An avian pathogenic Escherichia coli strain produces a hemolysin, the expression of which is dependent on cyclic AMP receptor protein gene function

An avian pathogenic Escherichia coli strain M1000 showed a clear zone of erythrocyte lysis on sheep blood agar plates. The hemolytic activity was not detected in the culture supernatant nor was any DNA sequence homologous to the E. coli -hemolysin gene detected in the chromosome or plasmid DNA of the strain, indicating that the observed hemolysis was different from -type. To identify the genetic determinant responsible for the hemolysis, we performed random Tn5 insertional mutagenesis and obtained one mutant, named M5005, that totally lacked the hemolytic activity. Cloning and nucleotide sequencing of the region flanking the transposon insertion site in the M5005 chromosome revealed that the transposon was inserted within an open reading frame of the cyclic AMP receptor protein (CRP) gene, which is involved in one of the global regulatory networks of gene expression in E. coli. Nucleotide sequence analysis of the intact crp gene of the strain M1000 showed that the CRP protein of M1000 is 99% identical to that of K-12. Introduction of the intact crp gene on a low copy plasmid into the mutant M5005 restored the hemolytic phenotype, confirming that the mutation site in M5005 was in the crp gene. CRP plays a central role in catabolite repression, the phenomenon by which the synthesis of many enzymes required to metabolize various sugars is repressed in the presence of glucose. When the hemolytic activity of E. coli M1000 grown in the presence of glucose was examined, the hemolysis was totally impaired. These results indicate that the avian pathogenic E. coli strain M1000 produces a hemolysin the expression of which is dependent on crp gene function.     

Development of immune assay system for both CpG and non-CpG DNA from lactic acid bacteria using a transfectant of swine Toll-like receptor 9

Bacterial DNA is expected to be a potent immune stimulating agent to Toll‐like receptor (TLR) 9 expressed cells such as macrophages, monocytes, B lymphocytes, NK cells and dendritic cells. In the present study, we constructed a transfectant of swine TLR9 with mammalian cells. We demonstrated that the transfectant, recognizing both CpG and non‐CpG oligonucleotides from lactic acid bacteria, induced NF‐κB activation by gene reporter assay. These findings indicate that the swine TLR9 transfectant will be highly useful for the screening of immunostimulatory DNA from lactic acid bacteria.     

Proteome analysis of cashmere

As an analysis of the cashmere proteins by Type IV 2‐DE, ten kinds of components, including three components with molecular mass 42–50 kDa whose expression level increased in the winter, were separated. In analyzing nine components of these ten using a mass spectrometer, the three components of molecular mass 70–120 kDa and pI 5.3 were identified as keratin type II microfibrillar (accession no. KRSHL2 ), keratin 48 k type I microfibrillar component 8c‐1 (accession no. KRSHL1 ) and cytosolic phospholipase A2 (accession no. O77793 ), respectively. The three components whose expression level increased in the winter, were identified as keratin type I microfibrillar 48 kDa component 8C‐1 (accession no. P02534 ) and keratin type I microfibrillar 47.6 kDa (accession no. P25690 ) (pI 5.2/42 kDa), keratin type II microfibrillar component 7C (accession no. P15241 ) and keratin typeII‐sheep (accession no. S34165 ) (pI 5.5/45 kDa), and the keratin type II microfibrillar component 5 (accession no. P25691 ) (pI 5.8–6.0/45 kDa), respectively. The three components of less than 17 kDa were identified as hair keratin type II intermediate filament (accession no. CAA51836 ) (pI 5.2) and keratin high‐sulfur matrix protein IIIB2 (accession no. P02447 ) with a different isoelectric point (pI 5.4 and 5.9), respectively.     

Effect of an Introduced Phytoene Synthase Gene Expression on Carotenoid Biosynthesis in the Marine Diatom Phaeodactylum tricornutum

Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy) to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and β-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase.     

Determination of the Chemical Structures of Tandyukisins B–D,Isolated from a Marine Sponge-Derived Fungus

Tandyukisins B–D (1–3), novel decalin derivatives, have been isolated from a strain of Trichoderma harzianum OUPS-111D-4 originally derived from the marine sponge Halichondria okadai, and their structures have been elucidated on the basis of spectroscopic analyses using 1D and 2D NMR techniques. In addition, their chemical structures were established by chemical transformation. They exhibited weak cytotoxicity, but selective growth inhibition on panel screening using 39 human cancer cell lines.     

Production of an antibacterial substance by Bacillus mojavensis strain F412 isolated from a Myanmar shrimp product fermented with boiled rice

An antibacterial-substance-producing bacterium, namely, strain F412, was isolated from a traditional Myanmar shrimp product fermented with boiled rice. It was a gram-positive, spore-forming, and rod-shaped bacterium, and identified as Bacillus mojavensis on the basis of the gyrA sequence. The antibacterial substance of this strain was partially purified from a culture supernatant using two steps of column chromatography. This substance was found to be widely effective against gram-positive bacteria, including Listeria monocytogenes. The antibacterial activity of this substance was not susceptible to treatments with several proteolytic enzymes. The antibacterial activity gradually decreased with increasing treatment temperature, but it remained even after heating for 15 min at 121 °C. This antibacterial substance showed different molecular weights, as shown by the results of gel filtration and electrophoresis analyses. Staining results after electrophoresis suggest that the antibacterial substance might be a glycopeptide with an estimated molecular weight between 3.5 and 8.5 kDa. From the decrease in optical density of a culture of the L. monocytogenes treated with this antibacterial substance, it was suggested that this substance might have bacteriolytic activity.

     

Comparison of prevalence of Felis catus papillomavirus type 2 in squamous cell carcinomas in cats between Taiwan and Japan

Felis catus papillomavirus (FcaPV), especially type 2 (FcaPV2) is considered as one of the causative agents in squamous cell carcinoma (SCC) in cats. However, our previous study detected FcaPV3 and FcaPV4, but not FcaPV2 in feline SCCs collected in Japan, suggesting that the prevalence of FcaPV2 in SCC may vary depending on geographic locations. To evaluate this hypothesis, two conventional PCR reactions targeting E1 and E7 genes were performed to detect FcaPV2 in feline SCC samples collected in Taiwan and Japan. While 46.9% (23/49) of feline SCC cases from Taiwan were PCR positive for FcaPV2, only 8.6% (3/35) cases from Japan were positive. Our result suggests that the prevalence of FcaPV2 in feline SCCs may depend on the region.     

Histological analysis of implanted embryos in large Japanese field mouse (Apodemus speciosus) and estimation of developmental stage

The large Japanese field mouse (Apodemus speciosus) is a small rodent species endemic to Japan. The genetic characteristics of A. speciosus include different chromosome numbers within the same species. Furthermore, A. speciosus has been used in radiation and genetic research. In the present study, a pregnant A. speciosus was obtained, and histochemical analysis of the implanted embryos was performed and compared with the developmental stages of the mouse (Mus musculus). Although there were some differences, the structures of the implanted embryos, including the primitive streak and placenta of A. speciosus were similar to those of mouse. Our study will be important for the construction of a developmental atlas of A. speciosus.