, suggesting that BmMFS is involved in the melanin synthesis pathway. PMID:22703239
Ito, Katsuhiko; Kidokoro, Kurako; Katsuma, Susumu; Shimada, Toru; Yamamoto, Kimiko; Mita, Kazuei; Kadono-Okuda, Keiko
Equilibrium and kinetic adsorption study of a cationic dye by a natural adsorbent--silkworm pupa.
In this work the use of silkworm pupa, which is the waste of silk spinning industries has been investigated as an adsorbent for the removal of C.I. Basic Blue 41. The amino acid nature of the pupa provided a reasonable capability for dye removal. Equilibrium adsorption isotherms and kinetics were investigated. The adsorption equilibrium data were analyzed by using various adsorption isotherm models and the results have shown that adsorption behavior of the dye could be described reasonably well by either Langmuir or Freundlich models. The characteristic parameters for each isotherm have been determined. The monolayer adsorption capacity was determined to be 555 mg/g. Kinetic studies indicated that the adsorption follows pseudo-second-order kinetics with a rate constant of 0.0434 and 0.0572 g/min mg for initial dye concentration of 200 mg/l at 20 and 40 degrees C, respectively. Kinetic studies showed that film diffusion and intra-particle diffusion were simultaneously operating during the adsorption process. The rate constant for intra-particle diffusion was estimated to be 1.985 mg/g min(0.5). PMID:16859827
Noroozi, B; Sorial, G A; Bahrami, H; Arami, M
Biosynthesis and cocoon-export of a recombinant globular protein in transgenic silkworms.
A gene construct was made by fusing the coding sequence of the red fluorescent protein (DsRed) to the exon 2 of the fibrohexamerin gene (fhx), that encodes a subunit of fibroin, the major silk protein of the silkworm Bombyx mori. The fusion gene was inserted into a piggyBac vector to establish a series of transgenic lines. The expression of the transgene was monitored during the course of larval life and was found restricted to the posterior silk gland cells as the endogenous fhx gene, in all the selected transgenic lines. The exogenous polypeptide was secreted into the lumen of the posterior silk gland together with fibroin, and further exported with the silk proteins as a foreign constituent of the cocoon fiber. The capacity of DsRed to emit fluorescence in the air-dried silk thread led to show that the recombinant protein was distributed over the whole length of the fiber. A remarkable property of the system lies in the localization of the globular protein at the periphery of the silk thread, allowing its rapid and easy recovery in aqueous solutions, without dissolving fibroin. The procedure represents a novel and promising strategy for the production of massive recombinant proteins of biomedical and pharmaceutical interest, with reduced cost. PMID:16201412
Royer, Corinne; Jalabert, Audrey; Da Rocha, Martine; Grenier, Anne-Marie; Mauchamp, Bernard; Couble, Pierre; Chavancy, G�rard
Induction of dauer pupae by fenoxycarb in the silkworm, Bombyx mori.
Topical application of fenoxycarb (1 &mgr;g per animal) at 129 or 132 h of the fifth instar larvae of the silkworm, Bombyx mori, did not induce morphological abnormalities in the pupal stage, but these animals became dauer (permanent) pupae. This condition of B. mori and the endocrine events leading to permanent pupae are discussed in this work. Application of fenoxycarb at 132 h of the fifth instar elicited a high ecdysteroid titre in the pharate pupal stage and a steadily high ecdysteroid titre in the pupal stage. The fenoxycarb-induced permanent pupae had non-degenerating prothoracic glands that secreted low amounts of ecdysteroid and did not respond to recombinant prothoracicotropic hormone (rPTTH) late in the pupal stage. The Bombyx PTTH titre in the haemolymph, determined by a time-resolved fluoroimmunoassay, was lower than that of controls at the time of pupal ecdysis, but higher than controls later in the pupal stage in fenoxycarb-treated animals. After application of fenoxycarb, its haemolymph level, measured by ELISA, reached a peak at pupal ecdysis, then remained low. These results suggest that the fenoxycarb-mediated induction of permanent pupae is only partially a brain-centred phenomenon. It also involves alterations in the hormonal interplay that govern both the initiation of pupal-adult differentiation and changes in the steroidogenic pathway of the prothoracic glands of B. mori. PMID:12770048
Dedos, S G.; Szurdoki, F; Sz�k�cs, A; Mizoguchi, A; Fugo, H
Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori
Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5?-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD+, thereby indicating ethanol as one of the substrates of BmADH.
Wang, Nan; Shi, Haifeng; Yao, Qin; Zhou, Yang; Kang, Lequn; Chen, Huiqin; Chen, Keping
Transgenic protein production in silkworm silk glands requires cathepsin and chitinase of Autographa californica multicapsid nucleopolyhedrovirus.
The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production. PMID:24477386
W�ltje, Michael; B�bel, Melanie; Rheinnecker, Michael; Tettamanti, Gianluca; Franzetti, Eleonora; Saviane, Alessio; Cappellozza, Silvia
Metabolic allometry during development and metamorphosis of the silkworm Bombyx mori: analyses, patterns, and mechanisms.
Intraspecific allometric (scaling) relationships for metabolism, which have received little examination compared to interspecific relationships, reflect a complex interplay of organogenesis, growth, and shifting physiologies. In this study of the silkworm Bombyx mori, we hypothesized that allometric relationships for metabolism both across all developmental stages and within each stage would not reflect conventional scaling coefficients (e.g., b not equal to 0.75). Histology, gross morphology, body surface and cross-sectional area, total lipid content, and cytochrome c oxidase activity levels (as evidence of the total metabolic potential of mitochondria) were determined across development. Also measured were oxygen consumption, carbon dioxide production, and the respiratory exchange ratio. The overall slope, b, in the allometric equation relating to body mass across all developmental stages was 0.82, not greatly different from the value of 0.75 typical of interspecific data. However, within larval instars II-V and in prepupae, b varied between 0.99 and 1.49, far higher than hypothesized. Thus, in B. mori, an analytical approach that lumps all developmental stages hides interinstar variability. Morphological and biochemical data suggest that observed scaling patterns in B. mori are likely correlated with changes in overall mitochondrial density rather than with specific changes in body proportion of tissues with higher intrinsic metabolic intensity. PMID:20105069
Blossman-Myer, Bonnie L; Burggren, Warren W
Wound healing properties of a 3-D scaffold comprising soluble silkworm gland hydrolysate and human collagen.
Biomaterials that serve as scaffolds for cell proliferation and differentiation are increasingly being used in wound repair. In this study, the potential regenerative properties of a 3-D scaffold containing soluble silkworm gland hydrolysate (SSGH) and human collagen were evaluated. The scaffold was generated by solid-liquid phase separation and a freeze-drying method using a homogeneous aqueous solution. The porosity, swelling behavior, protein release, cytotoxicity, and antioxidative properties of scaffolds containing various ratios of SSGH and collagen were evaluated. SSGH/collagen scaffolds had a high porosity of 61-81% and swelling behavior studies demonstrated a 50-75% increase in swelling, along with complete protein release in the presence of phosphate-buffered saline. Cytocompatibility of the SSGH/collagen scaffold was demonstrated using mesenchymal stem cells from human umbilical cord. Furthermore, SSGH/collagen efficiently attenuated oxidative stress-induced cell damage. In an in vivo mouse model of wound healing, the SSGH/collagen scaffold accelerated wound re-epithelialization over a 15-day period. Overall, the microporous SSGH/collagen 3-D scaffold maintained optimal hydration of the exposed tissues and decreased wound healing time. These results contribute to the generation of advanced wound healing materials and may have future therapeutic implications. PMID:24503353
Kim, Kyu-Oh; Lee, Youngjun; Hwang, Jung-Wook; Kim, Hojin; Kim, Sun Mi; Chang, Sung Woon; Lee, Heui Sam; Choi, Yong-Soo
Expression pattern of enzymes related to juvenile hormone metabolism in the silkworm, Bombyx mori L.
The physiological balance of juvenile hormone (JH) in insects depends on its biosynthesis and degradation pathway. Three key enzymes namely, juvenile hormone esterase (JHE), juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol kinase (JHDK) are required for degradation in insects. Our present results showed that JHE and JHEH exhibited expression in almost all the tissues. This indicated that JHE and JHEH might degrade JH simultaneously. In addition, the highest levels of JHDK were observed in the midgut, with trace level being found in the malpighian tubule and haemocytes. Since the midgut is a digestive organ and not a JH target, it was hypothesized that both JHE and JHEH hydrolyzed JH to JH diol (JHd) which was then transported to midgut and hydrolyzed further by JHDK, to be finally excreted�out of the body. Also the expression studies on JH degradation enzymes in different tissues and stages indicated that the activities of the three enzymes are specific and coincident with the JH functions in silkworm, Bombyx mori L. PMID:21107706
Hua-Jun, Yang; Fang, Zhou; Awquib, Sabhat; Malik, Firdose Ahmad; Roy, Bhaskar; Xing-Hua, Li; Jia-Biao, Hu; Chun-Guang, Sun; Niu, Yan-Shan; Yun-Gen, Miao
Programmed cell death: cytochemical and X-ray microanalytical characterization of calcium compartments in neuromuscular junctions during the normal breakdown of the intersegmental muscles in the giant silkmoth Antheraea polyphemus.
Calcium stores were cytochemically demonstrated using a combined oxalate-pyroantimonate method in the neuromuscular junctions of the degenerating intersegmental muscles in the giant silkmoth Antheraea polyphemus. The elemental composition of punctate precipitates of the reaction product was determined by electron probe X-ray microanalysis of unstained thin sections by energy-dispersive spectrometry and wavelength-dispersive spectrometry. The wavelength-dispersive spectra collected over terminal axons demonstrate a significant calcium signal and a trace of antimony. During the rapid lytic phase of spontaneous muscle degeneration, the calcium punctate deposits were detected in presynaptic terminals in the following sites: the synaptic vesicles and the mitochondria. Calcium precipitates were also found in the dense bodies and the mitochondria encountered in the glial convolutions. No calcium deposit was seen in the synaptic clefts and intercellular spaces of the subsynaptic reticulum of type I and type II. A comparison of calcium to antimony ratios between the terminal axons and the sarcoplasmic lysosomes revealed highly significant differences (P less than 0.001). Such a variability of the calcium to antimony ratio may be related to different conditions of precipitation or antimony diffusion in the different cell compartments. It was concluded that such synaptic terminals do not appear damaged in spite of the muscle degeneration and presumably continue to perform vital functions while the muscles are no longer contractile 20 h after adult ecdysis. PMID:3410737
Beaulaton, J
Molecular cloning and functional characterization of the dual oxidase (BmDuox) gene from the silkworm Bombyx mori.
Reactive oxygen species (ROS) from nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and their related dual oxidases are known to have significant roles in innate immunity and cell proliferation. In this study, the 5,545 bp cDNA of the silkworm Bombyx mori dual oxidase (BmDuox) gene containing a full-length open reading frame was cloned. It was shown to include an N-terminal signal peptide consisting of 28 amino acid residues, a 240 bp 5'-terminal untranslated region (5'-UTR), an 802 bp 3'-terminal region (3'-UTR), which contains nine ATTTA motifs, and a 4,503 bp open reading frame encoding a polypeptide of 1,500 amino acid residues. Structural analysis indicated that BmDuox contains a typical peroxidase domain at the N-terminus followed by a calcium-binding domain, a ferric-reducing domain, six transmembrane regions and binding domains for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD). Transcriptional analysis revealed that BmDuox mRNA was expressed more highly in the head, testis and trachea compared to the midgut, hemocyte, Malpighian tube, ovary, fat bodies and silk glands. BmDuox mRNA was expressed during all the developmental stages of the silkworm. Subcellular localization revealed that BmDoux was present mainly in the periphery of the cells. Some cytoplasmic staining was detected, with rare signals in the nucleus. Expression of BmDuox was induced significantly in the larval midgut upon challenge by Escherichia coli and Bombyx mori nucleopolyhedrovirus (BmNPV). BmDuox-deleted larvae showed a marked increase in microbial proliferation in the midgut after ingestion of fluorescence-labeled bacteria compared to the control. We conclude that reducing BmDuox expression greatly increased the bacterial load, suggesting BmDuox has an important role in inhibiting microbial proliferation and the maintenance of homeostasis in the silkworm midgut. PMID:23936382
Hu, Xiaolong; Yang, Rui; Zhang, Xing; Chen, Lin; Xiang, Xingwei; Gong, Chengliang; Wu, Xiaofeng
Molecular Cloning and Functional Characterization of the Dual Oxidase (BmDuox) Gene from the Silkworm Bombyx mori
Reactive oxygen species (ROS) from nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and their related dual oxidases are known to have significant roles in innate immunity and cell proliferation. In this study, the 5,545 bp cDNA of the silkworm Bombyx mori dual oxidase (BmDuox) gene containing a full-length open reading frame was cloned. It was shown to include an N-terminal signal peptide consisting of 28 amino acid residues, a 240 bp 5?-terminal untranslated region (5?-UTR), an 802 bp 3?-terminal region (3?-UTR), which contains nine ATTTA motifs, and a 4,503 bp open reading frame encoding a polypeptide of 1,500 amino acid residues. Structural analysis indicated that BmDuox contains a typical peroxidase domain at the N-terminus followed by a calcium-binding domain, a ferric-reducing domain, six transmembrane regions and binding domains for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD). Transcriptional analysis revealed that BmDuox mRNA was expressed more highly in the head, testis and trachea compared to the midgut, hemocyte, Malpighian tube, ovary, fat bodies and silk glands. BmDuox mRNA was expressed during all the developmental stages of the silkworm. Subcellular localization revealed that BmDoux was present mainly in the periphery of the cells. Some cytoplasmic staining was detected, with rare signals in the nucleus. Expression of BmDuox was induced significantly in the larval midgut upon challenge by Escherichia coli and Bombyx mori nucleopolyhedrovirus (BmNPV). BmDuox-deleted larvae showed a marked increase in microbial proliferation in the midgut after ingestion of fluorescence-labeled bacteria compared to the control. We conclude that reducing BmDuox expression greatly increased the bacterial load, suggesting BmDuox has an important role in inhibiting microbial proliferation and the maintenance of homeostasis in the silkworm midgut.
Production of classical swine fever virus envelope glycoprotein E2 as recombinant polyhedra in baculovirus-infected silkworm larvae.
Although, classical swine fever virus (CSFV) envelope glycoprotein E2 subunit vaccine has been developed using the baculovirus expression system, the expression of viral antigens in baculovirus-infected insect cells is often ineffective. Therefore, an alternative strategy to the traditional baculovirus expression system is needed that is more productive and effective. Here, we report a novel strategy for the large-scale production of a CSFV E2 in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (E2?C). BmNPV-E2?C-infected silkworm larvae expressed native polyhedrin and approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin were morphologically normal and contained CSFV E2?C. The CSFV E2?C antigen produced in BmNPV-E2?C-infected silkworm larvae reached 0.68�mg/ml of hemolymph and 0.53�mg/larva at 6-days post-infection. Six-week-old female BALB/c mice that were immunized with the E2?C protein purified from solubilized recombinant polyhedra elicited CSFV E2 antibodies, which indicated that the CSFV E2?C protein from recombinant polyhedra was immunogenic. The virus neutralization test showed that the serum from mice that were treated with E2?C protein from recombinant polyhedra contained significant levels of virus neutralization activity. These results demonstrate that this strategy can be used for the large-scale production of CSFV E2 antigen. PMID:21706129
Lee, Kwang Sik; Sohn, Mi Ri; Kim, Bo Yeon; Choo, Young Moo; Woo, Soo Dong; Yoo, Sung Sik; Je, Yeon Ho; Choi, Jae Young; Roh, Jong Yul; Koo, Hyun Na; Jin, Byung Rae
Structure-Function Relationship of SW-AT-1, a Serpin-Type Protease Inhibitor in Silkworm
Although SW-AT-1, a serpin-type trypsin inhibitor from silkworm (Bombyx mori), was identified in previous study, its structure-function relationship has not been studied. In this study, SW-AT-1 was cloned from the body wall of silkworm and expressed in E. coli. rSW-AT-1 inhibited both trypsin and chymotrypsin in a concentration-dependent manner. The association rate constant for rSW-AT-1 and trypsin is 1.31�10?5 M?1s?1, for rSW-AT-1 and chymotrpsin is 2.85�10?6 M?1s?1. Circular dichroism (CD) assay showed 33% ?-helices, 16% ?-sheets, 17% turns, and 31% random coils in the secondary structure of the protein. Enzymatic and CD analysis indicated that rSW-AT-1 was stable at wide pH range between 4�10, and exhibited the highest activity at weakly acidic or alkaline condition. The predicted three-dimensional structure of SW-AT-1 by PyMOL (v1.4) revealed a deductive reactive centre loop (RCL) near the C-terminus, which was extended from the body of the molecule. In addition to trypsin cleavage site in RCL, matrix-assisted laser desorption ionization time of flight mass spectrometry indicated that the chymotrypsin cleavage site of SW-AT-1 was between F336 and T337 in RCL. Directed mutagenesis indicated that both the N- and C-terminal sides of RCL have effects on the activity, and G327 and E329 played an important role in the proper folding of RCL. The physiological role of SW-AT-1 in the defense responses of silkworm were also discussed.
Liu, Cheng; Han, Yue; Chen, Xi; Zhang, Wei
A fluorescent single domain antibody against plumbagin expressed in silkworm larvae for fluorescence-linked immunosorbent assay (FLISA).
A fluorescent single-domain antibody (fluobody), a chimera of a green fluorescent protein (AcGFP) with a single chain variable fragment antibody (scFv), against plumbagin (5-hydorxy-2-methyl-1,4-naphthoquinone; PL) was successfully expressed in the hemolymph of silkworm larvae using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system to develop a rapid, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA). In this study, two kinds of fluobody, in which the PL-scFv was fused at the N-terminus (N-fluobody) or C-terminus of AcGFP (C-fluobody), were expressed in silkworm larvae for comparative purposes. Interestingly, both fluobodies expressed in the BmNPV bacmid DNA system retained both of their original functions as an AcGFP and a PL-scFv, although the functions of the N-fluobody were found to be inferior to those of C-fluobody when they were expressed in Escherichia coli. Moreover, an improvement in the limit of quantification for PL measurement was observed in FLISA (24 ng mL(-1)) compared with conventional ELISA (0.2 �g mL(-1)). Since both the C-fluobody and N-fluobody are useful probes for FLISA and the time-, cost-consuming refolding step required in the conventional bacterial expression system can be avoided when they are expressed in the BmNPV bacmid DNA system, the silkworm expression system is useful for expressing fluobodies when developing FLISA. PMID:21442099
Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Sasaki-Tabata, Kaori; Putalun, Waraporn; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi
DNA fingerprinting using AFLP markers to search for markers associated with yield attributes in the silkworm, Bombyx mori
This study was carried out on 11 Chinese and 12 Japanese silkworm strains maintained by the Center for the Technological Development of Sericulture (CDTS) germplasm bank, located in Pereira, Colombia. The goals were to determine the genetic population structure of the two groups and the association between molecular markers (AFLPs) and important productivity characters. Group analysis showed the separation of the strains according to their geographic origin. The molecular markers and the productivity characters were correlated by multiple variance analysis. The analysis permitted the identification of molecular markers associated with the cocoon weight or the shell weight separately. Some markers were associated with both characters.
Gaviria, Duverney A.; Aguilar, Enrique; Serrano, Herman J.; Alegria, Alvaro H.
Construction of new ligation-independent cloning vectors for the expression and purification of recombinant proteins in silkworms using BmNPV bacmid system.
A ligation independent cloning (LIC) system has been developed to facilitate the rapid and high-efficiency cloning of genes in a Bombyx mori expression system. This system was confirmed by the expression of human microsomal triglyceride transfer protein (hMTP) fused with EGFP in silkworm larvae and pupae. Moreover, hMTP and human protein disulfide isomerase (hPDI) genes were inserted into two LIC vectors harboring gcLINK sequences and were combined by using the LIC through gcLINK sequences. The constructed vector was incorporated into the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid, and injected into silkworm larvae. The expressed hMTP-hPDI complex was purified from the fat bodies of silkworm larvae. This LIC vector system was applied to express the E1, E2, and E3 subunits of human ?-ketoglutarate dehydrogenase (KGDH) in silkworm larvae. The expressed proteins were purified easily from fat bodies using three different affinity chromatography steps. The LIC vectors constructed as described in this report allow for the rapid expression and purification of recombinant proteins or their complexes by using the BmNPV bacmid system. PMID:23675518
Kato, Tatsuya; Thompson, James R; Park, Enoch Y
Enhancement of Protein Metabolism and Enzyme Activities in Haemolymph of Silkworm, Bombyx mori L. By Ultrasound Treatment During Fifth Instar Development
4 Abstract: Present study explains about the enhancement of protein metabolism and enzyme activities in haemolymph of the silkworm, Bombyx mori L during the fifth instar larva stage. The parameters of protein metabolism, such as the levels of soluble, total proteins and free amino acids and the activity levels of protease, glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase were assayed
P. Nagajyothi; K. Nagalakshmamma; Y. Suneetha; Phaninatha A. Sarma
Effects of added CeCl3 on resistance of fifth-instar larvae of silkworm to Bombyx mori nucleopolyhedrovirus infection.
One of the most important agents causing lethal disease in the silkworm is the Bombyx mori nucleopolyhedrovirus (BmNPV), while low-dose rare earths are demonstrated to increase immune capacity in animals. However, very little is known about the effects of added CeCl(3) on decreasing BmNPV infection of silkworm. The present study investigated the effects of added CeCl(3) to an artificial diet on resistance of fifth-instar larvae of silkworm to BmNPV infection. Our findings indicated that added CeCl(3) significantly decreased inhibition of growth and mortality of fifth-instar larvae caused by BmNPV infection. Furthermore, the added CeCl(3) obviously decreased lipid peroxidation level and accumulation of reactive oxygen species such as O(2)(-), H(2)O(2), (�)OH, and NO and increased activities of the antioxidant enzymes including superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase, ascorbate, and glutathione contents in the BmNPV-infected fifth-instar larvae. In addition, the added CeCl(3) could significantly promote acetylcholine esterase activity and attenuate the activity of inducible nitric oxide synthase in the BmNPV-infected fifth-instar larvae. These findings suggested that added CeCl(3) may relieve oxidative damage and neurotoxicity of silkworm caused by BmNPV infection via increasing antioxidant capacity and acetylcholine esterase activity. PMID:22076733
Li, Bing; Xie, Yi; Cheng, Zhe; Cheng, Jie; Hu, Rengping; Cui, Yaling; Gong, Xiaolan; Shen, Weide; Hong, Fashui
Electron Transport and Bulk-like Behavior of Wiedemann-Franz Law for Sub-7 nm-Thin Iridium Films on Silkworm Silk.
For ultrathin metallic films, either supported or free-standing, the inside nanocrystalline nature significantly reduces the electron and thermal transport. Quantum mechanical reflection of electrons at the grain boundary reduces the electrical conductivity further than the thermal conductivity, leading to a Lorenz number in the order of 7.0 � 10(-8) W ? K(-2), much higher than that of the bulk counterpart. We report on a finding that for ultrathin (0.6-6.3 nm) iridium films coated on degummed silkworm silk fibroin, the electron transport is around 100-200% higher than that of the same film on glass fiber, even though the grain size of Ir film on silkworm silk is smaller than that on glass fiber. At the same time, the thermal conductivity of the Ir film is smaller or close to that of the film on glass fiber. Its Lorenz number is found close to that of bulk crystalline Ir despite the nanocrystalline structure in the Ir films. This is similar to the behavior of metallic glasses. Our study of gold films on silkworm silk reveals the same trend of change as compared to that on glass fiber. Electron hopping and tunneling in silkworm silk is speculated to be responsible for the observed electron transport. The finding points out that silk could provide a better substrate for flexible electronics with significantly faster electron transport. PMID:24988039
Lin, Huan; Xu, Shen; Zhang, Yu-Qing; Wang, Xinwei
Protective effect of the silkworm protein 30Kc6 on human vascular endothelial cells damaged by oxidized low density lipoprotein (Ox-LDL).
Although the 30K family proteins are important anti-apoptotic molecules in silkworm hemolymph, the underlying mechanism remains to be investigated. This is especially the case in human vascular endothelial cells (HUVECs). In this study, a 30K protein, 30Kc6, was successfully expressed and purified using the Bac-to-Bac baculovirus expression system in silkworm cells. Furthermore, the 30Kc6 expressed in Escherichia coli was used to generate a polyclonal antibody. Western blot analysis revealed that the antibody could react specifically with the purified 30Kc6 expressed in silkworm cells. The In vitro cell apoptosis model of HUVEC that was induced by oxidized low density lipoprotein (Ox-LDL) and in vivo atherosclerosis rabbit model were constructed and were employed to analyze the protective effects of the silkworm protein 30Kc6 on these models. The results demonstrated that the silkworm protein 30Kc6 significantly enhanced the cell viability in HUVEC cells treated with Ox-LDL, decreased the degree of DNA fragmentation and markedly reduced the level of 8-isoprostane. This could be indicative of the silkworm protein 30Kc6 antagonizing the Ox-LDL-induced cell apoptosis by inhibiting the intracellular reactive oxygen species (ROS) generation. Furthermore, Ox-LDL activated the cell mitogen activated protein kinases (MAPK), especially JNK and p38. As demonstrated with Western analysis, 30Kc6 inhibited Ox-LDL-induced cell apoptosis in HUVEC cells by preventing the MAPK signaling pathways. In vivo data have demonstrated that oral feeding of the silkworm protein 30Kc6 dramatically improved the conditions of the atherosclerotic rabbits by decreasing serum levels of total triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC). Furthermore, 30Kc6 alleviated the extent of lesions in aorta and liver in the atherosclerotic rabbits. These data are not only helpful in understanding the anti-apoptotic mechanism of the 30K family proteins, but also provide important information on prevention and treatment of human cardiovascular diseases. PMID:23840859
Yu, Wei; Ying, Huihui; Tong, Fudan; Zhang, Chen; Quan, Yanping; Zhang, Yaozhou
Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori
Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD- dependent reaction. Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation.
Ciufo, Leonora F.; Murray, Patricia A.; Thompson, Anu; Rigden, Daniel J.; Rees, Huw H.
Precocious Metamorphosis in the Juvenile Hormone-Deficient Mutant of the Silkworm, Bombyx mori
Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several �moltinism� mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval�larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval�pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH�deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis.
Daimon, Takaaki; Kozaki, Toshinori; Niwa, Ryusuke; Kobayashi, Isao; Furuta, Kenjiro; Namiki, Toshiki; Uchino, Keiro; Banno, Yutaka; Katsuma, Susumu; Tamura, Toshiki; Mita, Kazuei; Sezutsu, Hideki; Nakayama, Masayoshi; Itoyama, Kyo; Shimada, Toru; Shinoda, Tetsuro
Retrotransposable elements on the W chromosome of the silkworm, Bombyx mori.
The sex chromosomes of the silkworm, Bombyxmori, are designated ZW(XY) for females and ZZ(XX) for males. The W chromosome of B. mori does not recombine with the Z chromosome and autosomes and no genes for morphological characters have been mapped to the W chromosome as yet. Furthermore, femaleness is determined by the presence of a single W chromosome, regardless of the number of autosomes or Z chromosomes. To understand these interesting features of the W chromosome, it is necessary to analyze the W chromosome at the molecular biology level. Initially to isolate DNA sequences specific for the W chromosome as randomly amplified polymorphic DNA (RAPD) markers, we compared the genomic DNAs between males and females by PCR with arbitrary 10-mer primers. To the present, we have identified 12 W-specific RAPD markers, and with the exception of one RAPD marker, all of the deduced amino acid sequences of these W-specific RAPD markers show similarity to previously reported amino acid sequences of retrotransposable elements from various organisms. After constructing a genomic DNA lambda phage library of B. mori we obtained two lambda phage clones, one containing the W-Kabuki RAPD sequence and one containing the W-Samurai RAPD sequence and found that these DNA sequences comprised nested structures of many retrotransposable elements. To further analyze the W chromosome, we obtained 14 W-specific bacterial artificial chromosome (BAC) clones from three BAC libraries and subjected these clones to shotgun sequencing. The resulting assembly of sequences did not produce a single contiguous sequence due to the presence of many retrotransposable elements. Therefore, we coupled PCR with shotgun sequencing. Through these analyses, we found that many long terminal repeat (LTR) and non-LTR retrotransposons, retroposons, DNA transposons and their derivatives, have accumulated on the W chromosome as strata. These results strongly indicate that retrotransposable elements are the main structural component of the W chromosome. PMID:16093666
Abe, H; Mita, K; Yasukochi, Y; Oshiki, T; Shimada, T
Gloverins of the silkworm Bombyx mori: Structural and binding properties and activities
Gloverins are basic, glycine-rich and heat-stable antibacterial proteins (~14-kDa) in lepidopteran insects with activity against Escherichia coli, Gram-positive bacteria, fungi and a virus. Hyalophora gloveri gloverin adopts a random coil structure in aqueous solution but has ?-helical structure in membrane-like environment, and it may interact with the lipid A moiety of lipopolysaccharide (LPS). Manduca sexta gloverin binds to the O-specific antigen and outer core carbohydrate of LPS. In the silkworm Bombyx mori, there are four gloverins with slightly acidic to neutral isoelectric points. In this study, we investigate structural and binding properties and activities of B. mori gloverins (BmGlvs), as well as correlations between structure, binding property and activity. Recombinant BmGlv1�4 were expressed in bacteria and purified. Circular dichroism (CD) spectra showed that all four BmGlvs mainly adopted random coli structure (>50%) in aqueous solution in regardless of pH, but contained ?-helical structure in the presence of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), smooth and rough mutants (Ra, Rc and Re) of LPS and lipid A. Plate ELISA assay showed that BmGlvs at pH 5.0 bound to rough mutants of LPS and lipid A but not to smooth LPS. Antibacterial activity assay showed that positively charged BmGlvs (at pH 5.0) were active against E. coli mutant strains containing rough LPS but inactive against E. coli with smooth LPS. Our results suggest that binding to rough LPS is the prerequisite for the activity of BmGlvs against E. coli.
Yi, Hui-Yu; Deng, Xiao-Juan; Yang, Wan-Ying; Zhou, Cong-Zhao; Cao, Yang; Yu, Xiao-Qiang
Effect of degumming time on silkworm silk fibre for biodegradable polymer composites
Recently, many studies have been conducted on exploitation of natural materials for modern product development and bioengineering applications. Apart from plant-based materials (such as sisal, hemp, jute, bamboo and palm fibre), animal-based fibre is a kind of sustainable natural materials for making novel composites. Silkworm silk fibre extracted from cocoon has been well recognized as a promising material for bio-medical engineering applications because of its superior mechanical and bioresorbable properties. However, when producing silk fibre reinforced biodegradable/bioresorbable polymer composites, hydrophilic sericin has been found to cause poor interfacial bonding with most polymers and thus, it results in affecting the resultant properties of the composites. Besides, sericin layers on fibroin surface may also cause an adverse effect towards biocompatibility and hypersensitivity to silk for implant applications. Therefore, a proper pre-treatment should be done for sericin removal. Degumming is a surface modification process which allows a wide control of the silk fibre's properties, making the silk fibre possible to be used for the development and production of novel bio-composites with unique/specific mechanical and biodegradable properties. In this paper, a cleaner and environmentally friendly surface modification technique for tussah silk in polymer based composites is proposed. The effectiveness of different degumming parameters including degumming time and temperature on tussah silk is discussed through the analyses of their mechanical and morphological properties. Based on results obtained, it was found that the mechanical properties of tussah silk are affected by the degumming time due to the change of the fibre structure and fibroin alignment.
Ho, Mei-po; Wang, Hao; Lau, Kin-tak
Translationally controlled tumor protein, a dual functional protein involved in the immune response of the silkworm, Bombyx mori.
Insect gut immunity is the first line of defense against oral infection. Although a few immune-related molecules in insect intestine has been identified by genomics or proteomics approach with comparison to well-studied tissues, such as hemolymph or fat body, our knowledge about the molecular mechanism underlying the gut immunity which would involve a variety of unidentified molecules is still limited. To uncover additional molecules that might take part in pathogen recognition, signal transduction or immune regulation in insect intestine, a T7 phage display cDNA library of the silkworm midgut is constructed. By use of different ligands for biopanning, Translationally Controlled Tumor Protein (TCTP) has been selected. BmTCTP is produced in intestinal epithelial cells and released into the gut lumen. The protein level of BmTCTP increases at the early time points during oral microbial infection and declines afterwards. In vitro binding assay confirms its activity as a multi-ligand binding molecule and it can further function as an opsonin that promotes the phagocytosis of microorganisms. Moreover, it can induce the production of anti-microbial peptide via a signaling pathway in which ERK is required and a dynamic tyrosine phosphorylation of certain cytoplasmic membrane protein. Taken together, our results characterize BmTCTP as a dual-functional protein involved in both the cellular and the humoral immune response of the silkworm, Bombyx mori. PMID:23894441
Wang, Fei; Hu, Cuimei; Hua, Xiaoting; Song, Liang; Xia, Qingyou
Identification of a functional element in the promoter of the silkworm (Bombyx mori) fat body-specific gene Bmlp3.
30K proteins are a group of structurally related proteins that play important roles in the life cycle of the silkworm Bombyx mori and are largely synthesized and regulated in a time-dependent manner in the fat body. Little is known about the upstream regulatory elements associated with the genes encoding these proteins. In the present study, the promoter of Bmlp3, a fat body-specific gene encoding a 30K protein family member, was characterized by joining sequences containing the Bmlp3 promoter with various amounts of 5' upstream sequences to a luciferase reporter gene. The results indicated that the sequences from -150 to -250bp and -597 to -675bp upstream of the Bmlp3 transcription start site were necessary for high levels of luciferase activity. Further analysis showed that a 21-bp sequence located between -230 and -250 was specifically recognized by nuclear factors from silkworm fat bodies and BmE cells, and could enhance luciferase reporter-gene expression 2.8-fold in BmE cells. This study provides new insights into the Bmlp3 promoter and contributes to the further clarification of the function and developmental regulation of Bmlp3. PMID:24853203
Xu, Hanfu; Deng, Dangjun; Yuan, Lin; Wang, Yuancheng; Wang, Feng; Xia, Qingyou
Bmdsx is an orthologue of the sex-determining gene doublesex (dsx) and known to be sex-specifically expressed in various tissues of the silkworm, Bombyx mori. Its pre-mRNA is sex-specifically spliced and encodes female-specific or male-specific polypeptides. The open reading frame of Bmdsx consists of 5 exons, of which exons 3 and 4 are female-specific and its pre-mRNA was known to undergo default processing to generate the female-type mRNA. Previous reports have shown that the mechanism of splicing of the doublesex gene is different in Drosophila melanogaster and Bombyx mori. However, intron 4 is so long that it is difficult to identify the intronic cis-element(s) required for male-specific splicing of Bmdsx pre-mRNA using Bmdsx minigenes whose introns are shortened in various manners. As a first step toward discovery of the cis-element, the Bmdsx mini gene, which consisted of exon 1 and 5 and internally shortened introns 2 to 4, was constructed, and transgenic silkworms expressing this construct were generated. Bmdsx pre-mRNA transcribed derived from transgene was sex-specifically spliced. This result shows that the mini gene contained the information necessary for the correct regulation of alternative splicing.
Funaguma, Shunsuke; Suzuki, Masataka G.; Tamura, Toshiki; Shimada, Toru
Isolation, Purification, and Identification of an Important Pigment, Sepiapterin, from Integument of the lemon Mutant of the Silkworm, Bombyx mori
Sepiapterin is the precursor of tetrahydrobiopterin, an important coenzyme of aromatic amino acid hydroxylases, the lack of which leads to a variety of physiological metabolic diseases or neurological syndromes in humans. Sepiapterin is a main pigment component in the integument of the lemon mutant of the silkworm, Bombyx mori (L.) (Lepidoptera: Bombycidae), and is present there in extremely high content, so lemon is a valuable genetic resource to extract sepiapterin. In this study, an effective experimental system was set up for isolation and purification of sepiapterin from lemon silkworms by optimizing homogenization solvent, elution buffer, and separation chromatographic column. The results showed that ethanol was the most suitable solvent to homogenize the integument, with a concentration of 50% and solid:liquid ratio of 1:20 (g/mL). Sepiapterin was purified successively by column chromatography of cellulose Ecteola, sephadex G-25-150, and cellulose phosphate, and was identified by ultraviolet-visible absorption spectrometry. A stable and accurate high performance liquid chromatography method was constructed to identify sepiapterin and conduct qualitative and quantitative analyses. Sepiapterin of high purity was achieved, and the harvest reached about 40 ug/g of integument in the experiments. This work helps to obtaining natural sepiapterin in large amounts in order to use the lemon B. mori mutant to produce BH4 in vitro.
Wang, Jing; Wang, Wenjing; Liu, Chaoliang; Meng, Yan
BmICE-2 is a novel pro-apoptotic caspase involved in apoptosis in the silkworm, Bombyx mori.
In this study we identified a potential pro-apoptotic caspase gene, Bombyx mori(B. mori)ICE-2 (BmICE-2) which encoded a polypeptide of 284 amino acid residues, including a (169)QACRG(173) sequence which surrounded the catalytic site and contained a p20 and a p10 domain. BmICE-2 expressed in Escherichia coli (E. coli) exhibited high proteolytic activity for the synthetic human initiator caspase-9 substrates Ac-LEHD-pNA, but little activity towards the effector caspase-3 substrates Ac-DEVD-pNA. When BmICE-2 was transiently expressed in BmN-SWU1 silkworm B. mori cells, we found that the high proteolytic activity for Ac-LEHD-pNA triggered caspase-3-like protease activity resulting in spontaneous cleavage and apoptosis in these cells. This effect was not replicated in Spodoptera frugiperda 9 cells. In addition, spontaneous cleavage of endogenous BmICE-2 in BmN-SWU1 cells could be induced by actinomycin D. These results suggest that BmICE-2 may be a novel pro-apoptotic gene with caspase-9 activity which is involved apoptotic processes in BmN-SWU1 silkworm B. mori cells. PMID:24491540
Yi, Hua-Shan; Pan, Cai-Xia; Pan, Chun; Song, Juan; Hu, Yan-Fen; Wang, La; Pan, Min-Hui; Lu, Cheng
A novel Rel protein and shortened isoform that differentially regulate antibacterial peptide genes in the silkworm Bombyx mori.
Two cDNAs encoding novel Rel proteins were cloned from the silkworm, Bombyx mori. These cDNA clones (BmRelA and BmRelB) showed identical nucleotide sequences except for the 5'-region. BmRelB cDNA derived probably from an alternatively spliced mRNA lacked 241 bp nucleotides at the 5'-region of the BmRelA cDNA, resulting in a loss of the first 52 amino acids. Expression of antibacterial peptide genes was strongly inhibited upon infection with Micrococcus luteus in transgenic silkworms in which BmRel gene expression was knocked down, suggesting that these two Rel proteins are involved in activation of antibacterial peptide genes. Co-transfection experiments indicated that BmRelB activated the Attacin gene strongly and other genes to a lesser extent, whereas BmRelA activated Lebocin 4 gene strongly and Attacin and Lebocin 3 genes very weakly. The Rel homology domain of BmRelA and BmRelB was shown to bind specifically to kappaB sites of antibacterial peptide genes. Proline-rich domains of the BmRels were necessary for activation of antibacterial peptide genes. These results illustrate that a minor structural change in Rel proteins can provoke a dramatic differential activation of antibacterial peptide genes, suggesting a novel regulatory mechanism for insect antibacterial peptide gene expression. PMID:16005991
Tanaka, Hiromitsu; Yamamoto, Masafumi; Moriyama, Yuko; Yamao, Masafumi; Furukawa, Seiichi; Sagisaka, Aki; Nakazawa, Hiroshi; Mori, Hajime; Yamakawa, Minoru
Use of Sleeve Nets to Improve Survival of the Boisduval Silkworm, Anaphe panda, in the Kakamega Forest of Western Kenya
Prospects for development of a wild silk industry in Africa would be improved if silkworm survival during mass production could be improved. A study on the survival of the Boisduval silkworm, Anaphe panda (Boisduval) (Lepidoptera: Thaumetopoeidae) was conducted with and without protection by net sleeves in two different forest habitats (natural and modified) in the Kakamega forest of western Kenya. Overall, cohort survival was significantly higher (P < 0.001) in the natural than in the modified forest, but larval survival was improved over three-fold by protection with net sleeves in both habitat types. In the modified forest, only 16.8% of unprotected larvae survived to the pupal stage and formed cocoons, whereas 62.3% survived in the same environment when they were protected with net sleeves. In the natural forest, 20.4% of unprotected larvae survived, whereas 67.7% survived in net sleeves. There was also a significant effect of season; cohorts of larvae that eclosed in the wet season had significantly lower survival than those eclosing in the dry season (P = 0.02). Sources of mortality appeared to be natural enemies (parasites, predators and diseases) and climatic factors.
Mbahin, N.; Raina, S. K.; Kioko, E. N.; Mueke, J. M.
Genome-wide identification and expression profiling of the fatty acid desaturase gene family in the silkworm, Bombyx mori.
Fatty acid desaturases exist in all living organisms and play important roles in many different biologic processes, such as fatty acid metabolism, lipid biosynthetic processes, and pheromone biosynthetic processes. Using the available silkworm genome sequence, we identified 14 candidate fatty acid desaturase genes. Eleven genes contain 3 conserved histidine cluster motifs and 4 transmembrane domains, but their N-terminal residues exhibit obvious diversity. Phylogenetic analysis revealed that there are 6 groups; Bmdesat1 and Bmdesat5-8 were clustered into group 2, which is involved in ?11 desaturation activity, and Bmdesat3-4 were grouped in group 1, which is involved in ?9 desaturation activity. Twelve of the 14 genes have expressed sequence tag evidence. Microarray data and reverse transcription polymerase chain reaction analysis demonstrated that Bmdesat3-4 and Bmdesat10 were expressed from the larval to moth stages and in multiple tissues on day 3 of 5th instar larvae. Bmdesat9, Bmdesat11, and Bmdesat14 were expressed during the pupal and late-embryonic stage, suggesting that they may take part in fatty acid metabolism to provide energy. These results provide some insights into the functions of individual fatty acid desaturases in silkworm. PMID:24854660
Chen, Q M; Cheng, D J; Liu, S P; Ma, Z G; Tan, X; Zhao, P
Development of a Foot-and-Mouth Disease Virus Serotype A Empty Capsid Subunit Vaccine Using Silkworm (Bombyx mori) Pupae
Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that inflicts severe economic losses in the livestock industry. In 2009, FMDV serotype A caused outbreaks of FMD in cattle in China. Although an inactivated virus vaccine has proven effective to control FMD, its use may lead to new disease outbreaks due to a possible incomplete inactivation of the virus during the manufacturing process. Here, we expressed the P1-2A and the 3C coding regions of a serotype A FMDV field isolate in silkworm pupae (Bombyx mori) and evaluated the immunogenicity of the expression products. Four of five cattle vaccinated with these proteins developed high titers of FMDV-specific antibody and were completely protected against virulent homologous virus challenge with 10,000 50% bovine infectious doses (BID50). Furthermore, the 50% bovine protective dose (PD50) test was performed to assess the bovine potency of the empty capsid subunit vaccine and was shown to achieve 4.33 PD50 per dose. These data provide evidence that silkworm pupae can be used to express immunogenic FMDV proteins. This strategy might be used to develop a new generation of empty capsid subunit vaccines against a variety of diseases.
Li, Zhiyong; Yi, Yongzhu; Yin, Xiangping; Zhang, Yun; Liu, Ming; Liu, Hang; Li, Xuerui; Li, Yinu; Zhang, Zhifang; Liu, Jixing
Isolation, Purification, and Identification of an Important Pigment, Sepiapterin, from Integument of the lemon Mutant of the Silkworm, Bombyx mori.
Abstract Sepiapterin is the precursor of tetrahydrobiopterin, an important coenzyme of aromatic amino acid hydroxylases, the lack of which leads to a variety of physiological metabolic diseases or neurological syndromes in humans. Sepiapterin is a main pigment component in the integument of the lemon mutant of the silkworm, Bombyx mori (L.) (Lepidoptera: Bombycidae), and is present there in extremely high content, so lemon is a valuable genetic resource to extract sepiapterin. In this study, an effective experimental system was set up for isolation and purification of sepiapterin from lemon silkworms by optimizing homogenization solvent, elution buffer, and separation chromatographic column. The results showed that ethanol was the most suitable solvent to homogenize the integument, with a concentration of 50% and solid:liquid ratio of 1:20 (g/mL). Sepiapterin was purified successively by column chromatography of cellulose Ecteola, sephadex G-25-150, and cellulose phosphate, and was identified by ultraviolet-visible absorption spectrometry. A stable and accurate high performance liquid chromatography method was constructed to identify sepiapterin and conduct qualitative and quantitative analyses. Sepiapterin of high purity was achieved, and the harvest reached about 40 ug/g of integument in the experiments. This work helps to obtaining natural sepiapterin in large amounts in order to use the lemon B. mori mutant to produce BH4 in vitro. PMID:24773269
Gao, Junshan; Wang, Jing; Wang, Wenjing; Liu, Chaoliang; Meng, Yan
A short-type peptidoglycan recognition protein from the silkworm: expression, characterization and involvement in the prophenoloxidase activation pathway.
Recognition of invading microbes as non-self is the first step of immune responses. In insects, peptidoglycan recognition proteins (PGRPs) detect peptidoglycans (PGs) of bacterial cell wall, leading to the activation of defense responses. Twelve PGRPs have been identified in the silkworm, Bombyx mori, through bioinformatics analysis. However, their biochemical functions are mostly uncharacterized. In this study, we found PGRP-S5 transcript levels were up-regulated in fat body and midgut after bacterial infection. Using recombinant protein isolated from Escherichia coli, we showed that PGRP-S5 binds to PGs from certain bacterial strains and induces bacteria agglutination. Enzyme activity assay confirmed PGRP-S5 is an amidase; we also showed it is an antibacterial protein effective against both Gram-positive and -negative bacteria. Additionally, we demonstrated that specific recognition of PGs by PGRP-S5 is involved in the prophenoloxidase activation pathway. Together, these data suggest the silkworm PGRP-S5 functions as a pattern recognition receptor for the prophenoloxidase pathway initiation and as an effecter to inhibit bacterial growth as well. We finally discussed possible roles of PGRP-S5 as a receptor for antimicrobial peptide gene induction and as an immune modulator in the midgut. PMID:24508981
Chen, Kangkang; Liu, Chen; He, Yan; Jiang, Haobo; Lu, Zhiqiang
Reducing blood glucose levels in TIDM mice with an orally administered extract of sericin from hIGF-I-transgenic silkworm cocoons.
In previous studies, we reported that the blood glucose levels of mice with type I diabetes mellitus (TIDM) was reduced with orally administered silk gland powder from silkworms transgenic for human insulin-like growth factor-I (hIGF-I). However, potential safety hazards could not be eliminated because the transgenic silk gland powder contained heterologous DNA, including the green fluorescent protein (gfp) and neomycin resistance (neo) genes. These shortcomings might be overcome if the recombinant hIGF-I were secreted into the sericin layer of the cocoon. In this study, silkworm eggs were transfected with a novel piggyBac transposon vector, pigA3GFP-serHS-hIGF-I-neo, containing the neo, gfp, and hIGF-I genes controlled by the sericin-1 (ser-1) promoter with the signal peptide DNA sequence of the fibrin heavy chain (Fib-H) and a helper plasmid containing the piggyBac transposase sequence under the control of the Bombyx mori actin 3 (A3) promoter, using sperm-mediated gene transfer to generate the transformed silkworms. The hIGF-I content estimated by enzyme-linked immunosorbent assay was approximately 162.7 ng/g. To estimate the biological activity of the expressed hIGF-I, streptozotocin-induced TIDM mice were orally administered sericin from the transgenic silkworm. The blood glucose levels of the mice were significantly reduced, suggesting that the extract from the transgenic hIGF-I silkworm cocoons can be used as an orally administered drug. PMID:24632065
Song, Zuowei; Zhang, Mengyao; Xue, Renyu; Cao, Guangli; Gong, Chengliang
Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey
2. ELEVATION OF THE OAK VIADUCT PONY TRUSS, LOOKING NORTH ON SAW MILL RUN BOULEVARD - Pittsburgh & Castle Shannon Railroad, Oak Viaduct, Overbrook Trolley Line, crossing Saw Mill Run Boulevard & Colerain Street, Pittsburgh, Allegheny County, PA
4. VIEW OF THE NORTHERN BENTS SUPPORTING OAK VIADUCT, LOOKING SOUTH ON SAW MILL RUN BOULEVARD - Pittsburgh & Castle Shannon Railroad, Oak Viaduct, Overbrook Trolley Line, crossing Saw Mill Run Boulevard & Colerain Street, Pittsburgh, Allegheny County, PA
3. ELEVATION OF THE OAK VIADUCT, LOOKING NORTH ON SAW MILL RUN BOULEVARD - Pittsburgh & Castle Shannon Railroad, Oak Viaduct, Overbrook Trolley Line, crossing Saw Mill Run Boulevard & Colerain Street, Pittsburgh, Allegheny County, PA
Oak Ridge National Laboratory: The Wigner Fellowship
Oak Ridge National Laboratory seeks applicants for its most prestigious Eugene P. Wigner Fellowship Program, established in 1975 to honor the Nobel Laureate (1963) and first Director of Research and Development at ORNL (1946-47). Fellows must be exceedingly well qualified, be no more than three years beyond the doctorate, and have not engaged in more than one post-doctoral position.
ORNL (Oak Ridge National Laboratory) 89
This is the inaugural issues of an annual publication about the Oak Ridge National Laboratory. Here you will find a brief overview of ORNL, a sampling of our recent research achievements, and a glimpse of the directions we want to take over the next 15 years. A major purpose of ornl 89 is to provide the staff with a sketch
T. D. Anderson; B. R. Appleton; J. W. Jefferson; J. R. Merriman; F. R. Mynatt; C. R. Richmond; M. W. Rosenthal
Oak Park Middle School and General Electric.
ERIC Educational Resources Information Center
Describes a partnership between Oak Park Middle School in Decatur, Alabama, and the General Electric Company that features the use of the quality circle approach. Not only were teachers, administrators, and parents organized into school quality circles, but students also formed quality circles to solve problems at the school. (CH)
Buckley, Anita
Oak Ridge Reservation Waste Management Plan
This report presents the waste management plan for the Oak Ridge Reservation facilities. The primary purpose is to convey what facilities are being used to manage wastes, what forces are acting to change current waste management systems, and what plans are in store for the coming fiscal year.
Turner, J.W. [ed.
Data Bank for Red Oak Lumber, 1992.
The 1992 Data Bank for Red Oak Lumber is a collection of fully described FAS, Selects, No. 1 Common, and No. 2A Common boards (a total of 1,578 at present). The data bank has two unique features to aid in sample selection. The first feature is the double ...
C. J. Gatchell J. K. Wiedenbeck E. S. Walker
Soil water dynamics in an oak stand
Soil water dynamics under a mixed stand of mature sessile and pedunculate oaks were studied both under natural conditions and during imposed water shortages in a lysimeter. Root densities of each species were described in situ by counting roots in the trench surrounding the dry plot. Soil water contents and potentials, and pre-dawn leaf water potentials (?wp) were monitored during
N. Br�da; A. Granier; F. Barataud; C. Moyne
Environmental Monitoring Plan for Waste Area Grouping 6 at Oak Ridge National Laboratory, Oak Ridge, Tennessee.
This document presents the Environmental Monitoring Plan (EMP) for Waste Area Grouping (WAG) 6 at the Oak Ridge National Laboratory (ORNL). Based on the results of the Resource Conservation and Recovery Act (RCRA) Facility Investigation (RFI) and on subse...
An oak (Quercus agrifolia) specialist (Neotoma macrotis) and a sympatric generalist (Neotoma lepida) show similar intakes and digestibilities of oak.
Dietary specialization is thought to be rare in mammalian herbivores as a result of either a limitation in their detoxification system to metabolize higher doses of plant secondary compounds or deficiencies in nutrients present in a diet composed of a single species of plant. Neotoma macrotis is an oak specialist, whereas Neotoma lepida is a dietary generalist when sympatric with N. macrotis. We hypothesized that N. macrotis would have a higher tolerance for and digestibility of oak. We determined the two species' tolerances for oak by feeding them increasing concentrations of ground oak leaves until they could no longer maintain body mass. The highest concentration on which both species maintained body mass was 75% oak. There were no differences between the species in their abilities to digest dry matter, nitrogen, or fiber in the oak diets. The species' similar tolerances for oak were probably due to their similar abilities to digest and potentially assimilate the ground oak leaves. PMID:18544017
Skopec, Michele M; Haley, Shannon; Torregrossa, Ann-Marie; Dearing, M Denise
Phase 1 report on the Bear Creek Valley treatability study, Oak Ridge Y-12 Plant, Oak Ridge, Tennessee.
Bear Creek Valley (BCV) is located within the US Department of Energy (DOE) Oak Ridge Reservation and encompasses multiple waste units containing hazardous and radioactive wastes associated with past operations at the adjacent Oak Ridge Y-12 Plant. The BC...
Oak ecosystem succession of the Northern Caucasus
English oak (Quercus robur L.) along with its well-known good properties has a high sanitary-hygienic and curative potential. Its volatile metabolites (VM) influence bacteriostatically Staphylococcus aureus 209r, oppressing it in vitro by 85% compared with the control, and Escherichia coli by 45%. There is the least amount of epiphytic microorganisms on the leaves of Q. robur L. compared with some tree species [1]. In addition, VM of Q. robur L. have direct milieu (hypotensive) effects on the organism under its canopy, lowering blood pressure by 20-25 mm Hg [2]. A.P. Kazankin (1993) [4] calculated the prehistoric formula of forest species composition of Caucasian Mineral Waters region (Northern Caucasus): 6Qr3Crp1Fr which was based on the theory of calcium-magnesium absorption complex. According to the theory, underground mineral water, soil, forest litter and the leaves of ground vegetation of the area have the same ratio of the cations Ca and Mg - calcium-magnesium index [3]. Hence oldgrowth in the region consisted of oak (Qr) by 60%, hornbeam (Carpinus-Crp) by 30% and ash (Fraxinus-Fr) only by 10%. Currently, the formula of the forests of the region has been changed by man: 5Fr3Crp2Qr. The proportion of oak forests has decreased to 20%, the proportion of ash has increased by 50%, but the proportion of hornbeam hasn't changed. So it is relevant to restore oak forests of the region in the former ratio to other forest-forming species - ash and hornbeam. Taking into consideration the change of economic formation of society in Russia, it is extremely important to restore natural seed oak forests. Therein the luminance of surface areal is a limiting factor. We have calculated that the natural recovery of oak forests is possible providing observation of optimal moisture standards and soil fertility in combination with solar light within 10-24% from the light of open space. Measures for promotion of oak natural regeneration in mountain resorts of the Northern Caucasus can be found in elaborate recommendations [5].
Chalaya, Elena; Slepykh, Victor; Efimenko, Natalia; Povolotskaya, Nina
Design assessment for the Bethel Valley FFA Upgrades at Oak Ridge National Laboratory, Oak Ridge, Tennessee
This report describes the proposed upgrades to Building 3025 and the Evaporator Area at Oak Ridge National Laboratory. Design assessments, specifications and drawings are provided. Building 3025 is a general purpose research facility utilized by the Materials and Ceramics Division to conduct research on irradiated materials. The Evaporator Area, building 2531, serves as the collection point for all low-level liquid wastes generated at the Oak Ridge National Laboratory.
Seismic hazard evaluation for Department of Energy Oak Ridge Reservations, Oak Ridge, Tennessee
This study presents the results of an investigation of seismic hazard at the Department of Energy Oak Ridge Reservations (K-25 Site, Oak Ridge National Laboratories, and Oak Ridge Y-12 Plant), located in Oak Ridge, Tennessee. Oak Ridge is located in eastern Tennessee, in an area of moderate to high historical seismicity. Results from two separate seismic hazard analyses are presented. The EPRI/SOG analysis uses the input data and methodology developed by the Electric Power Research Institute, under the sponsorship of several electric utilities, for the evaluation of seismic hazard in the central and eastern United States. The LLNL analysis uses the input data and methodology developed by the Lawrence Livermore National Laboratory for the Nuclear Regulatory Commission. Both the EPRI/SOG and LLNL studies characterize earth-science uncertainty on the causes and characteristics of earthquakes in the central and eastern United States. This is accomplished by considering multiple hypotheses on the locations and parameters of seismic source zones and by considering multiple attenuation functions for the prediction of ground shaking given earthquake size and location. These hypotheses were generated by multiple expert teams and experts. Furthermore, each team and expert was asked to generate multiple hypotheses in order to characterize his own internal uncertainty. The seismic-hazard calculations are performed for all hypotheses. Combining the results from each hypothesis with the weight associated to that hypothesis, one obtains an overall representation of the seismic hazard at the Oak Ridge site and its uncertainty.
McGuire, R.K.; Toro, G.F. [Risk Engineering, Inc., Golden, CO (United States); Hunt, R.J. [Martin Marietta Energy Systems, Inc., Oak Ridge, TN (United States). Center for Natural Phenomena Engineering
Sampling and Analysis Plan for White Oak Creek Watershed Remedial Investigation supplemental sampling, Oak Ridge National Laboratory, Oak Ridge, Tennessee
This Sampling and Analysis (SAP) presents the project requirements for proposed soil sampling to support the White Oak Creek Remedial Investigation/Feasibility Study at Oak Ridge National Laboratory. During the Data Quality Objectives process for the project, it was determined that limited surface soils sampling is need to supplement the historical environmental characterization database. The primary driver for the additional sampling is the need to identify potential human health and ecological risks at various sites that have not yet proceeded through a remedial investigation. These sites include Waste Area Grouping (WAG)3, WAG 4, WAG 7, and WAG 9. WAG 4 efforts are limited to nonradiological characterization since recent seep characterization activities at the WAG have defined the radiological problem there.
Hydrologic data summary for the White Oak Watershed at Oak Ridge National Laboratory, Oak Ridge, Tennessee, October 1990--December 1991
This report summarizes for the 15-month period of October 1990-- December 1991 the available dynamic hydrologic data collected, primarily on the White Oak Creek (WOC) watershed, along with information collected on the surface flow systems that affect the quality or quantity of surface water. The collection of hydrologic data is one component of numerous, ongoing Oak Ridge National Laboratory (ORNL) environmental studies and monitoring programs and is intended to: (1) characterize the quantity and quality of water in the flow systems; (2) assist with the planning and assessment of remedial action activities; and, (3) provide long-term availability of data and quality assurance. Characterization of the hydrology of the WOC watershed is critical for understanding the processes that drive contaminant transport in the watershed. Identification of spatial and temporal trends in hydrologic parameters and mechanisms that affect the movement of contaminants supports the development of interim corrective measures and remedial restoration alternatives. In addition, hydrologic monitoring supports long-term assessment of the effectiveness of remedial actions in limiting the transport of contaminants across Waste Area Grouping (WAG) boundaries and ultimately to the off-site environment. For these reasons, it is of paramount importance to the Environmental Restoration Program (ERP) to collect and report hydrologic data activities that contribute to the Site Investigations component of the ERP. (White Oak Creek is also referred to as ``Whiteoak`` Creek).
Borders, D.M.; Gregory, S.M.; Clapp, R.B.; Frederick, B.J.; Watts, J.A.
This report summarizes for the 15-month period of October 1990-- December 1991 the available dynamic hydrologic data collected, primarily on the White Oak Creek (WOC) watershed, along with information collected on the surface flow systems that affect the quality or quantity of surface water. The collection of hydrologic data is one component of numerous, ongoing Oak Ridge National Laboratory (ORNL) environmental studies and monitoring programs and is intended to: (1) characterize the quantity and quality of water in the flow systems; (2) assist with the planning and assessment of remedial action activities; and, (3) provide long-term availability of data and quality assurance. Characterization of the hydrology of the WOC watershed is critical for understanding the processes that drive contaminant transport in the watershed. Identification of spatial and temporal trends in hydrologic parameters and mechanisms that affect the movement of contaminants supports the development of interim corrective measures and remedial restoration alternatives. In addition, hydrologic monitoring supports long-term assessment of the effectiveness of remedial actions in limiting the transport of contaminants across Waste Area Grouping (WAG) boundaries and ultimately to the off-site environment. For these reasons, it is of paramount importance to the Environmental Restoration Program (ERP) to collect and report hydrologic data activities that contribute to the Site Investigations component of the ERP. (White Oak Creek is also referred to as Whiteoak'' Creek).
Rapid Detection of Infectious Flacherie Virus of the Silkworm, Bombyx mori, using RT-PCR and Nested PCR
In this study, a method for detection of an ssRNA viral pathogen that causes viral flacherie in the silkworm, Bombyx mori (L.) (Lepidoptera: Bombycidae), was used for the detection of B. mori infectious flacherie virus (BmIFV). A combination of nested and reverse transcriptase polymerase chain reaction was used for detection. Although BmIFV has been reported in almost all the sericultural regions of the world, there had been no reports of BmIFV incidence in India. Therefore, the confirmation of the presence of BmIFV in Karnataka, India, is of great significance. The present method is advantageous because it can be used to detect the virus by using samples from infected midgut tissues, thus simplifying and avoiding laborious genome isolation procedures. This method could help in early detection of BmIFV disease pathogens and help reduce crop losses.
Vootla, Shyam Kumar; Lu, Xing Meng; Kari, Neetha; Gadwala, Mallikarjun; Lu, Qineng
A complete full-length non-LTR retrotransposon, BMC1, on the W chromosome of the silkworm, Bombyx mori.
In the silkworm, Bombyx mori, a non-long terminal repeat (non-LTR) retrotransposon, BMC1, is considered to be a LINE (long interspersed nuclear element)-like element. So far, a BMC1 containing two intact open reading frames (ORFs) has not been found. However, we discovered a complete full-length BMC1 on the W chromosome. This BMC1 is 5091 bp and contains a 5' untranslated region (5'-UTR), two intact ORFs, and 3'-UTR which terminates in a poly(A) tail. ORF1 encodes a putative nucleic acid-binding protein, while ORF2 encodes a protein containing an endonuclease domain and a reverse transcriptase domain. PMID:10333566
Abe, H; Ohbayashi, F; Shimada, T; Sugasaki, T; Kawai, S; Oshiki, T
Juvenile Hormone Analogues, methoprene and fenoxycarb dose-dependently enhance certain enzyme activities in the silkworm Bombyx mori (L).
Use of Juvenile Hormone Analogues (JHA) in sericulture practices has been shown to boost good cocoon yield; their effect has been determined to be dose-dependent. We studied the impact of low doses of JHA compounds such as methoprene and fenoxycarb on selected key enzymatic activities of the silkworm Bombyx mori. Methoprene and fenoxycarb at doses of 1.0 microg and 3.0 fg/larvae/48 hours showed enhancement of the 5th instar B. mori larval muscle and silkgland protease, aspartate aminotransaminase (AAT) and alanine aminotransaminase (ALAT), adenosine triphosphate synthase (ATPase) and cytochrome-c-oxidase (CCO) activity levels, indicating an upsurge in the overall oxidative metabolism of the B.mori larval tissues. PMID:18678927
Mamatha, Devi M; Kanji, Vijaya K; Cohly, Hari H P; Rao, M Rajeswara
Juvenile Hormone Analogues, Methoprene and Fenoxycarb Dose-Dependently Enhance Certain Enzyme Activities in the Silkworm Bombyx Mori (L)
Use of Juvenile Hormone Analogues (JHA) in sericulture practices has been shown to boost good cocoon yield; their effect has been determined to be dose-dependent. We studied the impact of low doses of JHA compounds such as methoprene and fenoxycarb on selected key enzymatic activities of the silkworm Bombyx mori. Methoprene and fenoxycarb at doses of 1.0 ?g and 3.0fg/larvae/48 hours showed enhancement of the 5th instar B. mori larval muscle and silkgland protease, aspartate aminotransaminase (AAT) and alanine aminotransaminase (ALAT), adenosine triphosphate synthase (ATPase) and cytochrome-c-oxidase (CCO) activity levels, indicating an upsurge in the overall oxidative metabolism of the B.mori larval tissues.
Mamatha, Devi M.; Kanji, Vijaya K.; Cohly, Hari H.P.; Rao, M. Rajeswara
Isolation, purification, crystallization and preliminary X-ray studies of two 30?kDa proteins from silkworm haemolymph.
Juvenile hormone-binding protein (JHBP) and the low-molecular-mass lipoprotein PBMHP-12 belong to a group of 30?kDa proteins that comprise the major protein component of the haemolymph specific to the fifth-instar larvae stage of the mulberry silkworm Bombyx mori L. Proteins from this group are�often essential for the development of the insect. In a project aimed at crystallographic characterization of B. mori JHBP (BmJHBP), it was copurified together with PBMHP-12. Eventually, the two proteins were isolated and crystallized separately. The BmJHBP crystals were orthorhombic (space group C222(1)) and the PBMHP-12 crystals were triclinic. The crystals diffracted X-rays to 2.9?� (BmJHBP) and 1.3?� (PBMHP-12) resolution. PMID:21393846
Pietrzyk, Agnieszka J; Bujacz, Anna; ?ochy?ska, Ma?gorzata; Jask�lski, Mariusz; Bujacz, Grzegorz
Osteochondral Tissue Engineering In Vivo: A Comparative Study Using Layered Silk Fibroin Scaffolds from Mulberry and Nonmulberry Silkworms
The ability to treat osteochondral defects is a major clinical need. Existing polymer systems cannot address the simultaneous requirements of regenerating bone and cartilage tissues together. The challenge still lies on how to improve the integration of newly formed tissue with the surrounding tissues and the cartilage-bone interface. This study investigated the potential use of different silk fibroin scaffolds: mulberry (Bombyx mori) and non-mulberry (Antheraea mylitta) for osteochondral regeneration in vitro and in vivo. After 4 to 8 weeks of in vitro culture in chondro- or osteo-inductive media, non-mulberry constructs pre-seeded with human bone marrow stromal cells exhibited prominent areas of the neo tissue containing chondrocyte-like cells, whereas mulberry constructs pre-seeded with human bone marrow stromal cells formed bone-like nodules. In vivo investigation demonstrated neo-osteochondral tissue formed on cell-free multi-layer silk scaffolds absorbed with transforming growth factor beta 3 or recombinant human bone morphogenetic protein-2. Good bio-integration was observed between native and neo-tissue within the osteochondrol defect in patellar grooves of Wistar rats. The in vivo neo-matrix formed comprised of a mixture of collagen and glycosaminoglycans except in mulberry silk without growth factors, where a predominantly collagenous matrix was observed. Immunohistochemical assay showed stronger staining of type I and type II collagen in the constructs of mulberry and non-mulberry scaffolds with growth factors. The study opens up a new avenue of using inter-species silk fibroin blended or multi-layered scaffolds of a combination of mulberry and non-mulberry origin for the regeneration of osteochondral defects.
Saha, Sushmita; Kundu, Banani; Kirkham, Jennifer; Wood, David; Kundu, Subhas C.; Yang, Xuebin B.
The expression analysis of silk gland-enriched intermediate-size non-coding RNAs in silkworm Bombyx mori.
Small non-protein coding RNAs (ncRNAs) play important roles in development, stress response and other cellular processes. Silkworm is an important model for studies on insect genetics and control of Lepidopterous pests. We have previously identified 189 novel intermediate-size ncRNAs in silkworm Bombyx mori, including 40 ncRNAs that showed altered expression in different developmental stages. Here we characterized the functions of these 40 ncRNAs by measuring their expressions in six tissues of the fifth instar larvae using Northern blot and real-time polymerase chain reaction assays. We identified nine ncRNAs (four small nucleolar RNAs and five unclassified ncRNAs) that were enriched in silk gland, including four ncRNAs that showed silk gland-specific expression. We further showed that three of nine silk gland-enriched ncRNAs were predominantly expressed in the anterior silk gland, whereas another three ncRNAs were highly accumulated in the posterior silk gland, suggesting that they may play different roles in fibroin synthesis. Furthermore, an unclassified ncRNA, Bm-152, exhibited converse expression pattern with its antisense host gene gartenzwerg in diverse tissues, and might regulate the expression of gartenzwerg through RNA-protein complex. In addition, two silk gland-enriched ncRNAs Bm-102 and Bm-159 can be found in histone modification complex, which indicated that they might play roles through epigenetic modifications. Taken together, we provided the first expression and preliminary functional analysis of silk gland-enriched ncRNAs, which will help understand the molecular mechanism of silk gland-development and fibroin synthesis. PMID:24124013
Li, Dan-Dan; Liu, Zong-Cai; Huang, Lei; Jiang, Qian-Ling; Zhang, Kun; Qiao, Hui-Li; Jiao, Zhu-Jin; Yao, Lun-Guang; Liu, Ren-Yi; Kan, Yun-Chao
Genetic Analysis of the Electrophysiological Response to Salicin, a Bitter Substance, in a Polyphagous Strain of the Silkworm Bombyx mori
Sawa-J is a polyphagous silkworm (Bombyx mori L.) strain that eats various plant leaves that normal silkworms do not. The feeding preference behavior of Sawa-J is controlled by one major recessive gene(s) on the polyphagous (pph) locus, and several minor genes; moreover, its deterrent cells possess low sensitivity to some bitter substances including salicin. To clarify whether taste sensitivity is controlled by the pph locus, we conducted a genetic analysis of the electrophysiological characteristics of the taste response using the polyphagous strain Sawa-J�lem, in which pph is linked to the visible larval marker lemon (lem) on the third chromosome, and the normal strain Daiankyo, in which the wild-type gene of pph (+pph) is marked with Zebra (Ze). Maxillary taste neurons of the two strains had similar dose�response relationships for sucrose, inositol, and strychnine nitrate, but the deterrent cell of Sawa-J�lem showed a remarkably low sensitivity to salicin. The F1 generation of the two strains had characteristics similar to the Daiankyo strain, consistent with the idea that pph is recessive. In the BF1 progeny between F1 females and Sawa-J�lem males where no crossing-over occurs, the lem and Ze phenotypes corresponded to different electrophysiological reactions to 25 mM salicin, indicating that the gene responsible for taste sensitivity to salicin is located on the same chromosome as the lem and Ze genes. The normal and weak reactions to 25 mM salicin were segregated in crossover-type larvae of the BF1 progeny produced by a reciprocal cross, and the recombination frequency agreed well with the theoretical ratio for the loci of lem, pph, and Ze on the standard linkage map. These results indicate that taste sensitivity to salicin is controlled by the gene(s) on the pph locus.
Iizuka, Tetsuya; Tamura, Toshiki; Sezutsu, Hideki; Mase, Keisuke; Okada, Eiji; Asaoka, Kiyoshi
Applying fenoxycarb at the penultimate instar triggers an additional ecdysteroid surge and induces perfect extra larval molting in the silkworm.
When the juvenile hormone analog fenoxycarb was topically applied to the silkworm Bombyx mori at the beginning of the 3rd or 4th (penultimate) instar, an extra larval molt was induced. The 5th instar period was shortened to about 5 days and the extra 6th instar ranged from 8 to more than 20 days, depending on the dose applied. Starvation before fenoxycarb treatment strongly enhanced the incidence of extra molting up to 100%. When 1 ng was applied in the 4th instar after a 2-day starvation, most larvae underwent an extra molt, metamorphosed to pupae, then to fertile adults. Combining starvation and fenoxycarb application thus induces a perfect extra molt efficiently. In perfect extra molting larvae, profiles of total ecdysteroid titer during the 4th and 5th instars were similar to that during the 4th instar in the control, and the ecdysteroid profile during the extra 6th instar was similar to that during the control 5th (last) instar. At ecdysteroid peaks, 20-hydroxyecdysone (20E) and ecdysone (E), generally regarded as the active molting hormone and its precursor, had similar titers in the 6th instar, whereas E was much less than 20E in the 4th and 5th instars in the extra molting larvae. E was also abundant only in the last larval instar in the control. These results suggest that both 20E and E contents are important for regulation of larval molt and metamorphosis in silkworms and that fenoxycarb triggers the extra molt by inducing an additional larval molt type of ecdysteroid surge before the last larval instar. PMID:12392697
Kamimura, Manabu; Kiuchi, Makoto
Role of Bmbuffy in hydroxycamptothecine-induced apoptosis in BmN-SWU1 cells of the silkworm, Bombyx mori.
Bcl-2 family proteins have been reported previously to play important roles in the mitochondrial apoptotic pathway. Particularly, Bmbuffy has been identified as a key homologue of Bcl-2 in silkworm; however, its exact function is unknown. In this study, we investigated the role of Bmbuffy in hydroxycamptothecine (HCPT)-induced apoptosis of BmN-SWU1 cells. By conducting confocal microscopy studies, we found that Bmbuffy is located on the outer membrane of mitochondria and endoplasmic reticulum (ER). Furthermore, we discovered that the hydrophobic transmembrane domain at the COOH terminus is a putative anchor for the subcellular localization of Bmbuffy. Overexpression of Bmbuffy inhibited cytochrome c release, activation of caspase-3 and cell apoptosis, while RNAi-mediated silencing of Bmbuffy promoted apoptosis. In the absence of a hydrophobic membrane anchor, we revealed that Bmbuffy is unable to block apoptosis. These results indicate that Bmbuffy acts as an anti-apoptotic protein, located on the mitochondrial outer membrane and is involved in the mitochondrial apoptotic pathway. Moreover, in HCPT-induced apoptosis, we showed that the translocation of endogenous Bmp53 from the nucleus to the mitochondria is a slow and progressive process, followed by cytochrome c release. This suggests that mitochondrial Bmp53 accumulation may contribute to membrane permeability. The co-localization of Bmp53 and Bmbuffy suggests the interaction of the two proteins, which was further confirmed by Co-IP assay. In addition, overexpression of Bmp53 increased cytochrome c release and the cell apoptotic rate, whereas Bmbuffy overexpression blocked these. All the data suggest that Bmbuffy functions as an anti-apoptotic protein and interacts with Bmp53 in HCPT-induced apoptosis of silkworm cells. PMID:24690173
Pan, Chun; Hu, Yan-Fen; Yi, Hua-Shan; Song, Juan; Wang, La; Pan, Min-Hui; Lu, Cheng
Effects of Feeding Silkworm with Nanoparticulate Anatase TiO2 (TiO 2 NPs) on Its Feed Efficiency.
This study aimed to investigate the effects of titanium dioxide nanoparticles (TiO2 NPs) on feed efficiency of silkworm (Bombyx mori) (B. mori). The results showed that the amount of ingested food was increased by 3.31�%, the percentage of ingested food was increased by 16.36�%, the instar was prolonged by 6.77�h, the body mass of mature silkworm was increased by 11.41�%, the cocoon mass was increased by 5.47�%, the cocoon shell mass was increased by 9.80�%, and the ratio of cocoon shell was increased by 3.54�%. Average cocoon filament length, reelability, and neatness were increased by 3.15, 17.57, and 1.72�%, respectively, whereas the cocoon filament size was decreased by 0.19�%. The gene expression profiles were also examined with digital gene expression (DGE). The results showed that among 4,011 genes detected, 127 genes were differentially expressed including 57 genes whose expression was upregulated and 70 genes whose expression was downregulated. The expression levels of five genes related to metabolism were verified with quantitative real-time PCR, and the results were consistent with the DGE data. The activities of three major digestive enzymes in the midgut were also assayed; the results showed that the activities of trehalase, protease, and lipase in TiO2 NP-fed group were increased by 42.55, 78.13, and 33.33�%, respectively. These results indicate that feeding B. mori with TiO2 NPs can stimulate the transcriptional level of genes related to digestion and absorption of nutrients in the midgut and increase the activities of trehalase, protease, and lipase and, thus, increase the feed efficiency of B. mori. PMID:24789477
Zhang, Hua; Ni, Min; Li, Fanchi; Xu, Kaizun; Wang, Binbin; Hong, Fashui; Shen, Weide; Li, Bing
A single female-specific piRNA is the primary determiner of sex in the silkworm.
The silkworm Bombyx mori uses a WZ sex determination system that is analogous to the one found in birds and some reptiles. In this system, males have two Z sex chromosomes, whereas females have Z and W sex chromosomes. The silkworm W chromosome has a dominant role in female determination, suggesting the existence of a dominant feminizing gene in this chromosome. However, the W chromosome is almost fully occupied by transposable element sequences, and no functional protein-coding gene has been identified so far. Female-enriched PIWI-interacting RNAs (piRNAs) are the only known transcripts that are produced from the sex-determining region of the W chromosome, but the function(s) of these piRNAs are unknown. Here we show that a W-chromosome-derived, female-specific piRNA is the feminizing factor of B. mori. This piRNA is produced from a piRNA precursor which we named Fem. Fem sequences were arranged in tandem in the sex-determining region of the W chromosome. Inhibition of Fem-derived piRNA-mediated signalling in female embryos led to the production of the male-specific splice variants of B. mori doublesex (Bmdsx), a gene which acts at the downstream end of the sex differentiation cascade. A target gene of Fem-derived piRNA was identified on the Z chromosome of B. mori. This gene, which we named Masc, encoded a CCCH-type zinc finger protein. We show that the silencing of Masc messenger RNA by Fem piRNA is required for the production of female-specific isoforms of Bmdsx in female embryos, and that Masc protein controls both dosage compensation and masculinization in male embryos. Our study characterizes a single small RNA that is responsible for primary sex determination in the WZ sex determination system. PMID:24828047
Kiuchi, Takashi; Koga, Hikaru; Kawamoto, Munetaka; Shoji, Keisuke; Sakai, Hiroki; Arai, Yuji; Ishihara, Genki; Kawaoka, Shinpei; Sugano, Sumio; Shimada, Toru; Suzuki, Yutaka; Suzuki, Masataka G; Katsuma, Susumu
Oak Ridge National Laboratory Waste Management Plan
The objective of the Oak Ridge National Laboratory Waste Management Plan is to compile and to consolidate information annually on how the ORNL Waste Management Program is conducted, which waste management facilities are being used to manage wastes, what forces are acting to change current waste management systems, what activities are planned for the forthcoming fiscal year (FY), and how all of the activities are documented.
Not Available
Processing of Oak Ridge Mixed Waste Labpacks
The Oak Ridge Site Treatment Plan (STP) issued under a Tennessee Commissioner's Order includes a compliance milestone related to treatment of mixed waste labpacks on the Oak Ridge sites. The treatment plan was written and approved in Fiscal Year 1997. The plan involved approximately 1,100 labpacks and 7,400 on-the-shelf labpackable items stored at three Department of Energy (DOE) sites on the Oak Ridge Reservation (ORR). The labpacks and labpack items consist of liquids and solids with various chemical constituents and radiological concerns. The waste must be processed for shipment to a commercial hazardous waste treatment facility or treatment utilizing a Broad Spectrum mixed waste treatment contract. This paper will describe the labpack treatment plan that was developed as required by the Site Treatment Plan and the operations implemented to process the labpack waste. The paper will discuss the labpack inventory in the treatment plan, treatment and disposal options, processing strategies, project risk assessment, and current project status.
Estes, C. H.; Franco, P.; Bisaria, A.
Stability analysis of White Oak Dam
White Oak Dam is located in the White Oak Creek watershed which provides the primary surface drainage for Oak Ridge National Laboratory. A stability analysis was made on the dam by Syed Ahmed in January 1994 which included an evaluation of the liquefaction potential of the embankment and foundation. This report evaluates the stability of the dam and includes comments on the report prepared by Ahmed. Slope stability analyses were performed on the dam and included cases for sudden drawdown, steady seepage, partial pool and earthquake. Results of the stability analyses indicate that the dam is stable and failure of the structure would not occur for the cases considered. The report prepared by Ahmed leads to the same conclusions as stated above. Review of the report finds that it is complete, well documented and conservative in its selection of soil parameters. The evaluation of the liquefaction potential is also complete and this report is in agreement with the findings that the dam and foundation are not susceptible to liquefaction.
Environmental restoration: Oak Ridge National Laboratory perspective
The Oak Ridge National Laboratory (ORNL), located in Oak Ridge, Tennessee, is a multipurpose research facility managed by Martin Marietta Energy Systems, Inc. (Energy Systems) for the US Deparment of Energy-Oak Ridge Operations (DOE). The operation of ORNL has resulted in a legacy of contaminated and potentially contaminated facilities, research areas, and waste management areas that may require remediation. The most recent inventory of remediation sites has identified approximately 400 individual sites that will require investigation and possibly remediation. The Remedial Action program (RAP) was established at ORNL in 1985 to conduct the investigations, studies,and remediation necessary to prevent unacceptable risks to the environment and to the public from this legacy of contaminated sites. Then, in 1989 a central Environmental Restoration program (ERP) was established that consolidates the previous RAPs at all five sites managed by Energy Systems for DOE. This paper describes how a program was developed to solve the large and diverse problems associated with the environmental restoration of the ORNL. 3 figs., 1 tab.
Garland, S.B. II.
Partial release of aminopeptidase N from larval midgut cell membranes of the silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C.
1. The membrane anchor of aminopeptidase N associated with larval midgut cell membranes of the silkworm, Bombyx mori, was investigated by using phosphatidylinositol-specific phospholipase C (PIPLC) and proteases. 2. Aminopeptidase N, which was virtually all localized in the brush border membrane, was solubilized by PIPLC but not by papain or trypsin. 3. Detergent-solubilized amphiphilic aminopeptidase N was converted into a hydrophilic form by PIPLC but not by papain. 4. Either of these effects of PIPLC on aminopeptidase N was maximally 40%. 5. These results suggest that in larval midgut cells of the silkworm, B. mori, at least 40% aminopeptidase N is anchored in the brush border membrane via glycosyl-phosphatidylinositol. PMID:1356082
Takesue, S; Yokota, K; Miyajima, S; Taguchi, R; Ikezawa, H; Takesue, Y
The gustatory receptor family in the silkworm moth Bombyx mori is characterized by a large expansion of a single lineage of putative bitter receptors.
The gustatory receptor (Gr) family of insect chemoreceptors includes receptors for sugars and bitter compounds, as well as cuticular hydrocarbons and odorants such as carbon dioxide. We have annotated a total of 65 Gr genes from the silkworm Bombyx mori genome. The Gr family in the silkworm moth includes putative carbon dioxide receptors and sugar receptors, as well as duplicated orthologues of the orphan DmGr43a receptor. Most prominent in this 65-gene family, however, is a single large expansion of 55 Grs that we propose are predominantly 'bitter' receptors involved in perception of the large variety of secondary plant chemicals that caterpillars and moths encounter. These Grs might therefore mediate food choice and avoidance as well as oviposition site preference. PMID:19133074
Wanner, K W; Robertson, H M
Effect of Induced Oxidative Stress and Herbal Extracts on Acid Phosphatase Activity in Lysosomal and Microsomal Fractions of Midgut Tissue of the Silkworm, Bombyx mori
Lysosomal and microsomal acid phosphatase activity was estimated in midgut tissue of silkworm larvae, Bombyx mori L. (Lepidoptera: Bombycidae), after induced oxidative stress by D-galactose. The larvae were simultaneously were treated with ethanolic extracts of Bacopa monniera and Lactuca sativa to study their antioxidant properties. Lipid peroxidation and fluorescence was measured to analyze extent of oxidative stress. The ethanolic extract of Lactuca sativa was found to be more effective in protecting membranes against oxidative stress than Bacopa monniera.
Gaikwad, Y. B.; Gaikwad, S. M.; Bhawane, G. P.
Manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L enhances the NK activity and splenocyte proliferation against Sarcoma 180 tumor cells in�vivo
Natural killer cell (NK) is known as a major immune system in body through mediating cell death via several possible pathways,\\u000a and one of three subpopulations of lymphocytes functioning as scavenger of tumor, virus infected cells etc. Our present results\\u000a found that the SOD-contained silkworm larvae powder caused an enhancement of the effect on NK cell cytotoxicity, which implied\\u000a this
Wan-Fu Yue; Min-Li Yao; Jian-Mei Liu; Guang-Li Li; Xing-Hua Li; Xiao-Feng Wu; Wen Deng; Hong-Xiang Sun; Ji-Yong Zhou; Chuan-Xi Zhang; Yun-Gen Miao
Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac\\/BmNPV Baculovirus expression system
Superoxide dismutase (SODs) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and, thus, form a crucial part of the cellular antioxidant defense mechanism. In this paper, we used the total fat body RNA of silkworm, Bombyx mori L. to clone and sequence a 648-bp Mn-SOD cDNA fragment through RT-PCR. Furthermore, a newly
Wanfu Yue; Yungen Miao; Xinghua Li; Xiaofeng Wu; Aichun Zhao; Masao Nakagaki
In Vivo Expression and Immunological Studies of the 42-Kilodalton Carboxyl-Terminal Processing Fragment of Plasmodium falciparum Merozoite Surface Protein 1 in the Baculovirus-Silkworm System
The 42-kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-142) is an anti-erythrocytic stage malaria vaccine candidate. In this study, MSP-142 was expressed by using the Bombyx mori nuclear polyhedrosis virus-silkworm expression system, and the antigenicity and immmunogenicity of the recombinant protein, Bmp42, were evaluated. The average yield of Bmp42, as deter- mined by a sandwich enzyme-linked
Alan L. Y. Pang; Caryn N. Hashimoto; Leslie Q. Tam; Z. Q. Meng; George S. N. Hui; Walter K. K. Ho
Specific expression of GFP uv-?1,3- N-acetylglucosaminyltransferase 2 fusion protein in fat body of Bombyx mori silkworm larvae using signal peptide
Bombyxin (bx) and prophenoloxidase-activating enzyme (ppae) signal peptides from Bombyx mori, their modified signal peptides, and synthetic signal peptides were investigated for the secretion of GFPuv-?1,3-N-acetylglucosaminyltransferase 2 (GGT2) fusion protein in B. mori Bm5 cells and silkworm larvae using cysteine protease deficient B. mori multiple nucleopolyhedrovirus (BmMNPV-CP?) and its bacmid. The secretion efficiencies of all signal peptides were 15�30% in
Tatsuya Kato; Enoch Y. Park
Expression of metabolic enzyme genes and heat-shock protein genes during embryonic development in diapause and non-diapause egg of multivoltine silkworm Bombyx mori
The expression of metabolic enzyme genes and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm Bombyx mori was quantified by semi-quantitative RT-PCR. The trehalase gene (Tre) was expressed in non-diapause eggs up-to nine days, while in diapause eggs was not up regulated. The glycogen phosphorylase\\u000a gene (GPase) was expressed in non-diapause eggs, whereas
Ragunathan Saravanakumar; Kangayam M. Ponnuvel; Syed M. Hussaini Qadri
Proteome analysis on differentially expressed proteins of the fat body of two silkworm breeds, Bombyx mori , exposed to heat shock exposure
Proteomes of heat tolerant (multivoltine) and heat susceptible (bivoltine) silkworms (Bombyx mori) in response to heat shock were studied. Detected proteins from fat body were identified by using MALDI-TOF\\/TOF spectrometer,\\u000a MS\\/MS, and MS analysis. Eight proteins, including small heat shock proteins (sHSPs) and HSP70, were expressed similarly in\\u000a both breeds, while 4 protein spots were expressed specifically in the bivoltine
S. Hossein Hosseini Moghaddam; Xin Du; Jun Li; Jinru Cao; Boxiong Zhong; YuYin Chen
Effect of Fenoxycarb on Leucine Uptake and Lipid Composition of Midgut Brush Border Membrane in the Silkworm, Bombyx mori (Lepidoptera, Bombycidae)
Topical or oral applications of fenoxycarb (FC) to fifth instar larvae of Bombyx mori were performed immediately after the fourth ecdysis. A clear dose-response relationship in the frass-failure assay was recorded after 48 h for topically treated silkworms, whereas the oral treatment caused the maximal decrease of frass production even at the lowest dose (2.5 fg\\/larva). Preincubation of midgut brush
M. G. Leonardi; P. Marciani; P. G. Montorfano; S. Cappellozza; B. Giordana; G. Monticelli
Morphological and molecular studies of Vairimorpha necatrix BM, a new strain of the microsporidium V. necatrix (microsporidia, burenellidae) recorded in the silkworm, Bombyx mori.
Vairimorpha sp. BM (2012) is a recent isolate of the microsporidia from the silkworm in Shandong, China. The ultrastructure, tissue pathology and molecular characterization of this isolate is described in this study. This pathogenic fungus causes pebrine disease in silkworms which manifests as a systemic infection. Meanwhile, the silkworm eggs produced by the infected moths were examined using a microscope and PCR amplification. Neither spores nor the expected PCR band were observed, suggesting that no vertical transmission occurred in Bombyx mori. In addition, the ultrastructure of the isolate was studied by light microscopy and transmission electron microscopy. Two types of spores were observed: diplokaroytic spores with 13-17 coils of polar tubes and monokaryotic spores with less coils of polar tubes which could form octospores; however, no sporophorous vesicles were observed. Finally, phylogenetic analysis of the small subunit rRNA genes of Vairimorpha species showed that this isolate has a closer relationship to Vairimorpha necatrix than the other species studied. This result also is supported by phylogenetic analysis based on their actin genes, heat shock protein 70 (HSP70) and RNA polymerase II (RPB1). Based on the information gained during this study, we propose that this microsporidian species infecting B. mori should be given the name V. necatrix BM. PMID:24818618
Luo, Bo; Liu, Handeng; Pan, Guoqing; Li, Tian; Li, Zeng; Dang, Xiaoqun; Liu, Tie; Zhou, Zeyang
Identification of a protein interacting with the spore wall protein SWP26 of Nosema bombycis in a cultured BmN cell line of silkworm.
Nosema bombycis is a silkworm parasite that causes severe economic damage to sericulture worldwide. It is the first microsporidia to be described in the literature, and to date, very little molecular information is available regarding microsporidian physiology and their relationships with their hosts. Therefore, the interaction between the microsporidia N. bombycis and its host silkworm, Bombyx mori, was analyzed in this study. The microsporidian spore wall proteins (SWPs) play a specific role in spore adherence to host cells and recognition by the host during invasion. In this study, SWP26 fused with enhanced green fluorescence protein (EGFP) was expressed in BmN cells by using a Bac-to-Bac expression system. Subsequently, the turtle-like protein of B. mori (BmTLP) was determined to interact with SWP26 via the use of anti-EGFP microbeads. This interaction was then confirmed by yeast two-hybrid analysis. The BmTLP cDNA encodes a polypeptide of 447 amino acids that includes a putative signal peptide of 27 amino acid residues. In addition, the BmTLP protein contains 2 immunoglobulin (IG) domains and 2 IGc2-type domains, which is the typical domain structure of IG proteins. The results of this study indicated that SWP26 interacts with the IG-like protein BmTLP, which contributes to the infectivity of N. bombycis to its host silkworm. PMID:23542093
Zhu, Feng; Shen, Zhongyuan; Hou, Jiange; Zhang, Jiao; Geng, Tao; Tang, Xudong; Xu, Li; Guo, Xijie
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. PMID:17525867
Zhang, Yuansong; Hu, Junhua; Miao, Yungen; Zhao, Aichun; Zhao, Tianfu; Wu, Dayang; Liang, Liefeng; Miikura, Ayumi; Shiomi, Kunihiro; Kajiura, Zenta; Nakagaki, Masao
Isolation of megastigmane sesquiterpenes from the silkworm (Bombyx mori L.) droppings and their promotion activity on HO-1 and SIRT1.
The silkworm (Bombyx mori L.) droppings were extracted with 80% aqueous MeOH, and the concentrated extract was partitioned in succession with EtOAc, n-BuOH, and H(2)O. From the EtOAc fraction, five megastigmane sesquiterpenes were isolated through repeated silica gel and ODS column chromatography. According to the results of spectroscopic data, such as NMR, MS, and IR, the chemical structures of the isolated compounds were determined as (3S,5R,8R)-3,5-dihydroxymegastigma-6,7-dien-9-one (1), (S)-dehydrovomifoliol (2), (6R,7E,9R) -9-hydroxy-4,7-megastigmadien-3-one (3), (3S,5R,6S,7E)-3,5,6-trihydroxy-7-megastigmen-9-one (4), (6R,9R)-9-hydroxy-4-megastigmen-3-one (5). Compounds 2 through 5 were isolated for the first time from silkworm droppings. GC/MS analysis indicated silkworm powder contained compound 3, and mulberry leaves contained compound 4. Compounds 1 and 5 increased the expression of heme oxygenase-1 and SIRT1 in HepG2 and HEK239 cells, respectively. Heme oxygenase-1 is considered to be an antioxidant enzyme that catabolizes heme to carbon monoxide, free iron and biliverdin, while SIRT1 is the mammalian homologue of the yeast silent information regulator (Sir)-2, which are involved in the suppression of inflammatory mediators or factors that may be used to improve atopy-related symptoms. PMID:21544718
Park, Ji-Hae; Lee, Do-Gyeong; Yeon, Seung-Woo; Kwon, Hyuk-Sang; Ko, Jong-Hee; Shin, Dong-Jin; Park, Han-Sol; Kim, Yong-Soon; Bang, Myun-Ho; Baek, Nam-In
Floodplain and wetlands assessment of the White Oak Creek Embayment
This report describes the proposed methods for dealing with contaminants that have accumulated in White Oak Creek, White Oak Lake, and the White Oak Creek Embayment as a result of process releases and discharges from the Oak Ridge National Laboratory. Alternative methods of cleaning up the area which were considered in accordance with regulatory guidelines are listed, and information supporting the selected methods is provided. Also included are results of a site survey conducted at the White Oak Creek Embayment and the expected effects of the proposed control structures on the floodplain and wetlands. The appendix contains figures showing the nine cross-sections of the stream channel surveyed during studies of the White Oak Creek area.
Oak Woodland Restoration: Understory Response to Removal of Encroaching Conifers
Oregon white oak (or Carry oak, Quercusgarryana) woodlands and savannas of the coastal Pacific Northwest are legacies of an anthropogenic fire regime that ended with European settlement in the mid-1 800s. Historically, these oak stands had a sparse overstory and an understory dominated by fire-tolerant grasses and forbs. Post-settlement fire suppression resulted in widespread invasion and subsequent overstory dominance by
Warren D. Devine; Constance A. Harrington; David H. Peter
? Abstract Oak gall wasps (Hymenoptera: Cynipidae, Cynipini) are characterized by possession of complex,cyclically parthenogenetic,life cycles and the ability to induce a wide,diversity of highly complex,species- and generation-specific galls on oaks and other Fagaceae. The galls support species-rich, closed communities of inquilines and parasitoids that have become,a model,system,in community,ecology. We review recent advances in the ecology of oak cynipids, with
Graham N. Stone; Karsten Schonrogge; Rachel J. Atkinson; David Bellido; Juli Pujade-Villar
Specific expression of GFP{sub uv}-{beta}1,3-N-acetylglucosaminyltransferase 2 fusion protein in fat body of Bombyx mori silkworm larvae using signal peptide
Bombyxin (bx) and prophenoloxidase-activating enzyme (ppae) signal peptides from Bombyx mori, their modified signal peptides, and synthetic signal peptides were investigated for the secretion of GFP{sub uv}-{beta}1,3-N-acetylglucosaminyltransferase 2 (GGT2) fusion protein in B. mori Bm5 cells and silkworm larvae using cysteine protease deficient B. mori multiple nucleopolyhedrovirus (BmMNPV-CP{sup -} ) and its bacmid. The secretion efficiencies of all signal peptides were 15-30% in Bm5 cells and 24-30% in silkworm larvae, while that of the +16 signal peptide was 0% in Bm5 cells and 1% in silkworm larvae. The fusion protein that contained the +16 signal peptide was expressed specifically in the endoplasmic reticulum (ER) and in the fractions of cell precipitations. Ninety-four percent of total intracellular {beta}1,3-N-acetylglucosaminyltransferase ({beta}3GnT) activity was detected in cell precipitations following the 600, 8000, and 114,000g centrifugations. In the case of the +38 signal peptide, 60% of total intracellular activity was detected in the supernatant from the 114,000g spin, and only 1% was found in the precipitate. Our results suggest that the +16 signal peptide might be situated in the transmembrane region and not cleaved by signal peptidase in silkworm or B. mori cells. Therefore, the fusion protein connected to the +16 signal peptide stayed in the fat body of silkworm larvae with biological function, and was not secreted extracellularly.
Kato, Tatsuya [Laboratory of Biotechnology, Faculty of Agriculture, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529 (Japan); Park, Enoch Y. [Laboratory of Biotechnology, Faculty of Agriculture, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529 (Japan) and Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529 (Japan)]. E-mail: email@example.com
Spatial genetic structure in populations of the green oak leaf roller, Tortrix viridana L. (Lepidoptera, Tortricidae)
The green oak leaf roller (Tortrix viridana L.) is a major pest on oaks. The green oak leaf roller is one reason within a disease complex for the oak decline, and it\\u000a leads to defoliation of oaks in spring. In Germany, Quercus robur L. is mainly affected. The partitioning of genetic variation within and among populations of forest pests is
Hilke Schroeder; Bernd Degen
Associations Among Breeding Birds and Gambel Oak in Southwestern Ponderosa Pine Forests
Ponderosa pine (Pinus ponderosa) forests with Gambel oak (Quercus gambelii) are associated with higher bird abundance and diversity than are ponderosa pine forests lacking Gambel oak. Little is known, however, about specific structural characteristics of Gambel oak trees, clumps, and stands that may be important to birds in ponderosa pine-Gambel oak (hereafter pine-oak) forests. We examined associations among breeding birds
Stephanie Jentsch; R. William Mannan; Brett G. Dickson; William M. Block
Relationships between advance oak regeneration and biotic and abiotic factors.
Relationships between advance regeneration of four tree species (red maple (Acer rubrum L.), white oak (Quercus alba L.), chestnut oak (Q. montana Willd.) and northern red oak (Q. rubra L.)) and biotic (non-tree vegetation and canopy composition) and abiotic (soil series and topographic variables) factors were investigated in 52, mature mixed-oak stands in the central Appalachians. Aggregate height was used as a composite measure of regeneration abundance. Analyses were carried out separately for two physiographic provinces. Associations with tree regeneration were found for all biotic and abiotic factors both in partial models and full models. Red maple was abundant on most of the sites, but high red maple abundance was commonly associated with wet north-facing slopes with little or no cover of mountain-laurel (Kalmia latifolia L.) and hay-scented fern (Dennstaedtia punctilobula (Michx.) Moore). Regeneration of the three oak species was greatly favored by the abundance of overstory trees of their own kind. White oak regeneration was most abundant on south-facing, gentle, lower slopes with soils in the Buchanan series. Chestnut oak regeneration was more common on south-facing, steep upper slopes with stony soils. There was a positive association between chestnut oak and huckleberry (Gaylussacia baccata (Wangh.) Koch) cover classes. Northern red oak was more abundant on north-facing wet sites with Hazleton soil, and was associated with low occurrence of mountain-laurel and hay-scented fern. PMID:18450575
Fei, Songlin; Steiner, Kim C
The Nuclear Materials Identification System (NMIS), developed by the Oak Ridge National Laboratory and Oak Ridge Y-12 Plant (Y-12), has been successfully used at Y-12 for nuclear material control and accountability (NMC&A). It is particularly useful in the high gamma-ray background of storage arrays and for shielded HEU. With three systems in use at Y-12, NMIS has enhanced the NMC&A capability for verification and for confirmation of materials in storage and for HEU receipts by providing capability not available or practical by other NDA methods for safeguards. It has recently cost-effectively quantified the HEU mass and enrichment of hundreds of HEU metal items to within a total spread of {+-} 5% (3 sigma) with and mean deviations for all HEU verified of + 0.2% for mass and {minus}0.2% for enrichment. Three cart portable systems are easily moved around with minimal impact on facility operations since no permanent dedicated floor space is required. The positive impact of NMIS at the Oak Ridge Y-12 Plant is improved and more cost effective NMC&A as well as the resolution of NMC&A findings. Its operation at the Y-12 Plant is essential for compliance with the NMC&A requirements of the US Department of Energy. NMIS portability has allowed one system to be moved temporarily to the former K-25 Gaseous Diffusion Plant for characterization of a large deposit of hydrated uranyl fluoride. The impact of this NMIS application was enhanced and verified nuclear criticality safety that led to the safe removal of a large deposit originally estimated by gamma-ray spectrometry and neutron counting to contain 1300 kg of 3.3 wt% {sup 235}U material. NMIS has also been operational at Los Alamos National Laboratory and Pantex.
Chiang, L.G.; Conger, M.; Hughes, S.S.; Mattingly, J.K.; McEvers, J.A.; Mihalczo, J.T.; Mullens, J.A.; Perez, R.B.; Turner, C.R.; Uckan, T.; Valentine, T.E.
Crush Testing at Oak Ridge National Laboratory
The dynamic crush test is required in the certification testing of some small Type B transportation packages. International Atomic Energy Agency regulations state that the test article must be subjected to a dynamic crush test by positioning the specimen on the target so as to suffer maximum damage. Oak Ridge National Laboratory (ORNL) Transportation Technologies Group performs testing of Type B transportation packages, including the crush test, at the National Transportation Research Center in Knoxville, Tennessee (United States). This paper documents ORNL s experiences performing crush tests on several different Type B packages.
Feldman, Matthew R [ORNL
3001 canal radiological characterization and waste removal report, Oak Ridge National Laboratory, Oak Ridge, Tennessee
An underground steel reinforced concrete transfer and storage canal was built in 1943 and operated as an integral part of the Oak Ridge Graphite Reactor Building (3001) until 1963 when the reactor was shutdown. During operation, the canal was used for under water transfer of irradiated materials and other metals from the reactor in Building 3001 to the Building 3019
Survey of protected vascular plants on the Oak Ridge Reservation, Oak Ridge, Tennessee.
Vascular plant surveys were initiated during fiscal year 1992 by the environmentally sensitive areas program to determine the baseline condition of threatened and endangered (T&E) vascular plant species on the Oak Ridge Reservation (ORR). T&E species rece...
D. J. Awl L. R. Pounds B. A. Rosensteel A. L. King P. A. Hamlett
Elution of Nitrate at the NABIR Field Research Center, Oak Ridge Reservation, Oak Ridge, TN
As part of a bioremediation project for the in situ bioreduction of uranium at the Department of Energy Natural and Accelerated Bioremediation Research (NABIR) Field Research Center (FRC) in Oak Ridge, TN, aquifer and groundwater conditioning is required before conducting the remediation experiment. One step includes flushing of the aquifer with pH-adjusted fresh water in order to remove extremely high
M. N. Fienen; C. S. Criddle; P. M. Jardine; P. K. Kitanidis; T. L. Mehlhorn; D. B. Watson; W. Wu
US Department of Energy Oak Ridge Operations Environmental Management Public Involvement Plan for the Oak Ridge Reservation
This document was prepared in accordance with CERCLA requirements for writing community relations plans. It includes information on how the DOE Oak Ridge Operations Office prepares and executes Environmental Management Community relations activities. It is divided into three sections: the public involvement plan, public involvement in Oak Ridge, and public involvement in 1995. Four appendices are also included: environmental management in Oak Ridge; community and regional overview; key laws, agreements, and policy; and principal contacts.
Fossil oak galls preserve ancient multitrophic interactions.
Trace fossils of insect feeding have contributed substantially to our understanding of the evolution of insect-plant interactions. The most complex phenotypes of herbivory are galls, whose diagnostic morphologies often allow the identification of the gall inducer. Although fossil insect-induced galls over 300Myr old are known, most are two-dimensional impressions lacking adequate morphological detail either for the precise identification of the causer or for detection of the communities of specialist parasitoids and inquilines inhabiting modern plant galls. Here, we describe the first evidence for such multitrophic associations in Pleistocene fossil galls from the Eemian interglacial (130000-115000 years ago) of The Netherlands. The exceptionally well-preserved fossils can be attributed to extant species of Andricus gallwasps (Hymenoptera: Cynipidae) galling oaks (Quercus), and provide the first fossil evidence of gall attack by herbivorous inquiline gallwasps. Furthermore, phylogenetic placement of one fossil in a lineage showing obligate host plant alternation implies the presence of a second oak species, Quercus cerris, currently unknown from Eemian fossils in northwestern Europe. This contrasts with the southern European native range of Q. cerris in the current interglacial and suggests that gallwasp invasions following human planting of Q. cerris in northern Europe may represent a return to preglacial distribution limits. PMID:18559323
Stone, Graham N; van der Ham, Raymond W J M; Brewer, Jan G
Fossil oak galls preserve ancient multitrophic interactions
Trace fossils of insect feeding have contributed substantially to our understanding of the evolution of insect�plant interactions. The most complex phenotypes of herbivory are galls, whose diagnostic morphologies often allow the identification of the gall inducer. Although fossil insect-induced galls over 300?Myr old are known, most are two-dimensional impressions lacking adequate morphological detail either for the precise identification of the causer or for detection of the communities of specialist parasitoids and inquilines inhabiting modern plant galls. Here, we describe the first evidence for such multitrophic associations in Pleistocene fossil galls from the Eemian interglacial (130?000�115?000 years ago) of The Netherlands. The exceptionally well-preserved fossils can be attributed to extant species of Andricus gallwasps (Hymenoptera: Cynipidae) galling oaks (Quercus), and provide the first fossil evidence of gall attack by herbivorous inquiline gallwasps. Furthermore, phylogenetic placement of one fossil in a lineage showing obligate host plant alternation implies the presence of a second oak species, Quercus cerris, currently unknown from Eemian fossils in northwestern Europe. This contrasts with the southern European native range of Q. cerris in the current interglacial and suggests that gallwasp invasions following human planting of Q. cerris in northern Europe may represent a return to preglacial distribution limits.
Stone, Graham N; van der Ham, Raymond W.J.M; Brewer, Jan G
The goal of the Oak Ridge National Laboratory (ORNL) Waste Management Program is the protection of workers, the public, and the environment. A vital aspect of this goal is to comply with all applicable state, federal, and DOE requirements. Waste management requirements for DOE radioactive wastes are detailed in DOE Order 5820.2A, and the ORNL Waste Management Program encompasses all elements of this order. The requirements of this DOE order and other appropriate DOE orders, along with applicable Tennessee Department of Environment and Conservation (TDEC) and US Environmental Protection Agency (EPA) rules and regulations, provide the principal source of regulatory guidance for waste management operations at ORNL. The objective of the Oak Ridge National Laboratory Waste Management Plan is to compile and to consolidate information annually on how the ORNL Waste Management is to compile and to consolidate information annually on how the ORNL Waste Management Program is conducted, which waste management facilities are being used to manage wastes, what forces are acting to change current waste management systems, what activities are planned for the forthcoming fiscal year (FY), and how all of the activities are documented.
Regional impacts of periodical cicadas on oak radial increment
We investigated the effects of periodical cicadas (Magicicada spp.) on growth of their hosts using records of radial tree-ring growth on oaks (Quercus spp., a common host genus) and pines (Pinus spp., a common nonhost genus) growing within the ranges of 13- and 17-year broods. Growth of oaks was significantly depressed during years of adult emergence by about 4%. Effects
Walter D. Koenig; Andrew M. Liebhold
Fair Oaks Dairy Farms Cellulosic Ethanol Technology Review Summary
At Fair Oaks Dairy, dried manure solids (''DMS'') are currently used as a low value compost. United Power was engaged to evaluate the feasibility of processing these DMS into ethanol utilizing commercially available cellulosic biofuels conversion platforms. The Fair Oaks Dairy group is transitioning their traditional ''manure to methane'' mesophilic anaerobic digester platform to an integrated bio-refinery centered upon thermophilic
Andrew Wold; Robert Divers
Clinical Investigation of Hyposensitization in Poison Oak Sensitive Individuals.
The purpose of this contract was to determine whether individuals in California who were sensitive to poison oak could be desensitized by the use of the purified active principle of poison oak, urushiol. Many adult males were skin-tested to determine sens...
W. L. Epstein
With the exception of small mammals, little research has been conducted in eastern oak forests on the influence of fire on mammals. Several studies have documented little or no change in relative abundance or community measures for non-volant small mammals in eastern oak (Quercus spp.) forests following fires despite reductions in leaf litter, small woody debris, and changes in understory
Patrick D. Keyser; W. Mark Ford
Cynipid Gall-Wasp Communities Correlate with Oak Chemistry
Host-plant association data, gathered from field surveys conducted throughout Florida and from the literature, were used to identify the specificity of cynipid gall inducers to one or more of six Quercus species that occur at Archbold Biological Station, Lake Placid, Florida, USA, including the red oaks Q. laevis, Q. myrtifolia, and Q. inopina, and the white oaks Q. chapmanii, Q.
Warren G. Abrahamson; Mark D. Hunter; George Melika; Peter W. Price
Galls are commonly found on urban trees. In- duced by oviposition of insects and other arthropods, galls develop from woody tree tissues, forming shelters for develop- ing larvae. Few galls are physiologically harmful to the tree. Some, like the mealy-oak galls on live oak, are not only harmless but may harbor beneficial arthropods long after the gall-maker has departed. Because
David L. Morgan; Gordon W. Frankie
Short Note: High Throughput Microsatellite Genotyping in Oak Species
Microsatellites are widely used markers for multiple purposes in oaks. We describe a complete procedure for cheap DNA extraction and fast microsatellites genotyp- ing by multiplex PCR. 10 loci were selected to form two multiplex kits including three loci that show a high dif- ferentiation between Quercus robur and Q. petraea. The loci were tested in three oak species and
Postconstruction report for the mercury tanks interim action at the Oak Ridge Y-12 Plant, Oak Ridge, Tennessee.
Three underground concrete settling tanks (tanks 2101-U, 2104-U, and 2100-U) at the Y-12 Plant on the Oak Ridge Reservation in Oak Ridge, Tennessee, contained contaminated sludges contributing mercury to the Upper East Fork Poplar Creek (UEFPC). These tan...
T. L. Voskuil
Superfund Record of Decision (EPA Region 4): Oak Ridge Reservation (USDOE), Operable Unit 14, Oak Ridge, TN., September 25, 1997.
This record of decision (ROD) presents the selected remedial action for the Surface Impoundments Operable Unit (SIOU) on the U.S. Department of Energy (DOE) Oak Ridge Reservation (ORR) in Oak Ridge, Tennessee. the selected remedy addresses the principal t...
Phase I remedial investigation report of Waste Area Grouping 2 at Oak Ridge National Laboratory, Oak Ridge, Tennessee.
This report presents the activities and findings of the first phase of a three-phase remedial investigation (RI) of Waste Area Grouping (WAG) 2 at Oak Ridge National Laboratory (ORNL) in Oak Ridge, Tennessee, and updates the scope and strategy for WAG-2-r...
D. E. Miller
Influence of genetic variation of oak trees as a nutrient substrate adaption of the pea green oak leaf-roller
The influence of the genetic variation of the DNA (RAPD polymerase chain reaction, primer OPA-14) of two oak species (Quercus pubescens L. and Q. petraea L.) on the variation of certain important fitnesslinked traits of the imago of the pea green oak leaf-roller (Tortrix viridana L.) in different classes of genotypes and allozyme loci is studied. The results obtained demonstrate
A. P. Simchuk
Genetic analysis of scattered populations of the Indian eri silkworm, Samia cynthia ricini Donovan: Differentiation of subpopulations
Deforestation and exploitation has led to the fragmentation of habitats and scattering of populations of the economically important eri silkworm, Samia cynthia ricini, in north-east India. Genetic analysis of 15 eri populations, using ISSR markers, showed 98% inter-population, and 23% to 58% intra-population polymorphism. Nei�s genetic distance between populations increased significantly with altitude (R2 = 0.71) and geographic distance (R2 = 0.78). On the dendrogram, the lower and upper Assam populations were clustered separately, with intermediate grouping of those from Barpathar and Chuchuyimlang, consistent with geographical distribution. The Nei�s gene diversity index was 0.350 in total populations and 0.121 in subpopulations. The genetic differentiation estimate (Gst) was 0.276 among scattered populations. Neutrality tests showed deviation of 118 loci from Hardy-Weinberg equilibrium. The number of loci that deviated from neutrality increased with altitude (R2 = 0.63). Test of linkage disequilibrium showed greater contribution of variance among eri subpopulations to total variance. D�2IS exceeded D�2ST, showed significant contribution of random genetic drift to the increase in variance of disequilibrium in subpopulations. In the Lakhimpur population, the peripheral part was separated from the core by a genetic distance of 0.260. Patchy habitats promoted low genetic variability, high linkage disequilibrium and colonization by new subpopulations. Increased gene flow and habitat-area expansion are required to maintain higher genetic variability and conservation of the original S. c. ricini gene pool.
Pradeep, Appukuttannair R.; Jingade, Anuradha H.; Singh, Choba K.; Awasthi, Aravind K.; Kumar, Vikas; Rao, Guruprasad C.; Prakash, N.B. Vijaya
Identification, characterization, and crystal structure of an aldo-keto reductase (AKR2E4) from the silkworm Bombyx mori.
A new member of the aldo-keto reductase (AKR) superfamily with 3-dehydroecdysone reductase activity was found in the silkworm Bombyx mori upon induction by the insecticide diazinon. The amino acid sequence showed that this enzyme belongs to the AKR2 family, and the protein was assigned the systematic name AKR2E4. In this study, recombinant AKR2E4 was expressed, purified to near homogeneity, and kinetically characterized. Additionally, its ternary structure in complex with NADP(+) and citrate was refined at 1.3� resolution to elucidate substrate binding and catalysis. The enzyme is a 33-kDa monomer and reduces dicarbonyl compounds such as isatin and 17?-hydroxy progesterone using NADPH as a cosubstrate. No NADH-dependent activity was detected. Robust activity toward the substrate inhibitor 3-dehydroecdysone was observed, which suggests that this enzyme plays a role in regulation of the important molting hormone ecdysone. This structure constitutes the first insect AKR structure determined. Bound NADPH is located at the center of the TIM- or (?/?)8-barrel, and residues involved in catalysis are conserved. PMID:24012638
Yamamoto, Kohji; Wilson, David K
Silkworm apolipophorin protein inhibits hemolysin gene expression of Staphylococcus aureus via binding to cell surface lipoteichoic acids.
We previously reported that a silkworm hemolymph protein, apolipophorin (ApoLp), binds to the cell surface of Staphylococcus aureus and inhibits expression of the saePQRS operon encoding a two-component system, SaeRS, and hemolysin genes. In this study, we investigated the inhibitory mechanism of ApoLp on S. aureus hemolysin gene expression. ApoLp bound to lipoteichoic acids (LTA), an S. aureus cell surface component. The addition of purified LTA to liquid medium abolished the inhibitory effect of ApoLp against S. aureus hemolysin production. In an S. aureus knockdown mutant of ltaS encoding LTA synthetase, the inhibitory effects of ApoLp on saeQ expression and hemolysin production were attenuated. Furthermore, the addition of anti-LTA monoclonal antibody to liquid medium decreased the expression of S. aureus saeQ and hemolysin genes. In S. aureus strains expressing SaeS mutant proteins with a shortened extracellular domain, ApoLp did not decrease saeQ expression. These findings suggest that ApoLp binds to LTA on the S. aureus cell surface and inhibits S. aureus hemolysin gene expression via a two-component regulatory system, SaeRS. PMID:23873929
Omae, Yosuke; Hanada, Yuichi; Sekimizu, Kazuhisa; Kaito, Chikara
Biosynthesis of major plasma proteins in the primary culture of fat body cells from the silkworm, Bombyx mori.
Plasma proteins termed "SP1" and "30K proteins" are synthesized by the fat body cells of the silkworm, Bombyx mori, in a sex- and stage-specific manner during larval development. We successfully established a primary culture of the fat body cells in order to investigate the regulatory mechanisms of plasma protein gene expression. The primary cultures of fat body cells contained at least two cell types: small oval cells, and large spherical cells. The cells adhered to and migrated on the cultured dish after plating. By the 7th day of cultivation, the cells clustered to form fat body-like structures, which were maintained for at least 3 months. Plasma proteins were actively synthesized in the primary cultures of the fat body cells isolated from the final instar larvae only when the cells tightly adhered to and clustered on the cultured dish. Immunocytochemical analysis revealed that only 10-15% of the clustered cells synthesized plasma proteins in our culture system, indicating that the primary culture comprises heterogeneous cells that are morphologically and functionally distinct. The patterns of SP1 syntheses in primary cultures faithfully reproduced their sex-dependency in vivo. PMID:10470503
Kishimoto, A; Nakato, H; Izumi, S; Tomino, S
Development of a method for long-term preservation of Bombyx mori silkworm strains using frozen ovaries.
Development of long-term preservation is essential for conservation of stocks of silkworm genetic resources. Thus far, a few methods have been reported, but more improvement is required for practical use. We have developed two effective modifications of a method for long-term preservation using frozen ovaries. One was slow cooling (1 �C per min) until -80 �C of the donor ovaries made possible by use of a BICELL freezing vessel. Using donor ovaries of 4th instar larvae, the average number of eggs laid per moth increased significantly from 110.7 � 53.4 eggs per moth by slow cooling with the BICELL vessel vs 12.3 � 10.3 eggs per moth by direct cooling in liquid nitrogen. A second improvement was connecting the thread bodies of the donor ovaries with those of the host in the transplantation step. Females operated on with the new method yielded a significantly higher percentage of moths that laid fertilized eggs than those transplanted with the standard procedure (70.4 � 21.6% vs 22.9 � 9.3%). PMID:23500076
Banno, Yutaka; Nagasaki, Kiyomi; Tsukada, Marino; Minohara, Yuko; Banno, Junko; Nishikawa, Kazuhiro; Yamamoto, Kazunori; Tamura, Kei; Fujii, Tsuguru
Genetic mapping of RAPD markers linked to the densonucleosis refractoriness gene, nsd-1, in the silkworm, Bombyx mori.
In the silkworm, Bombyx mori, nonsusceptibility to B. mori densonucleosis virus type-1 (BmDNV-1) is controlled by a recessive gene, nsd-1 (nonsusceptibility to DNV-1), located on the twenty-first chromosome. We investigated genetic linkage between five random amplified polymorphic DNA (RAPD) markers and the +nsd-1 gene. Initially, we constructed the CSD-1 strain (nsd-1/+) which is congenic to strain C137 (nsd-1/nsd-1) for the twenty-first chromosome, starting with a female of C137 and a male of strain J137 (+nsd-1/+nsd-1). For the crossing over experiment, a female of C137 was crossed with a male (nsd-1/+) of CSD-1. Segregation analysis showed that the most closely linked RAPD marker mapped 3.0 cM distant from +nsd-1. A more specific marker for +nsd-1 was made by converting this RAPD band into a sequence characterized amplified region (SCAR) using a series of newly designed primer pairs based on its DNA sequence. PMID:9880921
Abe, H; Harada, T; Kanehara, M; Shimada, T; Ohbayashi, F; Oshiki, T
Identification and genetic mapping of RAPD markers linked to the densonucleosis refractoriness gene, nsd-2, in the silkworm, Bombyx mori.
In the silkworm, Bombyx mori, nonsusceptibility to B. mori densonucleosis virus type-2 (BmDNV-2) is controlled by a recessive gene, nsd-2 (nonsusceptibility to DNV-2). We investigated the genetic linkage between two random-amplified polymorphic DNA (RAPD) markers and the +nsd-2 gene. Initially, we constructed the JSD-2 strain (nsd-2/+), which is congenic to strain J137 (nsd-2/nsd-2) with respect to the chromosome containing the +nsd-2 gene, starting with a female of strain J137 and a male of strain C137 (+nsd-2/+nsd-2). Genomic DNAs were compared between infected individuals of the JSD-2 strain and J137 by a polymerase chain reaction (PCR) with 700 arbitrary 10-mer primers. Two RAPD markers (OPH19R and OPP01R) linked to the +nsd-2 gene were found. For the crossing-over experiment, a female of J137 was crossed with a male (nsd-2/+) of JSD-2. Segregation analysis showed that the most closely linked RAPD marker (OPP01R) was mapped 4.7 cM distant from +nsd-2. PMID:10925787
Abe, H; Sugasaki, T; Kanehara, M; Shimada, T; Gomi, S J; Ohbayashi, F; Oshiki, T
Combined Effect of Cameo2 and CBP on the Cellular Uptake of Lutein in the Silkworm, Bombyx mori
Formation of yellow-red color cocoons in the silkworm, Bombyx mori, occurs as the result of the selective delivery of carotenoids from the midgut to the silk gland via the hemolymph. This process of pigment transport is thought to be mediated by specific cellular carotenoids carrier proteins. Previous studies indicated that two proteins, Cameo2 and CBP, are associated with the selective transport of lutein from the midgut into the silk gland in Bombyx mori. However, the exact roles of Cameo2 and CBP during the uptake and transport of carotenoids are still unknown. In this study, we investigated the respective contributions of these two proteins to lutein and ?-carotene transport in Bombyx mori as well as commercial cell-line. We found that tissues, expressed both Cameo2 and CBP, accumulate lutein. Cells, co-expressed Cameo2 and CBP, absorb 2 fold more lutein (P<0.01) than any other transfected cells, and the rate of cellular uptake of lutein was concentration-dependent and reached saturation. From immunofluorescence staining, confocal microscopy observation and western blot analysis, Cameo2 was localized at the membrane and CBP was expressed in the cytosol. What�s more, bimolecular fluorescence complementation analysis showed that these two proteins directly interacted at cellular level. Therefore, Cameo2 and CBP are necessarily expressed in midguts and silk glands for lutein uptake in Bombyx mori. Cameo2 and CBP, as the membrane protein and the cytosol protein, respectively, have the combined effect to facilitate the cellular uptake of lutein.
Dong, Xiao-Long; Chai, Chun-Li; Pan, Cai-Xia; Tang, Hui; Chen, Yan-Hong; Dai, Fang-Yin; Pan, Min-Hui; Lu, Cheng
Mutation of a Cuticular Protein, BmorCPR2, Alters Larval Body Shape and Adaptability in Silkworm, Bombyx mori.
Cuticular proteins (CPs) are crucial components of the insect cuticle. Although numerous genes encoding cuticular proteins have been identified in known insect genomes to date, their functions in maintaining insect body shape and adaptability remain largely unknown. In the current study, positional cloning led to the identification of a gene encoding an RR1-type cuticular protein, BmorCPR2, highly expressed in larval chitin-rich tissues and at the mulberry leaf-eating stages, which is responsible for the silkworm stony mutant. In the Dazao-stony strain, the BmorCPR2 allele is a deletion mutation with significantly lower expression, compared to the wild-type Dazao strain. Dysfunctional BmorCPR2 in the stony mutant lost chitin binding ability, leading to reduced chitin content in larval cuticle, limitation of cuticle extension, abatement of cuticle tensile properties, and aberrant ratio between internodes and intersegmental folds. These variations induce a significant decrease in cuticle capacity to hold the growing internal organs in the larval development process, resulting in whole-body stiffness, tightness, and hardness, bulging intersegmental folds, and serious defects in larval adaptability. To our knowledge, this is the first study to report the corresponding phenotype of stony in insects caused by mutation of RR1-type cuticular protein. Our findings collectively shed light on the specific role of cuticular proteins in maintaining normal larval body shape and will aid in the development of pest control strategies for the management of Lepidoptera. PMID:24514903
Qiao, Liang; Xiong, Gao; Wang, Ri-Xin; He, Song-Zhen; Chen, Jie; Tong, Xiao-Ling; Hu, Hai; Li, Chun-Lin; Gai, Ting-Ting; Xin, Ya-Qun; Liu, Xiao-Fan; Chen, Bin; Xiang, Zhong-Huai; Lu, Cheng; Dai, Fang-Yin
Purification, characterization and cloning of a chymotrypsin inhibitor (CI-9) from the hemolymph of the silkworm, Bombyx mori.
Hemolymph chymotrypsin inhibitor 9 (CI-9) from the hemolymph of the silkworm, Bombyx mori, was purified by ammonium sulfate precipitation, Butyl Toyopearl hydrophobic chromatography, gel filtration through Sephadex C-50 and chymotrypsin-sepharose 4B affinity chromatography. Checked by Native PAGE and SDS-PAGE in combination with silver staining, the final preparation appeared homogeneous. In tricine SDS-PAGE, CI-9 displayed a molecular weight of 7.5 kD, which was determined to be 7167 Da with the Voyager TOFMass analyser. The pI value for CI-9, revealed by 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis), was 4.3. CI-9 exhibited inhibitory activity at a temperature as high as 100 degrees C and a stability against a wide range of pH (1-12). In N-terminal amino-acid analysis of CI-9, 40 amino acid residues were obtained. The C-terminal 22 amino acid residues were deduced by subsequently cloned cDNA and genomic fragments. MW and pI of CI-9 were predicted to be 7170.98 Da and 4.61, respectively, on the website. Its low molecular weight, high stability, conserved active site and Kunitz domain showed that CI-9 is a Kunitz-type CI. The difference of sequence and pI between CI-9 and other Kunitz type CIs indicated that it is a novel chymotrypsin inhibitor. PMID:17503165
Zhao, Ping; Xia, Qingyou; Li, Juan; Fujii, Hiroshi; Banno, Yutaka; Xiang, Zhonghuai
Identification of a cis-regulatory element that directs prothoracicotropic hormone gene expression in the silkworm Bombyx mori.
In the silkworm Bombyx mori and other insects, prothoracicotropic hormone (PTTH) plays a central role in controlling molting and metamorphosis by stimulating the prothoracic glands to synthesize and release the molting hormone ecdysone. Using an AcNPV (Autographa californica nucleopolyhedrovirus)-mediated transient gene transfer system, we identified a cis-regulatory element that participates in the decision to switch expression of PTTH on or off in PTTH-producing neurosecretory cells (PTPCs). The nucleotide sequence of this cis-regulatory element is similar to a cis-regulatory element that participates in direction of expression of diapause hormone-pheromone biosynthesis activating neuropeptide gene (DH-PBAN) (Shiomi et�al., 2007). Furthermore, we found that B. mori Pitx (BmPitx), a bicoid-like homeobox transcription factor, binds the element and activates PTTH expression. Therefore, we propose that the cell-specific expression of two neuropeptide hormone genes, PTTH and DH-PBAN, is activated by the Pitx transcription factor, which may act as a pan-activator in the insect neuroendocrine system and in vertebrate pituitary cells. PMID:21324358
Ohtsuka, Kazunori; Atsumi, Tsutomu; Fukushima, Yoshihisa; Shiomi, Kunihiro
White Oak Creek embayment sediment retention structure: The Oak Ridge model in action
White Oak Creek is the major surface-water drainage through the Department of Energy (DOE) Oak Ridge National Laboratory (ORNL). Samples taken from the lower portion of the creek revealed high levels of Cesium-137, and lower levels of Cobalt-60 in near-surface sediment. Other contaminants present in the sediment included: lead, mercury, chromium, and PCBS. In October 1990, DOE, US Environmental Protection Agency (EPA), and Tennessee Department of Environment and Conservation (DEC) agreed to initiate a time-critical removal action in accordance with Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) to prevent transport of the contaminated sediments into the Clinch River system. This paper discusses the environmental, regulatory, design, and construction issues that were encountered in conducting the remediation work.
Van Hoesen, S.D.; Kimmel, B.L. [Oak Ridge National Lab., TN (United States); Page, D.G.; Hudson, G.R. [USDOE Oak Ridge Field Office, TN (United States); Wilkerson, R.B. [MK-Ferguson Co., Oak Ridge, TN (United States); Zocolla, M. [Corps of Engineers, Nashville, TN (United States). Nashville District; Kauschinger, J.L. [Ground Engineering Services, Manchester, NH (United States)
Van Hoesen, S.D.; Kimmel, B.L. (Oak Ridge National Lab., TN (United States)); Page, D.G.; Hudson, G.R. (USDOE Oak Ridge Field Office, TN (United States)); Wilkerson, R.B. (MK-Ferguson Co., Oak Ridge, TN (United States)); Zocolla, M. (Corps of Engineers, Nashville, TN (United States). Nashville District); Kauschinger, J.L. (Ground Engineering Services, Manchester, NH (United States))
Melton Valley Storage Tanks Capacity Increase Project, Oak Ridge National Laboratory, Oak Ridge, Tennessee
The US Department of Energy (DOE) proposes to construct and maintain additional storage capacity at Oak Ridge National Laboratory (ORNL), Oak Ridge, Tennessee, for liquid low-level radioactive waste (LLLW). New capacity would be provided by a facility partitioned into six individual tank vaults containing one 100,000 gallon LLLW storage tank each. The storage tanks would be located within the existing Melton Valley Storage Tank (MVST) facility. This action would require the extension of a potable water line approximately one mile from the High Flux Isotope Reactor (HFIR) area to the proposed site to provide the necessary potable water for the facility including fire protection. Alternatives considered include no-action, cease generation, storage at other ORR storage facilities, source treatment, pretreatment, and storage at other DOE facilities.
Source document for waste area groupings at Oak Ridge National Laboratory, Oak Ridge, Tennessee
This document serves as a source document for Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) and other types of documents developed for and pertaining to Environmental Restoration (ER) Program activities at Oak Ridge National Laboratory (ORNL). It contains descriptions of the (1) regulatory requirements for the ORR ER Program, (2) Oak Ridge Reservation (ORR) ER Program, (3) ORNL site history and characterization, and (4) history and characterization of Waste Area Groupings (WAGS) 1-20. This document was created to save time, effort, and money for persons and organizations drafting documents for the ER Program and to improve consistency in the documents prepared for the program. By eliminating the repetitious use of selected information about the program, this document will help reduce the time and costs associated with producing program documents. By serving as a benchmark for selected information about the ER Program, this reference will help ensure that information presented in future documents is accurate and complete.
Osborne, P.L.; Kuhaida, A.J., Jr.
Oak Ridge Reservation environmental report for 1989
The first two volumes of this report are devoted to a presentation of environmental data and supporting narratives for the US Department of Energy's (DOE's) Oak Ridge Reservation (ORR) and surrounding environs during 1989. Volume 1 includes all narrative descriptions, summaries, and conclusions and is intended to be a stand-alone'' report for the ORR for the reader who does not want to review in detail all of the 1989 data. Volume 2 includes the detailed data summarized in a format to ensure that all environmental data are represented in the tables. Narratives are not included in Vol. 2. The tables in Vol. 2 are addressed in Vol. 1. For this reason, Vol. 2 cannot be considered a stand-alone report but is intended to be used in conjunction with Vol. 1. 16 figs., 194 tabs.
Jacobs, V.A.; Wilson, A.R. (eds.)
Oak Ridge Reservation Environmental report for 1990
The first two volumes of this report are devoted to a presentation of environmental data and supporting narratives for the US Department of Energy's (DOE's) Oak Ridge Reservation (ORR) and surrounding environs during 1990. Volume 1 includes all narrative descriptions, summaries, and conclusions and is intended to be a stand-alone'' report for the ORR for the reader who does not want to review in detail all of the 1990 data. Volume 2 includes the detailed data summarized in a format to ensure that all environmental data are represented in the tables. Narratives are not included in Vol. 2. The tables in Vol. 2 are addressed in Vol. 1. For this reason, Vol. 2 cannot be considered a stand-alone report but is intended to be used in conjunction with Vol. 1.
Wilson, A.R. (ed.)
Pulsed reactor experiments at Oak Ridge
This paper describes dynamic experiments for 3 pulsed reactors. 1st reactor was pulsed from some average power by rotating a partial Be reflector past an unreflected core face; the other 2 reactors were pulsed by rapid insertion of a fuel rod into the unmoderated and unreflected reactor at essentially zero neutron level with no significant neutron source present. The first reactor was a mockup of an EURATOM design (never constructed) of the proposed SORA Reactor, and the other two were the Health Physics Research Reactor and the Army Pulse Radiation Facility Reactor (APRFR). This paper describes the experiments performed in initial testing of these systems, including destructive tests of APRFR, to set operating limits for this type of reactor in pulsed operation. All the experiments described were performed at the Oak Ridge Critical Experiments Facility.
Mihalczo, J.T.
Site descriptions of environmental restoration units at Oak Ridge National Laboratory, Oak Ridge, Tennessee
This report provides summary information on Oak Ridge National Laboratory (ORNL) Environmental Restoration (ER) sites as listed in the Oak Ridge Reservation Federal Facility Agreement (FFA), dated January 1, 1992, Appendix C. The Oak Ridge National Laboratory was built in 1943 as part of the World War II Manhattan Project. The original mission of ORNL was to produce and chemically separate the first gram-quantities of plutonium as part of the national effort to produce the atomic bomb. The current mission of ORNL is to provide applied research and development in support of the U.S. Department of Energy (DOE) programs in nuclear fusion and fission, energy conservation, fossil fuels, and other energy technologies and to perform basic scientific research in selected areas of the physical, life, and environmental sciences. ER is also tasked with clean up or mitigation of environmental impacts resulting from past waste management practices on portions of the approximately 37,000 acres within the Oak Ridge Reservation (ORR). Other installations located within the ORR are the Gaseous Diffusion Plant (K-25) and the Y-12 plant. The remedial action strategy currently integrates state and federal regulations for efficient compliance and approaches for both investigations and remediation efforts on a Waste Area Grouping (WAG) basis. As defined in the ORR FFA Quarterly Report July - September 1995, a WAG is a grouping of potentially contaminated sites based on drainage area and similar waste characteristics. These contaminated sites are further divided into four categories based on existing information concerning whether the data are generated for scoping or remedial investigation (RI) purposes. These areas are as follows: (1) Operable Units (OU); (2) Characterization Areas (CA); (3) Remedial Site Evaluation (RSE) Areas; and (4) Removal Site Evaluation (RmSE) Areas.
Kuhaida, A.J. Jr.; Parker, A.F.
Advances in dose reconstruction at Oak Ridge
The Oak Ridge Dose Reconstruction has seven tasks: (1) releases of radionuclides from X-10 radiolanthanum processing, (2) atmospheric and aquatic discharges of mercury from Y-12, (3) releases of PCB`s from all facilities, (4) aquatic releases of radionuclides from X-10 into the Clinch River, (5) a systematic search for classified and unclassified records of past releases (6) releases of uranium from all facilities, and (7) screening of contaminants and release events not previously evaluated in the study. The contaminants, exposure pathways, and release events requiring the most intensive analysis are identified first through screening level calculations and then through an iterative assessment approach based on a preliminary uncertainty analysis of more realistic sets of models and assumptions. Subjective probability distributions are developed for uncertain model components, reflecting the present state of knowledge about true but unknown values, and these uncertainties are propagated through to estimates of dose and health risk using Monte Carlo simulation. This procedure is effective in identifying the components of the dose reconstruction model of dominant importance. Efforts are focused on the review and improvement of the set of preliminary assumptions that may significantly impact the overall uncertainty in the final result. The results of the screening calculations and the preliminary uncertainty analysis are compared against established decision criteria to identify the need for resource re-allocation among tasks. To date, a 10{sup -4} life-time incidence of cancer incidence and a hazard index of one have been proposed as a decision criterion. Decisions about reallocation of resources among Tasks will be made by the Oak Ridge Health Agreement Steering Panel and the Tennessee Department of Health, which actively seek public involvement and participation.
Hoffman, F.O. [SENES Oak Ridge, Inc., TN (United States); Widner, T.E. [ChemRisk/McLaren Hart Environmental Services, Inc., Alameda, CA (United States)
Molecular Cloning and Characterization of Ecdysone oxidase and 3-dehydroecdysone-3?-reductase Involved in the Ecdysone Inactivation Pathway of Silkworm, Bombyx mori
Molting hormone (ecdysteroid) is one of the most important hormones in insects. The synthesis and inactivation of the ecdysteroid regulate the developmental process of insects. A major pathway of ecdysone inactivation is that ecdysone is converted to 3-dehydroecdysone, and then further to 3-epiecdysone in insects. Two enzymes (ecdysone oxidase: EO and 3DE-3?-reductase) participate in this pathway. In this study, based on the previously characterized cDNAs in Spodoptera littoralis, we cloned and characterized EO and 3DE-3?-reductase genes in the silkworm, Bombyx mori. The heterologously expressed proteins of the two genes in yeast showed the ecdysone oxidase and 3DE-3?-reductase activities, respectively. Expression of BmEO was only detected in the midgut at transcriptional and translational levels. We also localized EO within the midgut goblet cell cavities. For Bm3DE-3?-reductase gene, RT-PCR and western blot showed that it was expressed in the midgut and the Malpighian tubules. Moreover, we localized 3DE-3?-reductase within the midgut goblet cell cavities and the cytosol of principal cells of the Malpighian tubules. These two genes have similar expression profiles during different developmental stages. Both genes were highly expressed at the beginning of the 5th instar, and remained a relative low level during the feeding stage, and then were highly expressed at the wandering stage. All these results showed that the profiles of the two genes were well correlated with the ecdysteroid titer. The functional characterization of the enzymes participating in ecdysone inactivation in the silkworm provides hints for the artificial regulation of the silkworm development and biological control of pests.
Sun, Wei; Shen, Yi-Hong; Qi, Deng-Wei; Xiang, Zhong-Huai; Zhang, Ze
Cynipid gall-wasp communities correlate with oak chemistry.
Host-plant association data, gathered from field surveys conducted throughout Florida and from the literature, were used to identify the specificity of cynipid gall inducers to one or more of six Quercus species that occur at Archbold Biological Station, Lake Placid, Florida, USA, including the red oaks Q. laevis, Q. myrtifolia, and Q. inopina, and the white oaks Q. chapmanii, Q. geminata, and Q. minima. Quercus myrtifolia had the highest cynipid richness and diversity (37 cynipid species, Shannon H' = 3.61, Simpson's D = 0.97), followed by Q. chapmanii, Q. laevis, Q. inopina, Q. geminata, and finally Q. minima (10 species, H' = 2.30, D = 0.90). All cynipid species showed strong fidelity to a particular host plant or a restricted set of host plants. An ordination of gall-wasp host associations indicated that the cynipid communities of each oak species were distinct and specific to a given oak species. Leaf samples taken from each oak species were analyzed for condensed and hydrolyzable tannins, total phenolics, lignin, cellulose and hemicellulose, nitrogen, and carbon. All of these chemical traits, with the exception of carbon, differed by oak species, and the differences were strongly correlated with the axes of the cynipid-species ordination. These results suggest that gall-wasp occurrence is influenced by oak chemistry and imply that experimental studies of cynipid gall inducers that examine host-plant chemistry and female oviposition choice and larval performance will yield useful insights. PMID:12647863
Abrahamson, Warren G; Hunter, Mark D; Melika, George; Price, Peter W
Oak Ridge Dose Reconstruction Project Summary Report; Reports of the Oak Ridge Dose Reconstruction, Vol. 7
In the early 1990s, concern about the Oak Ridge Reservation's past releases of contaminants to the environment prompted Tennessee's public health officials to pursue an in-depth study of potential off-site health effects at Oak Ridge. This study, the Oak Ridge dose reconstruction, was supported by an agreement between the U.S. Department of Energy (DOE) and the State of Tennessee, and was overseen by a 12-member panel of individuals appointed by Tennessee's Commissioner of Health. The panel requested that the principal investigator for the project prepare the following report, ''Oak Ridge Dose Reconstruction Project Summary Report,'' to serve the following purposes: (1) summarize in a single, less technical report, the methods and results of the various investigations that comprised the Phase II of the dose reconstruction; (2) describe the systematic searching of classified and unclassified historical records that was a vital component of the project; and (3) summarize the less detailed, screening-level assessments that were performed to evaluate the potential health significance of a number of materials, such a uranium, whose priority did not require a complete dose reconstruction effort. This report describes each major step of the dose reconstruction study: (1) the review of thousands of historical records to obtain information relating to past operations at each facility; (2) estimation of the quantity and timing of releases of radioiodines from X-10, of mercury from Y-12, of PCB's from all facilities, and of cesium-137 and other radionuclides from White Oak Creek; (3) evaluation of the routes taken by these contaminants through the environment to nearby populations; and (4) estimation of doses and health risks to exposed groups. Calculations found the highest excess cancer risks for a female born in 1952 who drank goat milk; the highest non-cancer health risk was for children in a farm family exposed to PCBs in and near East Fork Poplar Creek. More detailed dose and risk estimates, and associated uncertainties, are presented in several technical reports. One way to easily locate them in OSTI's Information Bridge is by searching the ''report number field'' for the number DOE/OR/21981*. The asterisk placed after the base number will enable the search to list all of the related reports in this series.
Thomas E. Widner; et. al.
Hemolymph ecdysteroid titer and ecdysteroid-dependent developmental events in the last-larval stadium of the silkworm, Bombyx mori: role of low ecdysteroid titer in larval�pupal metamorphosis and a reappraisal of the head critical period
The endocrine regulation of larval�pupal metamorphosis was studied in the silkworm, Bombyx mori, by measuring the following changes: hemolymph ecdysteroid titer, the secretory activity of prothoracic glands and the responsiveness of larvae to ecdysteroids and prothoracicotropic hormone (PTTH), with regard to developmental events such as the occurrence of spinneret pigmentation, initiation of cocoon spinning and onset of wandering stage as
Sho Sakurai; Masae Kaya; Shin'Ichirol Satake
A study of root-feeding insects as potential vectors of the oak wilt fungus Cerutocystis figaceurum (T. W. Bretz) J. Hunt in live oaks, revealed root galls induced by the cynipid gall wasp Odontocynips nebulosu Kieffer. The incidence of the wasp on roots of four oak species and natural live oak hybrids at 14 root excavation sites in 7 counties throughout
Technical background information for the environmental and safety report, Volume 4: White Oak Lake and Dam
This report has been prepared to provide background information on White Oak Lake for the Oak Ridge National Laboratory Environmental and Safety Report. The paper presents the history of White Oak Dam and Lake and describes the hydrological conditions of the White Oak Creek watershed. Past and present sediment and water data are included; pathway analyses are described in detail.
Oakes, T.W.; Kelly, B.A.; Ohnesorge, W.F.; Eldridge, J.S.; Bird, J.C.; Shank, K.E.; Tsakeres, F.S.
The Oak Ridge Refrigerant Management Program
NASA Technical Reports Server (NTRS)
For many years, chlorofluorocarbons (CFC's) have been used by the Department of Energy's (DOE) Oak Ridge Y-12 Plant in air conditioning and process refrigeration systems. However, Title 6 of the Clean Air Act Amendments (CAAA) and Executive Order 12843 (Procurement Requirements and Policies for Federal Agencies for Ozone Depleting Substances) signed by President Clinton require, as policy, that all federal agencies maximize their use of safe, alternate refrigerants and minimize, where economically practical, the use of Class 1 refrigerants. Unfortunately, many government facilities and industrial plants have no plan or strategy in place to make this changeover, even though their air conditioning and process refrigeration equipment may not be sustainable after CFC production ends December 31, 1995. The Y-12 Plant in Oak Ridge, Tennessee, has taken an aggressive approach to complying with the CAAA and is working with private industry and other government agencies to solve tough manufacturing and application problems associated with CFC and hydrochlorofluorocarbon (HCFC) alternatives. Y-12 was the first DOE Defense Program (DP) facility to develop a long-range Stratospheric Ozone Protection Plan for refrigerant management for compliance with the CAAA. It was also the first DOE DP facility to complete detailed engineering studies on retrofitting and replacing all air conditioning and process refrigeration equipment to enable operation with alternate refrigerants. The management plan and engineering studies are models for use by other government agencies, manufacturing plants, and private industry. This presentation identifies some of the hidden pitfalls to be encountered in the accelerated phaseout schedule of CFC's and explains how to overcome and prevent these problems. In addition, it outlines the general issues that must be considered when addressing the phase-out of ozone depleting substances and gives some 'lessons learned' by Y-12 from its Refrigerant Management Program. Discussion topics include requirements for developing a refrigerant management plan and establishing priorities for cost-effective compliance with the CAAA, as well as ways in which employees can be empowered to develop a comprehensive refrigerant management plan. The result of this employee empowerment was a cooperative labor-management effort that is beneficial for Y-12, DOE, and the environment.
Kevil, Thomas H.
Functional Genomics Initiative at Oak Ridge National Laboratory.
The Functional Genomics Initiative at the Oak Ridge National Laboratory integrates outstanding capabilities in mouse genetics, bioinformatics, and instrumentation. The 50 year investment by the DOE in mouse genetics/mutagenesis has created a one-of-a-kind...
D. Johnson M. Justice K. Beattle M. Buchanan M. Ramsey
Reference Manual for Managing Sudden Oak Death in California.
This publication contains background information and guidance for resource management professionals and landowners to understand and manage sudden oak death (SOD) in California forests. The publication is divided into three chapters: Chapter 1 discusses t...
E. A. Bernhardt T. J. Swiecki
60 years of great science [Oak Ridge National Laboratory
This issue highlights Oak Ridge National Laboratory's contributions in more than 30 areas of research and related activities during the past 60 years and provides glimpses of current activities that are carrying on this heritage.
Oak Ridge National Laboratory's philosophy and approach to NEPA.
At Oak Ridge National Laboratory (ORNL), the overall responsibility for compliance with the National Environmental Policy Act (NEPA) resides with Environmental Review and Documentations Section that is within the Office of Environmental Compliance and Doc...
R. I. Van Hook H. M. Braunstein L. L. Sigal C. C. Trettin
Technical Specifications for the Oak Ridge Critical Experiments Facility.
These Technical Specifications for the Oak Ridge Critical Experiments Facility (CEF) delineate limiting conditions of operation for the facility. The CEF is used primarily for testing the High Flux Isotope Reactor (HFIR) fuel assemblies. Specifically, the...
R. M. Stinnett
19. DETAIL VIEW OF SKIFF BOW WITH OAK STEM AND FRAMES PLANKED IN CEDAR USING COPPER CLINCH NAILS. TRANSOM OF SECOND SKIFF CAN BE SEEN BACKGROUND. - Lowell's Boat Shop, 459 Main Street, Amesbury, Essex County, MA
Effects of Environmental Conditions on Isoprene Emission from Live Oak.
Live-oak plants (Quercus virginia) were subjected to various levels of CO2, water stress or photosynthetic photon flux density to test the hypothesis that isoprene biosynthesis occurred only under conditions of restricted CO2 availability. Isoprene emissi...
D. T. Tingey R. Evans M. Gumpertz
Science and Technology at Oak Ridge National Laboratory
ORNL Director Thom Mason explains the groundbreaking work in neutron sciences, supercomputing, clean energy, advanced materials, nuclear research, and global security taking place at the Department of Energy's Office of Science laboratory in Oak Ridge, Tenn.
Mason, Thomas
Prepare and Purify Urushiol from Poison Ivy and Poison Oak.
The active ingredients (urushiols) of poison ivy and poison oak were isolated in purified form by various chemical and physical procedures. Various properties were measured, including molecular weight, double bond value, refractive index and infrared spec...
M. D. Corbett
Preparation of Urushiol from Poison Ivy or Poison Oak.
A method for the preparation of urushiol from poison ivy or poison oak is disclosed. The method includes extraction with alcohol at low temperature and a second extraction with benzene, followed by chromatographic separation on a solid adsorbent. The meth...
The population biology of oak gall wasps (Hymenoptera: Cynipidae).
Oak gall wasps (Hymenoptera: Cynipidae, Cynipini) are characterized by possession of complex cyclically parthenogenetic life cycles and the ability to induce a wide diversity of highly complex species- and generation-specific galls on oaks and other Fagaceae. The galls support species-rich, closed communities of inquilines and parasitoids that have become a model system in community ecology. We review recent advances in the ecology of oak cynipids, with particular emphasis on life cycle characteristics and the dynamics of the interactions between host plants, gall wasps, and natural enemies. We assess the importance of gall traits in structuring oak cynipid communities and summarize the evidence for bottom-up and top-down effects across trophic levels. We identify major unanswered questions and suggest approaches for the future. PMID:11729087
Stone, Graham N; Schonrogge, Karsten; Atkinson, Rachel J; Bellido, David; Pujade-Villar, Juli
Licensing and CRADA's in Oak Ridge technology transfer.
In the belief that effective technology transfer is a ''contact sport,'' Martin Marietta Energy Systems (Energy Systems), the Department of Energy's (DOE's) management contractor in Oak Ridge, Tennessee, encourages its research and engineering employees t...
G. A. Prosser
An epidemic airborne disease caused by the oak processionary caterpillar.
The oak processionary caterpillar (Thaumetopoea processionea Lepidoptera) is found in several European countries. It usually lives in oak forests or on single-standing oak trees. The larva of from the 3rd to 6th developmental stage (instar) develops poisonous hair (setae), filled with an urticating toxin that may lead to serious dermatitis, conjunctivitis, and pulmonary affection (summarized as lepidopterism) on contact with the setae. In June 2004 more than 40 people including young children developed symptoms of lepidopterism after resting within 20 m of an infested oak tree. Only a few people had touched the caterpillars. All those with clinical symptoms of lepidopterism could be treated as outpatients, but several needed systemic steroids because of the severity of their complaints. Lepidopterism, an airborne disease caused by the setae of the processionary caterpillar, is a growing public health problem because of the increasing numbers of outbreaks. PMID:16445416
Gottschling, Sven; Meyer, Sascha
Deep hydrogeologic flow system underlying the Oak Ridge Reservation.
The deep hydrogeologic system underlying the Oak Ridge Reservation contains some areas contaminated with radionuclides, heavy metals, nitrates, and organic compounds. The groundwater at that depth is saline and has previously been considered stagnant. On ...
R. Nativ A. E. Hunley
Prominent down-regulation of storage protein genes after bacterial challenge in eri-silkworm, Samia cynthia ricini.
We constructed two independent cDNA libraries from the fat body of Escherichia coli- or Candida albicans-challenged eri-silkworm Samia cynthia ricini larvae. We performed comparative expressed sequence tag (EST) analysis of the two cDNA libraries and found that two putative storage protein genes, ScSP1 and ScSP2, were markedly repressed by E. coli injection as compared with C. albicans injection. By quantitative real-time RT-PCR analysis, we showed that ScSP1 mRNA significantly reduced to 1/32-1/3 in the fat body of the female larvae, and ScSP2 mRNA reduced to 1/7-1/3 and 1/22-1/5 in the females and males, respectively, 12-36 h after E. coli injection as compared with PBS injection. In addition, SDS-PAGE analysis revealed that the accumulation of both the ScSP proteins in the larval hemolymph apparently decreased up to 36 h after E. coli injection. However, the amounts of the two ScSP proteins returned to the same level as those in the larvae injected with PBS by 48 h after injection, showing that the reduction in ScSPs caused by the bacterial challenge was transient. Moreover, potential binding sites for the Drosophila Rel/NF-kappaB protein Dorsal were found in the 5' upstream regulatory regions of ScSP1 and ScSP2, suggesting the participation of the Rel/NF-kappaB proteins in controlling the bacterial suppression of the ScSP genes. These results suggested the hypothesis that S. c. ricini has a genetic program to shut down temporarily dispensable gene expression in order to induce an acute and efficient expression of immune-related genes. These findings may provide new insight into the innate immune system in lepidopteran insects. PMID:18064702
Meng, Yan; Omuro, Naoko; Funaguma, Shunsuke; Daimon, Takaaki; Kawaoka, Shinpei; Katsuma, Susumu; Shimada, Toru
Identification of key uric acid synthesis pathway in a unique mutant silkworm Bombyx mori model of Parkinson's disease.
Plasma uric acid (UA) levels decrease following clinical progression and stage development of Parkinson's disease (PD). However, the molecular mechanisms underlying decreases in plasma UA levels remain unclear, and the potential to apply mutagenesis to a PD model has not previously been discovered. We identified a unique mutant of the silkworm Bombyx mori (B.mori) op. Initially, we investigated the causality of the phenotypic "op" by microarray analysis using our constructed KAIKO functional annotation pipeline. Consequently, we found a novel UA synthesis-modulating pathway, from DJ-1 to xanthine oxidase, and established methods for large-scale analysis of gene expression in B. mori. We found that the mRNA levels of genes in this pathway were significantly lower in B. mori op mutants, indicating that downstream events in the signal transduction cascade might be prevented. Additionally, levels of B.mori tyrosine hydroxylase (TH) and DJ-1 mRNA were significantly lower in the brain of B. mori op mutants. UA content was significantly lower in the B. mori op mutant tissues and hemolymph. The possibility that the B. mori op mutant might be due to loss of DJ-1 function was supported by the observed vulnerability to oxidative stress. These results suggest that UA synthesis, transport, elimination and accumulation are decreased by environmental oxidative stress in the B. mori op mutant. In the case of B. mori op mutants, the relatively low availability of UA appears to be due both to the oxidation of DJ-1 and to its expenditure to mitigate the effects of environmental oxidative stress. Our findings are expected to provide information needed to elucidate the molecular mechanism of decreased plasma UA levels in the clinical stage progression of PD. PMID:23894418
Tabunoki, Hiroko; Ono, Hiromasa; Ode, Hiroaki; Ishikawa, Kazuhiro; Kawana, Natsuki; Banno, Yutaka; Shimada, Toru; Nakamura, Yuki; Yamamoto, Kimiko; Satoh, Jun-Ichi; Bono, Hidemasa
Repression of tyrosine hydroxylase is responsible for the sex-linked chocolate mutation of the silkworm, Bombyx mori.
Pigmentation patterning has long interested biologists, integrating topics in ecology, development, genetics, and physiology. Wild-type neonatal larvae of the silkworm, Bombyx mori, are completely black. By contrast, the epidermis and head of larvae of the homozygous recessive sex-linked chocolate (sch) mutant are reddish brown. When incubated at 30 degrees C, mutants with the sch allele fail to hatch; moreover, homozygous mutants carrying the allele sch lethal (sch(l)) do not hatch even at room temperature (25 degrees C). By positional cloning, we narrowed a region containing sch to 239,622 bp on chromosome 1 using 4,501 backcross (BC1) individuals. Based on expression analyses, the best sch candidate gene was shown to be tyrosine hydroxylase (BmTh). BmTh coding sequences were identical among sch, sch(l), and wild-type. However, in sch the approximately 70-kb sequence was replaced with approximately 4.6 kb of a Tc1-mariner type transposon located approximately 6 kb upstream of BmTh, and in sch(l), a large fragment of an L1Bm retrotransposon was inserted just in front of the transcription start site of BmTh. In both cases, we observed a drastic reduction of BmTh expression. Use of RNAi with BmTh prevented pigmentation and hatching, and feeding of a tyrosine hydroxylase inhibitor also suppressed larval pigmentation in the wild-type strain, pnd(+) and in a pS (black-striped) heterozygote. Feeding L-dopa to sch neonate larvae rescued the mutant phenotype from chocolate to black. Our results indicate the BmTh gene is responsible for the sch mutation, which plays an important role in melanin synthesis producing neonatal larval color. PMID:20615980
Liu, Chun; Yamamoto, Kimiko; Cheng, Ting-Cai; Kadono-Okuda, Keiko; Narukawa, Junko; Liu, Shi-Ping; Han, Yu; Futahashi, Ryo; Kidokoro, Kurako; Noda, Hiroaki; Kobayashi, Isao; Tamura, Toshiki; Ohnuma, Akio; Banno, Yutaka; Dai, Fang-Ying; Xiang, Zhong-Huai; Goldsmith, Marian R; Mita, Kazuei; Xia, Qing-You
Functional and pharmacological characterization of a beta-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 beta-adrenergic-like octopamine (OA) receptor (DmOct beta 2R). 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. [(3)H]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 microM. These findings indicate that the cloned receptor is a beta-adrenergic-like OA receptor with unique functional and pharmacological properties. PMID:20417278
Chen, Xi; Ohta, Hiroto; Ozoe, Fumiyo; Miyazawa, Kazuya; Huang, Jia; Ozoe, Yoshihisa
Genetic analysis and gene mapping of two novel quail-like mutants from the silkworm (Bombyx mori).
Two novel body marking mutants were discovered during silkworm (Bombyx mori) breeding. The mutants have no obvious eye-spots compared with normal marking (+) individuals, but their star spots and semilunar markings on dorsal sides are normal, and there are dots and lines with longitudinal wave markings on dorsal sides of the 6th to 7th abdominal segments which consist quail markings in between star spots and semilunar markings. The whole body markings are very similar to that of quail mutant (q); thus these mutants are named as quail-like mutants (q-l). Young larvae of one mutant are in brown color, and develop normally. Their cocoons are regular and uniform in size. Thus, this mutant is designated as brown quail-like (q-lb). Another mutant's larvae are in light purple skin; thus this mutant is named as purple quail-like (q-lp). They take little amount of mulberry leaves, and are weak and develop slowly and unevenly. Their larval bodies and cocoons are small. Genetic analysis revealed that both q-lb and q-lp were recessive genes, and they were allelic, with q-lb recessive to q-lp. These genes are different from quail mutant (q) and located on the chromosome 8 after tested by the morphological markers, P3(2), p(2), Ze(3), L(4), re(5), E(6), q(7), I-a(9), ms(12), ch(13), oa(14), cts(16), mln(18), msn(19), rb(21) and so(26) and SSR markers. PMID:24846982
Qiaoling, Zhao; Wenbo, Wang; Anli, Chen; Zhiyong, Qiu; Dingguo, Xia; Heying, Qian; Xingjia, Shen
Identification of Key Uric Acid Synthesis Pathway in a Unique Mutant Silkworm Bombyx mori Model of Parkinson's Disease
Plasma uric acid (UA) levels decrease following clinical progression and stage development of Parkinson�s disease (PD). However, the molecular mechanisms underlying decreases in plasma UA levels remain unclear, and the potential to apply mutagenesis to a PD model has not previously been discovered. We identified a unique mutant of the silkworm Bombyx mori (B.mori) op. Initially, we investigated the causality of the phenotypic �op� by microarray analysis using our constructed KAIKO functional annotation pipeline. Consequently, we found a novel UA synthesis-modulating pathway, from DJ-1 to xanthine oxidase, and established methods for large-scale analysis of gene expression in B. mori. We found that the mRNA levels of genes in this pathway were significantly lower in B. mori op mutants, indicating that downstream events in the signal transduction cascade might be prevented. Additionally, levels of B.mori tyrosine hydroxylase (TH) and DJ-1 mRNA were significantly lower in the brain of B. mori op mutants. UA content was significantly lower in the B. mori op mutant tissues and hemolymph. The possibility that the B. mori op mutant might be due to loss of DJ-1 function was supported by the observed vulnerability to oxidative stress. These results suggest that UA synthesis, transport, elimination and accumulation are decreased by environmental oxidative stress in the B. mori op mutant. In the case of B. mori op mutants, the relatively low availability of UA appears to be due both to the oxidation of DJ-1 and to its expenditure to mitigate the effects of environmental oxidative stress. Our findings are expected to provide information needed to elucidate the molecular mechanism of decreased plasma UA levels in the clinical stage progression of PD.
Tabunoki, Hiroko; Ono, Hiromasa; Ode, Hiroaki; Ishikawa, Kazuhiro; Kawana, Natsuki; Banno, Yutaka; Shimada, Toru; Nakamura, Yuki; Yamamoto, Kimiko; Satoh, Jun-ichi; Bono, Hidemasa
Clumped distribution of oak leaf miners between and within plants
Leaf miners typically show non-random distributions both between and within plants. We tested the hypothesis that leaf miners on two oak species were clumped on individual host trees and individual branches and addressed whether clumping was influenced by aspects of plant quality and how clumping and\\/or interactions with other oak herbivores affected leaf-miner survivorship. Null models were used to test
Tatiana Cornelissen; Peter Stiling
The Oak Ridge Institute for Science and Education (ORISE) has completed the independent verification survey of the Building 3550 Slab. The results of this effort are provided. The objective of this verification survey is to provide independent review and field assessment of remediation actions conducted by Safety and Ecology Corporation (SEC) to document that the final radiological condition of the slab meets the release guidelines. Verification survey activities on the Building 3550 Slab that included scans, measurements, and the collection of smears. Scans for alpha, alpha plus beta, and gamma activity identified several areas that were investigated.
Weaver, Phyllis C.
Project plan for the Background Soil Characterization Project on the Oak Ridge Reservation, Oak Ridge, Tennessee
The Background Soil characterization Project (BSCP) will provide background concentration levels of selected metals, organic compounds, and radionuclides in soils from uncontaminated on-site areas at the Oak Ridge Reservation (ORR), and off-site in the western part of Roane County and the eastern part of Anderson County. The BSCP will establish a database, recommend how to use the data for contaminated site assessment, and provide estimates of the potential human health and environmental risks associated with the background level concentrations of potentially hazardous constituents.
Radioisotope production and management at Oak Ridge National Laboratory
The production of radioisotopes has been one of the basic activities at Oak Ridge since the end of World War II. The importance of this work was best described by Alvin Weinberg, former Laboratory Director, when he wrote ``... If God has a golden book and writes down what it is that Oak Ridge National Laboratory did that had the biggest influence on science, I would guess that was the production and distribution of isotopes.`` Radioisotopes production continues to be an important aspect of Oak Ridge programs today and of those planned for the future. Past activities, current projects, and future plans and potentials will be described briefly in this paper. Also, some of the major issues facing the continued production of radioisotopes will be described. The scope of the program has always been primarily that of process development, followed by special batch-type productions, where no other supply exists. The technology developed has been available for adoption by US commercial corporations, and in cases where this has occurred, Oak Ridge has withdrawn as a supplier of the particular isotopes involved. One method of production that will not be described is that of target bombardment with an accelerator. This method was used at Oak Ridge prior to 1978 in the 86-inch Cyclotron. However, this method has not been used at Oak Ridge since then for radioisotope production, except as a research tool.
Collins, E.D.; Aaron, W.S.; Alexander, C.W.; Bigelow, J.E.; Parks, J.T.; Tracy, J.G.; Wham, R.M.
Generation of hybrid transgenic silkworms that express Bombyx mori prolyl-hydroxylase alpha-subunits and human collagens in posterior silk glands: Production of cocoons that contained collagens with hydroxylated proline residues.
Prolyl 4-hydroxylase (P4H) is a heterotetramer enzyme consisting of alpha-subunits (P4Halpha) and beta-subunits (P4Hbeta), and is required for collagen biosynthesis. Previously, we generated transgenic silkworms that produced human type III collagen fragments (mini-collagens) in the posterior silk gland (PSG). However, prolyl 4-hydroxylation did not occur on the mini-collagens, because in spite of an abundant expression of P4Hbeta in PSGs, P4Halpha expression was quite low there, thus resulting in an insufficient activity of P4H. In this study we aimed at generating hybrid transgenic silkworms whose PSGs are capable of producing mini-collagens and enough P4H for their prolyl 4-hydroxylation. Isolated PSGs were bombarded with fibroin L-chain gene promoter-driven vectors containing Bombyx mori P4Halpha (BmP4Halpha) cDNAs and were transplanted into the hemolymphatic cavity. The P4H activity in the PSG cells significantly increased, indicating that the expressed BmP4Halpha formed active tetramers with endogenous BmP4Hbeta. Using germ-line transgenesis technology, silkworms were generated that synthesized BmP4Halpha in PSG cells. The P4H activity in the transgenic silkworms was 130-fold higher than that of wild-type counterparts. Finally, we generated hybrid transgenic silkworms that expressed cDNAs of both BmP4Halpha and mini-collagen in PSG cells. They spun cocoons that contained mini-collagens whose appropriate proline residues had been adequately hydroxylated. PMID:16766075
Adachi, Takahiro; Tomita, Masahiro; Shimizu, Katsuhiko; Ogawa, Shingo; Yoshizato, Katsutoshi
76 FR 80433 - In the Matter of Royal Oak Capital Management, LLC, 6173 Bellevue Road, Royal Oak, MD 21662...
...Bellevue Road, Royal Oak, MD 21662; Notice of Intention To Cancel Registration Pursuant to Section 203(H) of the Investment...adviser under section 203A, the Commission shall by order, cancel the registration of such person. The registrant indicated...
Contingency plan for the Old Hydrofracture Facility Tanks Sluicing Project at Oak Ridge National Laboratory, Oak Ridge, Tennessee.
This revised contingency plan addresses potential scenarios involving the release of radioactively contaminated waste from the Old Hydrofracture Facility Tanks Contents Removal project to the environment. The tanks are located at the Oak Ridge National La...
Interim action proposed plan: Mercury tank remediation in the Oak Ridge Y-12 Plant, Oak Ridge, Tennessee
The Oak Ridge Y-12 Plant is located adjacent to the city of Oak Ridge in Anderson County, Tennessee. The plant was constructed in the 1940s and primarily makes nuclear weapons components. In 1953, the Y-12 Plant was involved in the first production-scale separation of lithium isotopes for the development of hydrogen bombs. The lithium separation process involved separating the ⁶Li
Van Ryn
Microgravity survey of the Oak Ridge K-25 Site, Oak Ridge, Tennessee
Karst features are known to exist within the carbonate bedrock of the Oak Ridge K-25 Site and may play an important role in groundwater flow and contaminant migration. This report discusses the results of a microgravity survey of the Oak Ridge K-25 Site. The main objective of the survey is to identify areas containing bedrock cavities. Secondary objectives included correlating the observed gravity to the geology and to variations in overburden thickness. The analysis includes 11 profile lines that are oriented perpendicular to the geologic strike and major structures throughout the K-25 Site. The profile lines are modeled in an effort to relate gravity anomalies to karst features such as concentrations of mud-filled cavities. Regolith thickness and density data provided by boreholes constrain the models. Areally distributed points are added to the profile lines to produce a gravity contour map of the site. In addition, data from the K-901 area are combined with data from previous surveys to produce a high resolution map of that site. The K-25 Site is located in an area of folded and faulted sedimentary rocks within the Appalachian Valley and Ridge physiographic province. Paleozoic age rocks of the Rome Formation, Knox Group, and Chickamauga Supergroup underlie the K-25 Site and contain structures that include the Whiteoak Mountain Fault, the K-25 Fault, a syncline, and an anticline. The mapped locations of the rock units and complex structures are currently derived from outcrop and well log analysis.
Kaufmann, R.D.
Characterization plan for the Oak Ridge National Laboratory Area-Wide Groundwater Program, Oak Ridge, Tennessee
This characterization plan has been developed as part of the U.S. Department of Energy`s (DOE`s) investigation of the Groundwater Operable Unit (GWOU) at Oak Ridge National Laboratory (ORNL) located near Oak Ridge, Tennessee. The first iteration of the characterization plan is intended to serve as a strategy document to guide subsequent GWOU remedial investigations. The plan provides a rationale and organization for groundwater data acquisition, monitoring, and remedial actions to be performed during implementation of environmental restoration activities associated with the ORNL GWOU. It is important to note that the characterization plan for the ORNL GWOU is not a prototypical work plan. As such, remedial investigations will be conducted using annual work plans to manage the work activities, and task reports will be used to document the results of the investigations. Sampling and analysis results will be compiled and reported annually with a review of data relative to risk (screening level risk assessment review) for groundwater. This characterization plan outlines the overall strategy for the remedial investigations and defines tasks that are to be conducted during the initial phase of investigation. This plan is presented with the understanding that more specific addenda to the plan will follow.
Quality assurance plan for Waste Area Grouping 6 at Oak Ridge National Laboratory, Oak Ridge, Tennessee
This Quality Assurance Plan (QAP) is concerned with design and construction (Sect. 2) and characterization and monitoring (Sect. 3). The basis for Sect. 2 is the Quality Assurance Plan for the Design and Construction of Waste Area Grouping 6 Closure at Oak Ridge National Laboratory, Oak Ridge, Tennessee, and the basis for Sect. 3 is the Environmental Restoration Quality Program Plan. Combining the two areas into one plan gives a single, overall document that explains the requirements and from which the individual QAPs and quality assurance project plans can be written. The Waste Area Grouping (WAG) 6 QAP establishes the procedures and requirements to be implemented for control of quality-related activities for the WAG 6 project. Quality Assurance (QA) activities are subject to requirements detailed in the Martin Marietta Energy Systems, Inc. (Energy Systems), QA Program and the Environmental Restoration (ER) QA Program, as well as to other quality requirements. These activities may be performed by Energy Systems organizations, subcontractors to Energy Systems, and architect-engineer (A-E) under prime contract to the US Department of Energy (DOE), or a construction manager under prime contract to DOE. This plan specifies the overall Energy Systems quality requirements for the project. The WAG 6 QAP will be supplemented by subproject QAPs that will identify additional requirements pertaining to each subproject.
Influence of cattle grazing on the density of oak seedlings and saplings in a Tabor oak forest in Israel
The effects of cattle grazing on the density of seedlings and saplings in a Tabor oak forest ( Quercus ithaburensis subsp. ithaburensis) are investigated. The Tabor oak forest studied is located in a Nature Reserve in the Mediterranean region of Israel. Cattle graze at a stocking density of 0.71 head/ha for 6 months a year. The cattle grazing in the Nature Reserve is a beneficial management measure because it enhances plant species richness and reduces shrub encroachment. The impact of grazing on the densities of seedlings and young saplings was quantified in 46 large sampling plots (333 m 2 each) distributed over two experimental sites; the first being used as a rangeland for decades while the second is a forest patch totally free from grazing. The density and the height of Tabor oak individuals in each sampling plot were recorded. Four height categories were distinguished with a special focus on young seedlings (<0.15 m), established seedlings and young saplings (0.15 m-1 m). The density of seedlings and young sapling in the grazed Tabor oak forest were, respectively, 61% to 67% lower than in the ungrazed treatment. Implications on the continuity of the entire Tabor oak forest ecosystem are discussed. Three management measures that enable to prevent a decrease in young oak densities are proposed - reduction of stocking rate, deferment of the commencement of grazing, and fencing young seedlings.
Dufour-Dror, Jean-Marc
In Vitro Studies of Poison Oak Immunity
Studies were performed to ascertain the effect of urushiol analogues on the in vitro lymphocyte blastogenesis elicited by urushiol in peripheral blood lymphocytes taken from individuals sensitized to poison oak or ivy. Urushiol is a mixture of alkylcatechols composed of a catechol ring coupled to mono-, di-, or tri-unsaturated C-15 or C-17 carbon side chains. Each of these two moieties, catechol ring and side chain, was tested for its role in eliciting reactivity. Analogues tested represented the catechol ring (3-methylcatechol), the mono- or di-unsaturated side chain (oleic or linoleic acid), and the saturated side chain coupled to a catechol ring (pentadecylcatechol), a blocked catechol ring (heptadecylveratrole), or a resorcinol (pentadecylresorcinol). Urushiol with a blocked catechol ring (urushiol dimethyl ether) was also included. Of these, only pentadecylcatechol evoked reactivity in sensitized lymphocytes, and this reactivity was only a fraction of that evoked by urushiol. This suggested that the system has some requirement for the side chain, and that the catechol ring is critical for reactivity. This was further investigated by testing the ability of some of these analogues to inhibit urushiol-specific blastogenesis. No inhibition was noted with compounds bearing the saturated side chain with modified ring structures (pentadecylresorcinol and heptadecylveratrole). However, both 3-methylcatechol and pentadecylcatechol (at equimolar concentrations) blocked reactivity. The results of our experiments suggested that although both the side chain and the catechol ring are required for reactivity, the latter is most critical. Unsaturation in the side chain is important for maximal reactivity because the saturated catechols were only partially as active as the urushiol oil. There may be a greater dose requirement for the catechol ring than for the side chain.
Byers, Vera S.; Castagnoli, Neal; Epstein, William L.
White Oak Creek Watershed: Melton Valley Area Remedial Investigation Report, Oak Ridge National Laboratory, Oak Ridge, Tennessee: Volume 3 Appendix C
This report provides details on the baseline ecological risk assessment conducted in support of the Remedial Investigation (RI) Report for the Melton Valley areas of the White Oak Creek watershed (WOCW). The RI presents an analysis meant to enable the US Department of Energy (DOE) to pursue a series of remedial actions resulting in site cleanup and stabilization. The ecological risk assessment builds off of the WOCW screening ecological risk assessment. All information available for contaminated sites under the jurisdiction of the US Department of Energy`s Comprehensive Environmental Response, Compensation, and Liability Act Federal Facilities Agreement within the White Oak Creek (WOC) RI area has been used to identify areas of potential concern with respect to the presence of contamination posing a potential risk to ecological receptors within the Melton Valley area of the White Oak Creek watershed. The risk assessment report evaluates the potential risks to receptors within each subbasin of the watershed as well as at a watershed-wide scale. The WOC system has been exposed to contaminant releases from Oak Ridge National Laboratory and associated operations since 1943 and continues to receive contaminants from adjacent waste area groupings.
Description of Vegetation of the Oak Openings of Northwestern Ohio at the Time of Euro-American Settlement 1
Original land survey notes were used to produce a map of the Oak Openings of northwestern Ohio showing the vegetation at the time of Euro-American settlement (1817-1832). For that period, the area of the Oak Openings was 43% Oak Savanna, 27% Wet Prairie, 23% Oak Woodland, 7% Oak Barrens, and <1% Floodplain Forest. The composition of the tree layer was
Genetic Diversity Increases Insect Herbivory on Oak Saplings
A growing body of evidence from community genetics studies suggests that ecosystem functions supported by plant species richness can also be provided by genetic diversity within plant species. This is not yet true for the diversity-resistance relationship as it is still unclear whether damage by insect herbivores responds to genetic diversity in host plant populations. We developed a manipulative field experiment based on a synthetic community approach, with 15 mixtures of one to four oak (Quercus robur) half-sib families. We quantified genetic diversity at the plot level by genotyping all oak saplings and assessed overall damage caused by ectophagous and endophagous herbivores along a gradient of increasing genetic diversity. Damage due to ectophagous herbivores increased with the genetic diversity in oak sapling populations as a result of higher levels of damage in mixtures than in monocultures for all families (complementarity effect) rather than because of the presence of more susceptible oak genotypes in mixtures (selection effect). Assemblages of different oak genotypes would benefit polyphagous herbivores via improved host patch location, spill over among neighbouring saplings and diet mixing. By contrast, genetic diversity was a poor predictor of the abundance of endophagous herbivores, which increased with individual sapling apparency. Plant genetic diversity may not provide sufficient functional contrast to prevent tree sapling colonization by specialist herbivores while enhancing the foraging of generalist herbivores. Long term studies are nevertheless required to test whether the effect of genetic diversity on herbivory change with the ontogeny of trees and local adaptation of specialist herbivores.
Castagneyrol, Bastien; Lagache, Lelia; Giffard, Brice; Kremer, Antoine; Jactel, Herve
Genetically defined membrane-bound and soluble alkaline phosphatases of the silkworm: their discrete localization and properties.
In the midgut tissue of the silkworm, Bombyx mori, alkaline phosphatase isozymes, membrane-bound (m-ALP) and soluble (s-ALP) forms are controlled by non-allelic genes on the same chromosome. We purified and characterized both ALPs to elucidate their possible functions and to compare with mammalian ALPs. Both forms were found to be similar Mr = 68,000 in gel permeation chromatography and as a single subunit as a monomer in SDS-polyacrylamide gel electrophoresis with Mr = 58,000 for m-ALP and Mr = 61,000 for s-ALP. The pH optima of ALPs were 10.9 (m-ALP) and 9.8 (s-ALP), and the former was extremely stable even in pH 10-12 which accords with the physiological milieu in Bombyx midgut lumen. Both ALPs had similar substrate specificities. L-cysteine inhibited strongly both ALPs, but inhibitory effects of L-phenylalanine, L-homoarginine, and L-leucine were undetectable for s-ALP and very weak for m-ALP. The antibody raised against purified m-ALP recognized m-ALP but not purified s-ALP and vice versa. Rocket-immunoassay showed that m-ALP was distributed in similar levels along the length of midgut except for the most anterior portion. Seventy percent of s-ALP activity existed in the last one-third of midgut. Immunohistochemical study revealed that the m-ALP was localized at the brush border of columnar cells in the middle and posterior midgut epithelia. In contrast, the s-ALP was localized at the apical surface of goblet cells through the length of midgut. We detected ATPase activity in the purified s-ALP preparation; Mg2+ was essential for the ATPase activity and the activity also increased with KHCO3 but not with KCl. The solubilization test of m- ALP with various agents was attempted and the relationship between m-ALP and the digestive fluid-ALP was discussed. PMID:2392471
Eguchi, M; Azuma, M; Yamamoto, H; Takeda, S
RIP-seq of BmAgo2-associated small RNAs reveal various types of small non-coding RNAs in the silkworm, Bombyx mori
Background Small non-coding RNAs (ncRNAs) are important regulators of gene expression in eukaryotes. Previously, only microRNAs (miRNAs) and piRNAs have been identified in the silkworm, Bombyx mori. Furthermore, only ncRNAs (50-500nt) of intermediate size have been systematically identified in the silkworm. Results Here, we performed a systematic identification and analysis of small RNAs (18-50nt) associated with the Bombyx mori argonaute2 (BmAgo2) protein. Using RIP-seq, we identified various types of small ncRNAs associated with BmAGO2. These ncRNAs showed a multimodal length distribution, with three peaks at ~20nt, ~27nt and ~33nt, which included tRNA-, transposable element (TE)-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. The tRNA-derived fragments (tRFs) were found at an extremely high abundance and accounted for 69.90% of the BmAgo2-associated small RNAs. Northern blotting confirmed that many tRFs were expressed or up-regulated only in the BmNPV-infected cells, implying that the tRFs play a prominent role by binding to BmAgo2 during BmNPV infection. Additional evidence suggested that there are potential cleavage sites on the D, anti-codon and T?C loops of the tRNAs. TE-derived small RNAs and piRNAs also accounted for a significant proportion of the BmAgo2-associated small RNAs, suggesting that BmAgo2 could be involved in the maintenance of genome stability by suppressing the activities of transposons guided by these small RNAs. Finally, Northern blotting was also used to confirm the Bombyx 5.8�s rRNA-derived small RNAs, demonstrating that various novel small RNAs exist in the silkworm. Conclusions Using an RIP-seq method in combination with Northern blotting, we identified various types of small RNAs associated with the BmAgo2 protein, including tRNA-, TE-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. Our findings provide new clues for future functional studies of the role of small RNAs in insect development and evolution.
High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV) displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization
Background Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR) that displayed human (pro)renin receptor (hPRR) connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC). Results A rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR). A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 � 108 plaque forming unit (pfu) in hemolymph, which was 2.8 � 104 times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i.), but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa) to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i.. Conclusion The successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR.
Kato, Tatsuya; Manoha, Suganthi Lavender; Tanaka, Shigeyasu; Park, Enoch Y
Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein
Background Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens. Methodology/Principal Findings Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60�C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/? bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5?- and 3?-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense. Conclusions/Significance The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.
Patnaik, Bharat Bhusan; Kim, Dong Hyun; Oh, Seung Han; Song, Yong-Su; Chanh, Nguyen Dang Minh; Kim, Jong Sun; Jung, Woo-jin; Saha, Atul Kumar; Bindroo, Bharat Bhushan; Han, Yeon Soo
Management of spent nuclear fuel on the Oak Ridge Reservation, Oak Ridge, Tennessee: Environmental assessment
On June 1, 1995, DOE issued a Record of Decision [60 Federal Register 28680] for the Department-wide management of spent nuclear fuel (SNF); regionalized storage of SNF by fuel type was selected as the preferred alternative. The proposed action evaluated in this environmental assessment is the management of SNF on the Oak Ridge Reservation (ORR) to implement this preferred alternative of regional storage. SNF would be retrieved from storage, transferred to a hot cell if segregation by fuel type and/or repackaging is required, loaded into casks, and shipped to off-site storage. The proposed action would also include construction and operation of a dry cask SNF storage facility on ORR, in case of inadequate SNF storage. Action is needed to enable DOE to continue operation of the High Flux Isotope Reactor, which generates SNF. This report addresses environmental impacts.