Kosuke Kasai

Novel L-amino acid oxidase with antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from epidermal mucus of the flounder Platichthys stellatus

Fish produce mucus substances as a defensive outer barrier against environmental xenobiotics and predators. Recently, we found a bioactive protein in the mucus layer of the flounder Platichthys stellatus, which showed antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and methicillin‐resistant S. aureus. In this study, we isolated and identified the antibacterial protein from the mucus components of P. stellatus using a series of column chromatography steps. We then performed gel electrophoresis and cDNA cloning to characterize the protein. The antibacterial protein in the mucus had a molecular mass of approximately 52 kDa with an isoelectric point of 5.3, and cDNA sequencing showed that it corresponded completely with the peptide sequence of antibacterial protein from the gill. A BLAST search suggested that the cDNA encoded an antibacterial protein sharing identity with a number of l‐amino acid oxidases (LAAOs) and possessing several conserved motifs found in flavoproteins. RT‐PCR using a specific primer, and immunohistochemical analysis with anti‐LAAO IgG, demonstrated tissue‐specific expression and localization in the gill. Moreover, the anti‐LAAO IgG was able to neutralize the antibacterial activity of the protein against methicillin‐resistant S. aureus. Thus, we demonstrated that this antibacterial protein, identified from P. stellatus‐derived epidermal mucus, is a novel LAAO‐like protein with antibacterial activity, similar to snake LAAOs.

Structural features of GmIRCHS, candidate of the I gene inhibiting seed coat pigmentation in soybean: implications for inducing endogenous RNA silencing of chalcone synthase genes.

Most commercial soybean varieties have yellow seeds due to loss of pigmentation in the seed coat. The I gene inhibits pigmentation over the entire seed coat, resulting in a uniform yellow color of mature harvested seeds. We previously demonstrated that the inhibition of seed coat pigmentation by the I gene results from post-transcriptional gene silencing (PTGS) of chalcone synthase (CHS) genes. Little is known about the structure of the I gene and the mechanism by which it induces PTGS of CHS genes. Here, we report a candidate of the I gene, GmIRCHS, which consists of a 5′-portion of a DnaJ-like gene containing a promoter region and a perfect inverted repeat (IR) of 1.1-kb truncated CHS3 sequences (5′-ΔCHS3 and 3′-ΔCHS3). RT-PCRs and RNase protection assay indicated the existence of the read-through product from 5′-ΔCHS3 to 3′-ΔCHS3 and the dsRNA region of ΔCHS3, suggesting that dsRNA of ΔCHS3 could be transcribed from GmIRCHS and could induce PTGS of CHS genes. Moreover, the IR structure of ΔCHS3 in GmIRCHS was lost in the soybean mutants in which I was changed to i, supporting the conclusion that GmIRCHS is the I gene.

Anti-inflammatory effect of proteoglycan and progesterone on human uterine cervical fibroblasts.

AIMS The aim of this study was to compare the anti-inflammatory effect of proteoglycan (PG) with that of progesterone (P) in the cultured fibroblasts from human uterine cervix. MAIN METHODS After obtaining informed consent, the cervix was collected from normal women undergoing total hysterectomy. The cervix was cultured until fibroblasts proliferated and had grown to confluence, then, the fibroblasts were stimulated by lipopolysaccharide (LPS) with or without PG, P and a combination of both; they were cultured for 24-48 h. The anti-inflammatory effects of PG and P were evaluated by the suppression of IL-6 or IL-8 secretion. The expression of the IL-6 or IL-8 gene and the expression of their protein were determined by real-time PCR, and ELISA, respectively. Activation of Toll-like receptor (TLR) 4 was evaluated by Western blotting. KEY FINDINGS LPS markedly enhanced gene and protein expression of IL-6 and IL-8 in human uterine cervical fibroblasts. The up-regulation of the IL-6 or IL-8 gene and protein expression by LPS was significantly suppressed with PG, P and a combination of both. Western blotting revealed that combination of PG and P showed more potent inhibition on LPS-stimulated TLR4 induction than that seen by each. SIGNIFICANCE This study showed that both PG and P have an inhibitory effect on LPS-induced inflammation. This anti-inflammatory effect of PG and P was augmented by co-administration of both, suggesting for the first time that PG has an anti-inflammatory effect on human uterine cervical fibroblasts.

Antibacterial properties of l-amino acid oxidase: mechanisms of action and perspectives for therapeutic applications

Venom, the mucus layer covering the body surface, ink glands, mammary glands, milk, and various animal secretory functions as both a physical and chemical defense barrier against bacteria and virus infections. Previously, several studies reported that l-amino acid oxidases (LAAOs) present in animal secretary fluids have strong antimicrobial activities and selective cytotoxic activities against Gram-positive and Gram-negative bacteria, various pathogenic bacteria, viruses, and parasite species. These LAAOs catalyze oxidative deamination of an l-amino acid substrate with the generation of hydrogen peroxide. The antibacterial activity of LAAOs is completely inhibited by catalase; thus, LAAOs kill bacteria by the hydrogen peroxide generated from the oxidation of l-amino acid substrates. This review focuses on the selective, specific, and local antibacterial actions of various LAAOs that may be used as novel therapeutic agents against infectious diseases. LAAOs that are suitable leads for combating multidrug-resistant bacterial infections are also studied.

Identification of up-regulated and down-regulated cis-natural antisense transcripts in the human B lymphoblastic cell line IM-9 after X-ray irradiation

Ionizing radiation (IR) causes DNA injury and induces multiple signal mechanisms, including the regulation of DNA repair, the cell cycle and gene expression through the activation of p53-related pathways. Cis-natural antisense transcripts (cis-NATs), which are transcribed from the DNA strand opposite to that for mRNA of the gene, are recognized as important regulators of gene expression in eukaryotic cells, but the effects on cis-NAT expression by IR are unknown to date. Therefore, we investigated the effects of X-ray irradiation on cis-NAT expression together with mRNA expression using a human B lymphoblast cell line (IM-9), a custom-microarray and strand-specific RT-qPCR. Eighteen, 33 and 106 mRNAs were demonstrated to be differentially expressed in IM-9 cells after 1, 2 and 4 Gy irradiation, respectively, as compared to 0 Gy by microarray analysis (fold change, FC >2.0). On the other hand, 10, 22 and 43 NATs were demonstrated to be differentially expressed in IM-9 cells after 1, 2 and 4 Gy irradiation, respectively, as compared to 0 Gy by microarray analysis (FC >2.0). Among these mRNAs/NATs, the IR dose-dependent up-regulation of mRNAs and cis-NATs of MDM2 and CDKN1A were confirmed by strand-specific RT-qPCR. Additionally, the cis-NATs of MDM2 were indicated to be localized in the cytoplasm, while cis-NATs of CDKN1A were located in the nucleus and cytoplasm. In conclusion, the radiation-responsive cis-NATs in conjunction with mRNAs were identified for the first time in the present study. It is possible that these cis-NATs regulate the gene expression in a post-transcriptional fashion. The IR dose-dependent up- and down-regulation of these mRNAs/cis-NATs may be a marker for ionizing radiation.

UDP/P2Y6 receptor signaling regulates IgE-dependent degranulation in human basophils

BACKGROUND P2Y purinergic receptors (P2YR) are G protein-coupled receptors that are stimulated by extracellular nucleotides. They mediate cellular effects by regulating cAMP production, protein kinase C activation, inositol trisphosphate generation, and Ca2+ release from intracellular stores. The P2Y6 receptor of this family is selectively stimulated by UDP, and selectively inhibited by MRS2578. In the present study, we examined the effect of UDP/P2Y6 receptor signaling on IgE-dependent degranulation in human basophils. METHODS Basophils were purified from human peripheral blood. The mRNA expression of genes encoding P2YR and ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) was measured by RT-PCR. Intracellular Ca2+ influx via UDP/P2Y6 receptor signaling in basophils was detected using a calcium probe. The effect of UDP/P2Y6 receptor signaling on IgE-dependent degranulation in basophils was confirmed by measuring CD63 expression by flow cytometry. Autocrine secretion of nucleotides was detected by HPLC analysis. RESULTS We showed that purified basophils express P2Y6 mRNA and that UDP increased intracellular Ca2+, which was reduced by MRS2578 treatment. UDP promoted IgE-dependent degranulation. Furthermore, MRS2578 inhibited IgE-dependent degranulation in basophils. HPLC analysis indicated that basophils spontaneously secrete UTP. In addition, basophils expressed the extracellular nucleotide hydrolases ENTPDase2, ENTPDase3, and ENTPDase8. CONCLUSIONS This study showed that UDP/P2Y6 receptor signaling is involved in the regulation of IgE-dependent degranulation in basophils, which might stimulate the P2Y6 receptor via the autocrine secretion of UTP. Thus, this receptor represents a potential target to regulate IgE-dependent degranulation in basophils during allergic diseases.

Induction of genomic instability and activation of autophagy in artificial human aneuploid cells

Chromosome missegregation can lead to a change in chromosome number known as aneuploidy. Although aneuploidy is a known hallmark of cancer cells, the various mechanisms by which altered gene and/or DNA copy number facilitate tumorigenesis remain unclear. To understand the effect of aneuploidy occurring in non-tumorigenic human breast epithelial cells, we generated clones harboring artificial aneuploidy using microcell-mediated chromosome transfer. Our results demonstrate that clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. Also, the increased autophagy was observed in the artificially aneuploidy clones, and inhibition of autophagy resulted in increased genomic instability and DNA damage. In addition, the intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones, and inhibition of autophagy further increased the production of reactive oxygen species. Together, these results suggest that even a single extraneous chromosome can induce genomic instability, and that autophagy triggered by aneuploidy-induced stress is a mechanism to protect cells bearing abnormal chromosome number.

Analysis of the Effect of Chronic and Low-Dose Radiation Exposure on Spermatogenic Cells of Male Large Japanese Field Mice (Apodemus speciosus) after the Fukushima Daiichi Nuclear Power Plant Accident

In this study we analyzed the effect of chronic and low-dose-rate (LDR) radiation on spermatogenic cells of large Japanese field mice (Apodemus speciosus) after the Fukushima Daiichi Nuclear Power Plant (FNPP) accident. In March 2014, large Japanese field mice were collected from two sites located in, and one site adjacent to, the FNPP ex-evacuation zone: Tanashio, Murohara and Akogi, respectively. Testes from these animals were analyzed histologically. External dose rate from radiocesium (combined 134Cs and 137Cs) in these animals at the sampling sites exhibited 21 μGy/day in Tanashio, 304–365 μGy/day in Murohara and 407–447 μGy/day in Akogi. In the Akogi group, the numbers of spermatogenic cells and proliferating cell nuclear antigen (PCNA)-positive cells per seminiferous tubule were significantly higher compared to the Tanashio and Murohara groups, respectively. TUNEL-positive apoptotic cells tended to be detected at a lower level in the Murohara and Akogi groups compared to the Tanashio group. These results suggest that enhanced spermatogenesis occurred in large Japanese field mice living in and around the FNPP ex-evacuation zone. It remains to be elucidated whether this phenomenon, attributed to chronic exposure to LDR radiation, will benefit or adversely affect large Japanese field mice.

A novel parameter, cell-cycle progression index, for radiation dose absorbed estimation in the premature chromosome condensation assay.

The calyculin A-induced premature chromosome condensation (PCC) assay is a simple and useful method for assessing the cell-cycle distribution in cells, since calyculin A induces chromosome condensation in various phases of the cell cycle. In this study, a novel parameter, the cell-cycle progression index (CPI), in the PCC assay was validated as a novel biomarker for biodosimetry. Peripheral blood was drawn from healthy donors after informed consent was obtained. CPI was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation ((60)Co-gamma rays: ∼0.6 Gy min(-1), or X ray: 1.0 Gy min(-1); 0-10 Gy) model. The calyculin A-induced PCC assay was performed for chromosome preparation. PCC cells were divided into the following five categories according to cell-cycle stage: non-PCC, G1-PCC, S-PCC, G2/M-PCC and M/A-PCC cells. CPI was calculated as the ratio of G2/M-PCC cells to G1-PCC cells. The PCC-stage distribution varied markedly with irradiation doses. The G1-PCC cell fraction was significantly reduced, and the G2/M-PCC cell fraction increased, in 10-Gy-irradiated PBL after 48 h of culture. CPI levels were fitted to an exponential dose-response curve with gamma-ray irradiation [y = 0.6729 + 0.3934 exp(0.5685D), r = 1.0000, p < 0.0001] and X-ray irradiation [y = -0.3743 + 0.9744 exp(0.3321D), r = 0.9999, p < 0.0001]. There were no significant individual (p = 0.853) or gender effects (p = 0.951) on the CPI in the human peripheral blood ex vivo irradiation model. Furthermore, CPI measurements are rapid (< 15 min per case). These results suggest that the CPI is a useful screening tool for the assessment of radiation doses received ranging from 0 to 10 Gy in radiation exposure early after a radiation event, especially after a mass-casualty radiological incident.

Nonclonal growth of preneoplastic cells positive for glutathione S‐transferase P‐form in the rat liver

We investigated the process of induction of preneoplastic cells positive for glutathione S‐transferase P‐form (GST‐P) in the rat liver. AAF (2‐Acetylaminofluorene) mixed with normal rat chow at high concentration (0.04%) induced 517 000 ± 86 000 GST‐P+ single hepatocytes/g liver after 2 weeks followed by induction of a few foci and nodules after 4–6 weeks. Overproduction of GST‐P+ single hepatocytes was dose‐ and time‐dependent, and the induction kinetics were typical of first‐order consecutive reaction, by which induction of the positive cells was nongenetic. Quantitative analysis indicated that the estimated numbers of cells in foci and nodules at 4–6 weeks after exposure to AAF ranged from 2.7 × 104 (214.7) to 3.6 × 106 (221.7) cells, and 2.0 × 104 (214.3) to 2.7 × 106 (221.4) cells, respectively, when analyzed by using two equations. According to the initiated cell theory of Farber, foci and nodules are formed through sequential cell division of 14 to 21‐times or more within a short time period. The rapid growth exceeded the rate of cell division, indicating that the growth of preneoplastic cells is based on a nonclonal penetration mechanism. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02325.x, 2012)