New Pyrene Derivatives Having Rigid Imidazole Moiety for Blue Emitters.

Pyrene, imidazole and dibenzofuran were used to synthesize new blue emitters of 1-(4-(dibenzo[b,d]furan-4-yl)phenyl)-2-(pyren-1-yl)-1H-phenanthro[9,10-d]imidazole (BFP-PI) and 1-(4-(dibenzo[b,d]furan-4-yl)phenyl)-4,5-diphenyl-2-(pyren-1-yl)-1H-imidazole (BFP-DPI). In the film state, BFP-PI and BFP-DPI show photoluminescence (PL) maximum values of 462 nm and 459 nm. The relative PL quantum efficiency (PLQY) of BFP-PI and BFP-DPI is 89.16% and 79.2% by using reference compound of 9,10-diphenylanthracene. The device using BFP-PI in the non-doped state as emitting material showed current efficiency (C.E.) of 3 cd/A and external quantum efficiency (E.Q.E.) of 2.15%, and the device using BFP-DPI as emitting material exhibited C.E. of 2.64 cd/A and E.Q.E. of 1.6%.

Silk fibroin and 4-hexylresorcinol incorporation membrane for guided bone regeneration.

The objective of this study was to demonstrate that a silk fibroin (SF) and 4-hexylresorcinol (4-HR) incorporation membrane could be used for a guided bone regeneration technique. Fourier transform infrared measurements were obtained to determine change of physical property of SF membrane by 4-HR incorporation. Two peri-implant defects, 3.0 × 5.0 mm (width × length), were prepared on the lateral side of the implant hole in the tibia of New Zealand white rabbits (n = 8). The peri-implant defect was left unfilled in the control group. Silk fibroin + 4-HR membrane was applied to the peri-implant defect in the experimental group. The 8 animals were killed at 8 weeks after implantation. Subsequently, removal torque test and histomorphometric evaluation were done. Fourier transform infrared spectroscopy showed no specific chemical interaction between 4-HR and SF. In the histomorphometric analysis, the mean bone regeneration was 18.3 ± 1.9 mm(2) in the experimental group and 9.3 ± 0.9 mm(2) in the control group (P = 0.004). In conclusion, the SF and 4-HR incorporation membrane successfully regenerated bone in the rabbit tibia peri-implant bone defect model.

Long-term preservation, regeneration, and cultivation of Paecilomyces tenuipes (Peck) Samson (Ascomycetes), an entomopathogenic fungus inoculated into the silkworm larva of Bombyx mori.

Paecilomyces tenuipes reportedly have anticancer and immune activities, along with various other medicinal uses. Cultured products with P. tenuipes are certified for use in food in South Korea, and processed goods containing this fungus have been developed in many countries, particularly South Korea, Japan, and China. Research on mass production technology-procured raw materials for the manufacture of P. tenuipes is very important; however, cultures of the fungus have been unstable. This study identified stable cultivation conditions, focusing on growth inhibition and revitalization. Moisture regulation and preservation of pupae inoculated with P. tenuipes were used to control growth inhibition and revitalization. When inoculated silkworm pupae were dehydrated to 4% moisture and preserved freeze-dried or at -70 degrees C, -20 degrees C, or 4 degrees C, the mycelia in their bodies were able to survive for 14 d. Inoculated silkworm pupae were rehydrated for 3 h and the mycelia within their bodies were recovered at 94.3-96.3%. Silkworm pupae at 4% moisture were able to survive for 135 d at temperatures < 4 degrees C and for 1 y after freeze-drying. Optimal conditions for synnemata induction were 25 degrees C and 100-300 1x.

Comparison of methods for the repair of acute tympanic membrane perforations: Silk patch vs. paper patch.

We investigated the effects of repairing large tympanic membrane (TM) perforations in rats with a thin silk patch compared with the commonly used paper patch. We performed bilateral myringotomies of 1.8 mm in diameter on 50 adult Sprague-Dawley rats with intact TMs. The perforations in the right ears of 40 rats were treated with a silk patch, and the perforations in the left ears of the same rats were treated with a paper patch. Ten rats acted as controls. The mean healing times of the TM perforations on the silk-patch-treated ears and the paper-patch-treated ears were 7.2+/-1.48 and 9.1+/-1.11 days, respectively (control 10.38+/-1.70 days). The difference between silk-patch- and paper-patch-treated ears was statistically significant, with a mean difference of 1.9 days (0.6-4.5 days). The mean perforation closure times were significantly shorter in silk-patch- and paper-patch-treated ears than in the control animals. The endoscopic and histological findings of this study provide evidence that silk-patch treatment accelerates wound healing and shortens TM perforation closure time. We suggest that the silk patch may prove to be an effective material for repairing TM perforations in human patients in an outpatient clinical setting.

Production of recombinant human granulocyte macrophage colony-stimulating factor from silkworm Bombyx mori Bm5 cells.

Using silkworm Bombyx mori Bm5 cells, we established a stable cell line expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF), which gets its name from the Bm5-hGM-CSF cell in which the glycoprotein of the hGM-CSF is secreted in the cell culture supernatant (CCS). It was demonstrated that secreted hGM-CSF had in vivo biological activity and the white blood cell (WBC) value increased two times that of the control. We expect to produce useful human recombinant glycoproteins from silkworm cultured cells for a low price and a large quantity.

Two gap junction channel (innexin) genes of the Bombyx mori and their expression.

Gap junctions are clusters of intercellular channels that are associated with embryonic development and neural signaling. Innexins, invertebrate gap junction proteins, have been identified in Drosophila and Caenorhabditis. Here, we report the isolation and characterization of two novel members of the insect innexin family, Bm inx2 and Bm inx4, from embryos of the silkworm, Bombyx mori, during the germ-band formation stage. Bm inx2 is a single copy gene with one exon, while Bm inx4 is a single copy gene with four exons and three introns. The predicted proteins show structural similarities with other innexin family members, including four transmembrane (TM) domains, two extracellular loops (ELs), one cytoplasmic loop (CL), and typical conserved amino acids. Bm inx2 is phylogenetically orthologous to the other insect inx2 genes, but Bm inx4 is not orthologous to any known innexin including Dm inx4. Interestingly, Northern blotting and in situ hybridization showed that Bm inx2 was variously expressed across all developmental stages and in various tissues, with high expression seen in the nervous system at the time of embryogenesis. In contrast, Bm inx4 was transiently expressed at the germ-band formation stage of embryogenesis, and was specifically expressed in the ovary and testis during the larval and pupal stages. The isolation and characterization of these novel genes should form the basis for further study of the functional events that occur during development and neuronal communication in B. mori.

Structure and expression analysis of the cecropin-E gene from the silkworm, Bombyx mori.

Cecropins belong to the antibacterial peptides family and are induced after injection of bacteria or their cell-wall components. By silkworm cDNA microarray analysis, a novel type of Cecropin family gene was identified as a cDNA up-regulated in early embryo, 1 day after oviposition. The cDNA isolated was 394 bp with 198 ORF translating 65 amino acids, encoding BmCecropin-E (BmCec-E). Using Southern hybridization and genome search analysis, the number of BmCec-E gene was estimated to be at least two per haploid, which consisted of two exons, as in other Cecropin family members. BmCec-E mRNA was expressed transiently 1 day after egg-laying (AEL, germ-band formation stage), and was specifically expressed in the degenerating intestine during the pre-pupal and pupal stages, unlike other Cecropin family genes. Immune challenge analysis showed that BmCec-E gene expression was more strongly induced by Escherichia coli (gram-negative) than by Micrococus luteus (gram-positive), and not by virus injection. By bacterial challenge, expression of BmCec-E mRNA was induced 12 h after injection, and was maintained for 24 h. Expression of BmCec-E after immune challenge was observed strongly in excretory organs, such as hindgut and malphigian, slightly in fat body, skin, and midgut.

Domain a' of Bombyx mori protein disulfide isomerase has chaperone activity.

Protein disulfide isomerase (PDI) is an endoplasmic reticulum (ER)-localized multifunctional enzyme that can function as a disulfide oxidase, a reductase, an isomerase, and a chaperone. The domain organization of PDI is abb'xa'c, with two catalytic (CxxC) motifs and a KDEL ER retention motif. The members of the PDI family exhibit differences in tissue distribution, specificity, and intracellular localization. We previously identified and characterized the PDI of Bombyx mori (bPDI) as a thioredoxin-like protein that shares primary sequence homology with other PDIs. Here we compare the reactivation of inactivated rRNase and sRNase by bPDI and three bPDI mutants, and show that bPDI has mammalian PDI-like activity. On its own, the N-terminal a domain does not retain this activity, but the a' domain does. This is the first report of chaperone activity only in the a' domain, but not in the a domain.

Secretion of the antibacterial recombinant protein enbocin.

The insect baculovirus expression vector system (BEVS) is useful for the production of biologically active recombinant proteins. However, the overexpression of foreign proteins in this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we have developed a versatile baculovirus expression and secretion system using the Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion improved the secretion and antibacterial activity of recombinant enbocin proteins. Thus, bPDI gene fusion is a useful addition to the BEVS for the large-scale production of bioactive recombinant proteins.

Analysis of the immune-inducible genes of Plutella xylostella using expressed sequence tags and cDNA microarray.

In the present study, the complex gene expression responses of Plutella xylostella to microbial challenges and injury were surveyed using a newly constructed expressed sequence tag (EST) clone collection and cDNA microarray analysis. A total of 1132 P. xylostella ESTs were cloned, annotated and categorized by their putative functions; these included proteases, protease inhibitors, recognition molecules and anti-microbial peptides. GeneOntology revealed that 4% of the P. xylostella ESTs corresponded to immunity-related genes potentially involved in innate immunity. We then used microarray analysis to identify 44 genes that were differentially expressed with at least a two-fold expression difference in P. xylostella before and after pathogen challenge. Together, our EST categorization and microarray profiling analyses allowed us to identify 70 genes that should be considered candidate immune response genes, providing important new insights into the molecular events that occur during the innate immune response in P. xylostella.

Gene expression profiling in the silkworm, Bombyx mori, during early embryonic development.

We prepared a cDNA library for a microarray from eggs of the silkworm, Bombyx mori, at the germ-band formation (24 hours after fertilization) stage. Using a microarray constructed with 2,445 ESTs, we screened gene expression profiles during germ-band formation at six specific time points in the early embryonic stages (from the unfertilized egg to the formation of abdominal leg appendages), and determined 241 of these cDNAs to represent genes that were expressed differentially during the germ-band formation stage. These differentially expressed genes grouped into two clusters. In the early and late clusters, 203 and 38 genes were upregulated, respectively. In the upregulated clusters, we isolated several genes that were associated with development and cell communication, including egalitarian, RAD23b, innexin 2, and senescence-associated protein. Northern blot hybridization revealed that the expression patterns of 14 genes had changed in each of the stages. In this study, we assessed changes in the levels of gene expression in relation to the germ-band formation stages in whole Bombyx embryos.

Characterization of a novel repetitive secretory protein specifically expressed in the modified salivary gland of Hydropsyche sp. (Trichoptera; Hydropsychidae).

Here, we report the cloning and characterization of a common salivary gland-specific gene, Nf-1, from late instar Hydropsyche sp. larvae, and show that the corresponding gene product is translated in the gland and secreted to the gland lumen. The deduced Nf-1 protein is primarily composed of five repetitive sequence units of 63-65 amino acids, and contains a putative signal sequence composed of 19 amino acids. Secreted Nf-1 (approximately 37 kDa) was localized to the gland lumen by Western blotting of gland and lumen fractions. Together, the structure, expression pattern and protein localization of Nf-1 indicate that this protein is likely to be a major component of the silk shields and nets produced by the aquatic insect, Trichoptera.

Characterization of a silkworm thioredoxin peroxidase that is induced by external temperature stimulus and viral infection.

A thioredoxin peroxidase (TPx) that reduces H(2)O(2) was firstly characterized in the lepidopteran insect, silkworm Bombyx mori. The B. mori TPx (BmTPx) cDNA contains an open reading frame of 585 bp 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% identity to Drosophila melanogaster (DmTPx-1), 73% to Aedes aegypti (AaTPx), and 54-48% to other insect 2-Cys TPx. The cDNA encoding BmTPx was expressed as a 25-kDa polypeptide in baculovirus-infected insect Sf9 cells. The purified recombinant BmTPx was shown to reduce H(2)O(2) in the presence of electrons donated by dithiothreitol and shown to be active in the presence of thioredoxin as electron donor. Northern blot analysis revealed the presence of BmTPx transcripts in all tissues examined. Western blot analysis showed the presence of the BmTPx in the fat body and midgut, but not in the hemolymph, suggesting the BmTPx is not secretable. When H(2)O(2) was injected into body cavity of B. mori larva, BmTPx mRNA expression was up-regulated in the fat body tissues. Interestingly, the expression levels of BmTPx enzyme in the fat body were particularly high when B. mori larva was exposed at low (4 degrees C) and high (37 degrees C) temperatures or baculovirus infection, suggesting that the BmTPx seems to play a protective role against oxidative stress caused by temperature stimuli and viral infection.

Functional recovery guided by an electrospun silk fibroin conduit after sciatic nerve injury in rats.

The aim of this study was to evaluate the regenerative capacity of a newly developed nerve guidance conduit using electrospun silk fibroin (SFNC) implanted in a 10-mm defect of the sciatic nerve in rats. After evaluating the physical properties and cytocompatibility of SFNC in vitro, rats were randomly allocated into three groups: defect only, autograft and SFNC. To compare motor function and abnormal sensation among groups, ankle stance angle (ASA) and severity of autotomy were observed for 10 weeks after injury. Immunostaining with axonal neurofilament (NF) and myelin basic protein (MBP) antibodies were performed to investigate regenerated nerve fibres inside SFNC. ASA increased significantly in the SFNC group at 1, 7 and 10 weeks after injury compared to the defect only group (p<0.05). At one week, mean ASA of the SFNC group was significantly higher than that of the autograft group (p<0.05). Onset and severity of autotomy decreased significantly in the SFNC group compared to other groups (p<0.05). Autotomy in the SFNC group started at 4 weeks and maximally reached toe level. However, the defect only and autograft groups first showed autotomy at 2 and 1 weeks following injury, respectively, and then reached the sole level. Well myelinated nerve fibres stained with NF and MBP were found inside SFNC. In conclusion, SFNC could be helpful in restoring motor function and preventing abnormal sensations after nerve injury.

Molecular characterization of a Bombyx mori protein disulfide isomerase (bPDI).

We have isolated a complementary deoxyribonucleic acid clone that encodes the protein disulfide isomerase of Bombyx mori (bPDI). This protein has a putative open reading frame of 494 amino acids and a predicted size of 55.6 kDa. In addition, 2 thioredoxin active sites, each with a CGHC sequence, and an endoplasmic reticulum (ER) retention signal site with a KDEL motif were found at the C-terminal. Both sites are typically found in members of the PDI family of proteins. The expression of bPDI messenger ribonucleic acid (mRNA) was markedly increased during ER stress induced by stimulation with calcium ionophore A23187, tunicamycin, and dithiothreitol, all of which are known to cause an accumulation of unfolded proteins in the ER. We also examined the tissue distribution of bPDI mRNA and found pronounced expression in the fat body of insects. Hormonal regulation studies showed that juvenile hormone, insulin, and a combination of juvenile hormone and transferrin (although not transferrin alone) affected bPDI mRNA expression. A challenge with exogenous bacteria also affected expression, and the effect peaked 16 hours after infection. These results suggest that bPDI is a member of the ER-stress protein group, that it may play an important role in exogenous bacterial infection of the fat body, and that its expression is hormone regulated.

Evidence for brain-derived neurotrophic factor-like neuropeptide in brain of the silk moth Bombyx mori during postembryonic periods.

Brain-derived neurotrophic factor-like neuropeptide is produced in the brain of the silk moth, Bombyx mori. Immunocytochemical studies of brain and retrocerebral complex of larvae, prepupae, pupae and adults showed that four pairs of median neurosecretory cells and six pairs of lateral neurosecretory cells which had different immunoreactivities to BDNF peptide. Day-1 adult brains showed no evidence of neurons stained by anti-BDNF antibodies. Those reactivities, which were much stronger in median cells than in lateral cells, were the weakest in an earliest larval stage and a latest pupal stage but the strongest in late larval stage. Median neurosecretory cells projected their axons into the contralateral corpora allata by decussation in the median region, nerve corpora cardiaca (NCC) I, and nerve corpora allata (NCA) I, whereas lateral neurosecretory cells extended their axons to the ipsilateral corpora allata via NCC II and NCA I.

Molecular characterization of a cDNA from the silk moth Bombyx mori encoding Manduca sexta allatotropin peptide.

Allatotropin is a 13-residue amidated neuropeptide isolated from pharate adult heads of the tobacco hornworm, Manduca sexta and strongly stimulates biosynthesis of juvenile hormones in adults, but not larval, lepidopteran corpora allata. From a Bombyx mori midgut cDNA library, a cDNA that encodes a 130-amino-acid polypeptide containing M. sexta allatotropin sequence was isolated. The B. mori allatotropin cDNA consists of 1196 nucleotides. The encoded allatotropin peptide is identical to that isolated from M. sexta and that predicted from Pseudaletia unipuncta, with 84% and 81% identity in the amino acid sequence of the allatotropin peptide precursor, respectively. M. sexta allatotropin is flanked by two different endoproteolytic cleavage sites within the precursor of the B. mori allatotropin peptide. Evidence from northern blotting of B. mori tissues showed that the allatotropin gene is expressed in the cells of midgut, head and integument with different transcription amount, but not in the fat body and silk gland. Midgut has also a number of allatotropin-immunoreactive cells and nerve fibers. These results will provide valuable information in understanding the AT gene of insects.

Effects of 20-hydroxyecdysone and serotonin on neurite growth and survival rate of antennal lobe neurons in pupal stage of the silk moth Bombyx mori in vitro.

Effects of 20-hydroxyecdysone and serotonin on the morphological development and the survival of antennal lobe neurons from day-2 pupal brains of the silk moth Bombyx mori were investigated in vitro. Four morphologically distinct neuronal types could be identified in the cultured antennal lobe neurons: unipolar, bipolar, multi-polar and projection neurons. Antennal lobe neurons in culture with 20-hydroxyecdysone and serotonin showed different patterns of the morphological development from those described in Manduca sexta. Projection neurons extend their neurites remarkably by 20-hydroxyecdysone in B. mori, but there is no extension from antennal lobe neurons in M. sexta. Multi-polar neurons conspicuously increase only formation of new branches from their primary neurites by serotonin in B. mori, but there are both extension and branching of the neurites in M. sexta. On day-5, antennal lobe neurons in lower titers of 20-hydroxyecdysone had significantly higher survival rates than those in higher titers. Neurons cultured for 7 days at different levels of 20-hydroxyecdysone generally showed significantly lower survival rates than neurons cultured for 5 days under the same conditions.

FMRFamide-expressing efferent neurons in eighth abdominal ganglion innervate hindgut in the silkworm, Bombyx mori.

The tetrapeptide FMRFamide is known to affect both neural function and gut contraction in a wide variety of invertebrates and vertebrates, including insect species. This study aimed to find a pattern of innervation of specific FMRFamide-labeled neurons from the abdominal ganglia to the hindgut of the silkworm Bombyx mori using the immunocytochemical method. In the 1st to the 7th abdominal ganglia, labeled efferent neurons that would innervate the hindgut could not be found. However, in the 8th abdominal ganglion, three pairs of labeled specific efferent neurons projected axons into the central neuropil to eventually innervate the hindgut. Both axons of two pairs of labeled cell bodies in the lateral rind and axons of one pair of labeled cell bodies in the posterior rind extended to the central neuropil and formed contralateral tracts of a labeled neural tract with a semi-circular shape. These labeled axons ran out to one pair of bilateral cercal nerves that extended out from the posterior end of the 8th abdominal ganglion and finally to the innervated hindgut. These results provide valuable information for detecting the novel function of FMRFamide-related peptides in metamorphic insect species.

Association of reticular cells with CD34+/Sca-1+ apoptotic cells in the hemopoietic organ of grasshopper, Euprepocnemis shirakii.

Hemopoiesis in orthopteran insects occurs in a hemopoietic organ that is located bilaterally along the aorta. This organ is also known as a reticulo-hemopoietic organ because of the rich presence of reticular cells. This study was performed to further elucidate hemopoiesis in the reticulo-hemopoietic organ of an orthopteran, Euprepocnemis shirakii. We focused on the question why reticular cells are so abundant (35% of cells in hemopoietic organ). Interestingly, 21% of these reticular cells surrounded hemocytes with their reticular cytoplasm. The surrounded hemocytes were distinguished by their different size and darkly stained nucleus. These cells were characterized by immunostaining using antibodies against several types of hemocytes: 45% of the surrounded hemocytes were CD34+, and these positive cells were double stained (over 85%) when immunostained by another hemopoietic pluripotent cell marker, Sca-1. Transmission electron microscopic analysis showed that reticular cells surrounded hemocytes containing large nuclei and poorly developed cytoplasmic organelles. This strongly suggests that the reticular cells surround hemopoietic stem cells. Additionally, surrounded hemopoietic progenitor cells are undergoing apoptosis as indicated by the TUNEL assay. The enclosed apoptotic cells are engulfed and then phagocytosed by reticular cells. Our results suggest that reticular cells are related to the differentiation and apoptosis of hemopoietic stem cells.