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In insect cell culture, a fetal bovine serum is often used to maintain the cellular physiological conditions, irrespective of its relatively high cost and unstable supply. Several serum-free media for insects were developed, but it is challenging to adopt some insect cells to these serum-free media. Silkworm serum was used as a replacement of fetal bovine serum for a long time but not used widely due to the melanization of insect serum catalyzed by phenoloxidase (PO). PO inhibitors reported for insect serum preparation have a significantly negative effect on the long-term culture of healthy insect cells. In this study, we evaluated several kinds of PO inhibitors and found the method to prepare silkworm serum, which is suitable for the maintenance of insect cells and the production of the recombinant proteins by the baculovirus expression system. When L-Glutathione (reduced form) or L-Cysteine is used as a PO inhibitor at the time of recovery of silkworm serum, it is possible to suppress the browning of the silkworm serum. However, in this state of silkworm serum, browning is observed in long-term cell culture use. Therefore, it is preferable for cell culture to add 10% of silkworm serum treated at 50°C for 30 minutes to the medium with 2.7 mM L-Glutathione. Our results suggest that silkworm serum can be an alternative of mammalian serum for cell culture and used for producing the proteins for clinical application without any risk of contamination of zoonotic infectious disease viruses. [ABSTRACT FROM AUTHOR]

Virus-like particles (VLPs) are multi-protein complex, which mimic viral particles without viral genomes. Recently various applications of VLPs for medical and pharmaceutical fields are developed. Furthermore, surface modification of VLPs is expected to extend their functional versatility. As an enzymatic strategy for the modification, microbial transglutaminase (MTG) that catalyzes the covalent bond between a glutamine residue and a lysine residue or a primary amine is suitable for efficient protein ligation. In the previous study, we demonstrated the efficient production of immunogenic VLPs of porcine circovirus type 2 (PCV2) using silkworm-baculovirus expression vector system (silkworm-BEVS). Herein, we established the display system of exotic molecules to VLPs by enzymatic covalent cross-linking using MTG. For the target modification, Q-tag (YPLQMRG) that is MTG reactive sequence were introduced into the N-terminus, C-terminus, or loop BC of capsid protein of PCV2 and successfully expressed as VLPs in silkworm pupae. Of these, PCV2 VLPs with Q-tag at the loop BC and C-terminus were efficiently conjugated with FITC-cadaverine and K-tagged (MRHKGS) EGFP by MTG. These results showed that flexible surface modification of VLPs mediated by MTG could be used for development of several therapeutic tools such as multivalent vaccines. [ABSTRACT FROM AUTHOR]

Recent advances in bacterial characterization methodologies have made taxonomic categorization significantly more accurate. Here, we re-evaluated the position of bacterial strains (532 and 652) belonging to the genus Hafnia, isolated from flacherie-diseased silkworms in 1965. Phylogenetic analysis based on the 16S rRNA gene sequences of these strains suggests that they belong to genus Enterobacter. Using multilocus sequence analysis (MLSA), these strains were further classified to MLSA group A, which is a "core" group of Enterobacter containing E. cloacae (the type species of the genus). Although these strains were closely related to E. mori, E. tabaci, and E. asburiae, they also had other MLSA characteristics that distinguished them from these neighboring bacterial species. These data were supported by further biochemical analysis. Thus, it appears that the 532 and 652 strains isolated almost half a century ago belong to genus Enterobacter, and their unique characteristics strongly suggest that they are a novel bacterial species. [ABSTRACT FROM AUTHOR]

We previously found that rRNA of BM-N cells derived from the silkworm Bombyx mori undergoes rapid and extensive degradation through site-specific cleavage during abortive infection with nucleopolyhedroviruses (NPVs) of Autographa californica (AcMNPV), Hyphantria cunea (HycuMNPV), Spodoptera exigua and S. litura. Here, we demonstrated that rRNA degradation also occurs in Bme21 and Bm-aff3 cells, which are derived from B. mori embryo and fat body, respectively, during infection with AcMNPV. rRNA degradation in Bme21 cells was also observed following HycuMNPV infection, but was not detected in Bm-aff3 cells. We further showed that rRNA in a cell line derived from B. mandarina, an ancestor of B. mori, underwent degradation in response to cellular infection with AcMNPV and HycuMNPV. In contrast, no rRNA degradation was observed in a cell line derived from Antheraea pernyi. Taken together, these results indicate that NPV-triggered rRNA degradation represents a mechanism of innate antiviral immunity that is unique to B. mori and B. mandarina cells. [ABSTRACT FROM AUTHOR]

DNA replication is one of key event in cell-cycle progression, yet due to their importance and lethality, the chronological phenotypes of DNA synthesis machineries after the depletion of corresponding genes have proved difficult to study. In the present study, mRNAs for three DNA polymerases, a clamp, and three clamp loaders were gradually depleted from cultured silkworm cells by soaking RNAi. Interestingly, the depletion of these DNA synthesis factors had different effects on the cell growth rate and arrest of cell-cycle progression during timelapse observation. The depletion of DNA polymerases immediately arrested the cell-cycle progression at the S phase, while that of PCNA, a DNA clamp, required more time to slow cell growth and finally induced apoptosis. Surprisingly, silkworm cells continued to undergo several rounds of cell division when the components of clamp loaders were knocked down. [ABSTRACT FROM AUTHOR]

We aimed to identify virulence-associated genes of Serratia liquefaciens FK01 against Bombyx mori. Among 1,200 transconjugants from a transposon library, 4 (ET0234, ET0373, ET0418, and ET0964) showed decreased virulence towards B. mori. Southern hybridization revealed that the transposon was inserted at a single site of the S. liquefaciens genome. The flanking sequences of the transposon indicated that it disrupted the lipopolysaccharide (LPS) synthesis gene in all mutants. The complemented strain restored virulence completely or partially. Thus, LPS contributes to the virulence of S. liquefaciens against B. mori. Serum killing assays indicated that the bacterium was probably killed by the complement system. Since the innate immunity of a host is triggered by bacterial recognition, LPS might inhibit recognition by modifying the bacterial surface in B. mori. [ABSTRACT FROM AUTHOR]

N-glycosylation plays an important role in various biological activities and in the structural stability of serum glycoproteins. The baculovirus expression system (BES) is widely used to produce recombinant proteins but in some case it is not suitable for medical use because of the differences in N-linked glycans between insects and mammals. We reported that human serum protein alpha 1-acid protein (a1AGP) is effectively used as a model protein for evaluating the validity of engineering the insect-type N-glycosylation pathway. Using this protein, the productivity and N-linked glycan structures were compared among the 37 different silkworm strains. Interestingly, there was no difference in N-linked glycan structure among the silkworm strains, but there was difference in the degree of N-glycosylation. [ABSTRACT FROM AUTHOR]

The article discusses a study which reports the draft genome sequence of Paenibacillus popilliae, a rod-shaped endospore-forming bacterium, which causes milky disease in Japanese beetle Popillia japonica and related species. Topics discussed include preparation of the bacterial culture to extract genomic DNA from bacterial cells, phylogenetic analysis using previous data sets on microbial population, and comparison of the analysis with other entomo-pathogenic bacteria.

Human bone morphogenetic protein 4 (BMP4) fused to the collagen-binding domain (CBD) of fibronectin was constructed and expressed in a silkworm-baculovirus expression system. When recombinant BmNPV was injected into the silkworm larvae and harvested after approximately 4 days, 0.22 mg/ml (0.1 mg/larva) of recombinant CBD-BMP4 was secreted into the silkworm haemolymph. Interestingly, cultured silkworm cells infected with the same recombinant virus could not secrete the recombinant CBD-BMP4 into culture media. The purified rCBDBMP4 showed Smad- and MAPK-stimulating activities in human UET-13 cells. [ABSTRACT FROM AUTHOR]

The article discusses a study which reports the draft genome sequence of Paenibacillus popilliae, a rod-shaped endospore-forming bacterium, which causes milky disease in Japanese beetle Popillia japonica and related species. Topics discussed include preparation of the bacterial culture to extract genomic DNA from bacterial cells, phylogenetic analysis using previous data sets on microbial population, and comparison of the analysis with other entomo-pathogenic bacteria.

Using a cDNA microarray, we monitored global gene expression profiles in silkworm BmN4-SID1 cells after the knockdown of BmAGOI and BmAG02 genes. Interestingly, there was a significant overlap between the target genes up-regulated by BmAgol (292 out of 481) and BmAgo2 (292 out of 361). Of these, the Replicase gene of macula-like virus (BmMLV) was highly induced in both of BmAgol - and BmAgo2-depleted cells. RT-PCR analysis confirmed that the knockdown of silkworm RNAi pathway-related genes increased the expression of the Replicase gene. These data strongly suggested that the RNAi pathway negatively regulated BmMLV proliferation in silkworm BmN4 cells. [ABSTRACT FROM AUTHOR]

Four housekeeping genes including gapA, groEL, gyrA and pgi were used in order to reveal a phylogenetic relationship among Paenibacillus species. Phylogenetic analysis and end-to-end pairwise analysis were carried out using nucleotide and amino acid sequences of the housekeeping genes. A monophylogenetic clade including P. popilliae, P. thiaminolyticus and P. dendritiformis formed in all phylogenetic trees. The results in both analyses indicated that the relationship between P. larvae subsp. larvae and P. popilliae was comparatively far. The result of the end-to-end pairwise analysis also showed that, among the Paenibacillus species used in this study, the closest species to P. popilliae was P. dendritiformis. [ABSTRACT FROM AUTHOR]

Serratia liquefaciens FK01 produces two serralysin-like metalloproteases. One of the genes, serf, has been cloned before. Another gene, designated as ser2, was cloned in this study. The protein encoded by ser2 was a typical serralysin-like metalloprotease, since a zinc binding motif (HEXXHXUGUXH), glycine-rich repeats (GGXGXD), a Met-turn (SXMXY) and an ABC exporter motif (DXXX) were found in the deduced amino acid se-quence. In order to investigate the contribution of these proteases to the virulence in an insect, protease- defi-cient mutants were created. The mutant strains were inoculated to the silkworm, Bombyx mori. The result of the mortality test suggested that we could not show that deficiency in serl and ser2 caused a virulence attenuation in the silkworm under our experimental conditions used in this study. It also suggested that other virulence fac-tors existed in the infection in the silkworm. [ABSTRACT FROM AUTHOR]

We constructed pHERDHis vectors to express polyhistidine-tagged proteins based on the pHERD vectors. The oligonucleotides containing the hexahistidine sequence were designed to be able to introduce the tag at either the NH2- or COOH- terminal. To demonstrate the usefulness of the vectors, a pyocyanin biosynthetic protein, PhzS, was expressed in a pyocyanin-deficient mutant of Pseudomonas aeruginosa. In the mutant strain, both HisPhzS (tag at the NH2- terminal) and PhzSHis (tag at the COOH- terminal) worked as a functional protein and pyocyanin was produced. It was also demonstrated that HisPhzS was purified by standard affinity chromatography. [ABSTRACT FROM AUTHOR]