Tomomi Higashi

Phylogenetic analysis of atmospheric halotolerant bacterial communities at high altitude in an Asian dust (KOSA) arrival region, Suzu City.

The microbial communities transported by Asian desert dust (KOSA) events have attracted much attention as bioaerosols because the transported microorganisms are thought to influence the downwind ecosystems in Korea and Japan. However, the atmospheric microbial community has not been investigated at high altitude in the KOSA arrival area. In this study, to estimate the viability and diversity of atmospheric halotolerant bacteria, which are expected to resist to various environmental stresses as well as high salinities, bioaerosol samples were collected at 10 and 600 m above the ground within the KOSA arrival area, Suzu City, Japan, during KOSA events. During the sampling period, the particle numbers at 600 m were higher than those at 10 m, suggesting that large particles of aerosol fall from the high altitude of 600 m to the ground surface. The microorganisms in bioaerosol samples grew in media containing up to 15% NaCl concentrations demonstrating the viability of the halotolerant bacteria in bioaerosol samples. The PCR-DGGE analysis using 16S rDNA revealed that the bacterial species in NaCl-amended cultures were similar to the bacteria detected from the genomic DNA directly extracted from the bioaerosol samples. The 16S rDNA sequences of bacterial communities in bioaerosol samples were classified into 4 phylotypes belonging to the Bacilluscereus or Bacillussubtilis group. The bioaerosol samples collected at 600 m included 2 phylotypes belonging to B. subtilis, and one phylotype among all 4 phylotypes was identical between the samples at 10 and 600 m. In the atmosphere at 600 m, the halotolerant bacterial community was expected to remain viable, and the species composition was expected to include a few species of the genus Bacillus. During this investigation period, these atmospheric bacteria may have been vertically transported to the ground surface, where the long-range KOSA particle transport from China is frequently observed.

Overexpression of Latent Transforming Growth Factor‐βl (TGF‐βl) Binding Protein 1 (LTBP‐1) in Association with TGF‐β1 in Ovarian Carcinoma

Using the differential display method, latent transforming growth factor‐β (TGF‐β1) binding protein 1 (LTBP‐1) mRNA was identified as one of the enriched mRNAs in ovarian carcinoma tissues after isolation of genes responsible for the development of ovarian cancer. Semi‐quantitative reverse transcription (RT)‐PCR analysis showed that expression of LTBP‐1 and TGF‐β1 mRNAs was much higher in both serous and mucinous adenocarcinomas than in their benign counterparts, including serous and mucinous cystadenomas and cystadenomas of low malignant potential (LMPs). Immunohistochemical analysis demonstrated that only proliferating benign adenoma cells were immunoreactive for both LTBP‐1 and TGF‐β1 proteins. In contrast, most serous and mucinous adenocarcinoma cells and their surrounding stroma were intensely immunoreactive for LTBP‐1 and TGF‐β1. LTBP‐1 and TGF‐β1 proteins, and their complex forms were identified in ovarian carcinoma cell lines and in their culture media by western blot analysis, suggesting these products were produced in ovarian carcinoma cells. RT‐PCR analysis demonstrated that LTBP‐1L, one of the LTBP‐1 transcripts that has a strong activity in targeting the latent form of TGF‐β1 to extracellular matrix (ECM), was predominantly expressed in ovarian carcinomas. Taken together, the results suggest that upregulation of LTBP‐1 in ovarian carcinoma cells may have an important role in distributing TGF‐β1 in the stromal tissues surrounding carcinoma cells.

Behavioral abnormalities and apoptotic changes in neurons in mice brain following a single administration of allylnitrile

Abstract A single dose of allylnitrile in mice might induce persistent behavioral abnormalities, of which the mechanism is not yet known. The present study was undertaken to explore the relationship between behavioral abnormalities and pathological changes in the brain of mice following exposure to allylnitrile. Exposure to allylnitrile (63, 84, and 112 mg/kg, p.o.) resulted in dose-dependent changes in behavioral abnormalities, including increased locomotor activity, circling, retropulsion, head twitching, and alteration in reflexive behavior, which appeared at day 2 postdosing and were persistent throughout the experimental period (60 days) at the higher dose levels. Allylnitrile produced neuronal retraction including hyperchromasia of the nuclei in the raphe nuclei, cerebral cortex, hypothalamus, hippocampal CA1 and dentate gyrus later than 30 days. No gliosis was observed in these regions. Not all but a significant number of neurons in the hippocampal CA1, medial habenula and raphe nuclei were immunoreactive to CPP32 (Caspase-3) even at day 2. These neurons were also positive to Hoechst 33258 staining, indicating allylnitrile caused apoptotic changes in specific neurons when neuronal behaviors became apparent. These apoptotic changes were persistent even in the area without neuronal contraction such as medial habenula. However, almost all neurons in these areas were also positive to terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL). It is conceivable that allylnitrile caused apoptotic changes in neurons but did not always lead them to cell death immediately. Moreover, even when neuronal contraction resulted in retention of behavioral abnormalities, onset of these abnormalities seems to be associated with the impairment in the habenulo-raphe relay due to activation of apoptotic cascade in neurons.

Purpurin expression in the zebrafish retina during early development and after optic nerve lesion in adults

Purpurin, a retina-specific protein, is known to play a role in cell adhesion during development of the chicken retina. Although purpurin has been significantly detected in adult chicken retina, its function in the matured retina is not well understood. Therefore, to determine the expression pattern of purpurin in the retina, we simultaneously investigated expression patterns of purpurin in the zebrafish retina during development in larvae and optic nerve regeneration after nerve transection in adults. In early development, levels of purpurin suddenly increased in the zebrafish retina 3 to 5 days after fertilization, and purpurin-positive immunoreactivity was diffusely located in all retinal layers. In contrast, levels of purpurin mRNA rapidly increased in the adult retina 1-3 days after optic nerve transection, and rapidly declined by 10 days after injury. Signal for purpurin mRNA was seen only in photoreceptors. Immunohistochemistry showed that levels of purpurin protein were also increased in the retina 1-3 days after nerve injury, but positive staining was located in photoreceptors and ganglion cells, and the staining in ganglion cells was stronger than that in photoreceptors. Thus, the transient expression of purpurin protein was greatly different during development and optic nerve regeneration. In the former, purpurin may be required in all retinal layers, whereas in the latter, purpurin may be required for injured ganglion cells.

Purpurin is a key molecule for cell differentiation during the early development of zebrafish retina

Recently, we cloned purpurin cDNA as an upregulated gene in the axotomized fish retina. The retina-specific protein was secreted from photoreceptors to ganglion cell layer during an early stage of optic nerve regeneration in zebrafish retina. The purpurin worked as a trigger molecule for axonal regrowth in adult injured fish retina. During zebrafish development, purpurin mRNA first appeared in ventral retina at 2 days post-fertilization (dpf) and spread out to the outer nuclear layer at 3 dpf. Here, we investigated the role of purpurin for zebrafish retinal development using morpholino gene knockdown technique. Injection of purpurin morpholino into the 1-2 cell stage of embryos significantly inhibited the transcriptional and translational expression of purpurin at 3 dpf. In the purpurin morphant, the eyeball was significantly smaller and retinal lamination of nuclear and plexiform layers was not formed at 3 dpf. Retinal cells of purpurin morphants were still proliferative and undifferentiated at 3 dpf. The visual function of purpurin morphant estimated by optomotor response was also suppressed at 5 dpf. By contrast, the control morphants with random sequence morpholino showed retinal lamination with distinct layers and differentiated cells at 3 dpf. These results strongly suggest that purpurin is a key molecule for not only optic nerve regeneration in adult but also cell differentiation during early development in embryo.

Novel Functional Single Nucleotide Polymorphisms in the Latent Transforming Growth Factor-β Binding Protein-1L Promoter: Effect on Latent Transforming Growth Factor-β Binding Protein-1L Expression Level and Possible Prognostic Significance in Ovarian Cancer

Latent transforming growth factor (TGF)binding proteins (LTBPs) play important roles in the secretion and activation of TGF. We previously reported that LTBP-1L is overexpressed in some patients with ovarian cancer. To clarify the molecular mechanism of LTBP-1L regulation, we analyzed DNA sequences in the promoter region of LTBP-1L and identified two novel single nucleotide polymorphisms, 202G/C and 20A/C. While the alleles with 202C and 20C were initially reported, our data demonstrated that 202G and 20A are common in both ovarian cancer patients and healthy patients in the Japanese population. Luciferase reporter assays revealed that the G-A haplotype induced transcriptional activation in a Sp1-dependent manner. Electrophoretic mobility shift assays showed that increased binding affinity of Sp1 to the promoter with 202G and 20A. Interestingly , ovarian cancer patients (n 42) with G-A/G-A homozygous genotype had increased expression of LTBP-1 and apparently poorer survival than those with other genotypes (P 0.02). These findings suggest that the single nucleotide polymorphisms 202G/C and 20A/C on the LTBP-1L promoter may affect the clinical outcome of ovarian cancer patients , probably via up-regulating protein expression. Further studies using a larger number of samples will definitively determine the correlation between LTBP-1 haplotype and clinical behavior of ovarian cancer. (J Mol Diagn 2006, 8:342–350; DOI: 10.2353/jmoldx.2006.050133) Transforming growth factor (TGF)is a potent growth inhibitor for most cell types, including epithelial cells. The secretion of TGFby tumor cells may contribute to tumor suppression by autocrine growth inhibition, but on the other hand, it may also promote tumor progression by stimulating tumor invasion and angiogenesis, and inhibiting immune response. TGFis synthesized as latent high-molecular weight complexes, composed of TGF, the NH2-terminal part of the TGFprecursor, and the latent TGFbinding protein (LTBP). LTBP is a glycoprotein with a molecular weight of more than 190 kd; it possesses 16 to 18 epidermal growth factor (EGF)-like domains and several repeats of a unique motif containing eight cysteine residues. Four isoforms of LTBP have been found in mammalian species (LTBP-1 to LTBP4). LTBP-1 binds to TGF1 through one of the eight cysteine motifs and facilitates assembly and secretion and activation of TGF1. Immunoelectronmicroscopic observations indicate that LTBP-1 is one of the extracellular microfibrillar components; it targets TGF1 to extracellular structures and participates in the activation of latent TGF1, perhaps by concentrating latent TGF1 on the cell surface where activation occurs. LTBP-1S (short) and LTBP-1L (long) are derived from independent promoters and alternative splicing between codons 145 and 146 of LTBP-1S. LTBP-1L has a N-terminal extension of 346 amino acids that is not found in the LTBP-1S. The N-terminal extension contains an EGF-like domain that facilitates matrix incorporation, and LTBP-1L has been confirmed to associate more efficiently than LTBP-1S with the extracellular matrix.

Preconditioning with subneurotoxic allyl nitrile: Protection against allyl nitrile neurotoxicity

High-dose cruciferous allyl nitrile can induce behavioral abnormalities in rodents, while repeated exposure to allyl nitrile at subneurotoxic levels can increase phase 2 detoxification enzymes in many tissues, although the brain has not been investigated yet. In the present study, we examined the effect of 5 days repeated exposure to subneurotoxic allyl nitrile (0-400 micromol/kg/day) on the brain. Elevated glutathione S-transferase activity was recorded in the striatum, hippocampus, medulla oblongata plus pons, and cortex. Enhancement of quinone reductase activity was observed in the medulla oblongata plus pons, hippocampus, and cortex. In the medulla oblongata plus pons, elevated glutathione levels were recorded. Following repeated subneurotoxic allyl nitrile exposure (0-400 micromol/kg/day), mice were administered a high-dose allyl nitrile (1.2 mmol/kg) which alone led to appearance of behavioral abnormalities. Compared with the 0 micromol/kg/day group, animals in the 200 and 400 micromol/kg/day pre-treatment groups exhibited decreased behavioral abnormalities and elevated GABA-positive cell counts in the substantia nigra pars reticulata and the interpeduncular nucleus. These data suggest that repeated exposure to subneurotoxic levels of allyl nitrile can induce phase 2 enzymes in the brain, which together with induction in other tissues, may contribute to protection against allyl nitrile neurotoxicity.

Fos induction in the brain of mice exhibiting behavioral abnormalities following administration of allylnitrile or crotononitrile

Allylnitrile and crotononitrile induce behavioral abnormalities in mice. To explore the possible involvement of the vestibular system in these behavioral abnormalities, the expression of Fos protein, used as an indicator of neuronal activity, was examined within various brain structures in allylnitrile-, crotononitrile- and vehicle-treated mice. In each nitrile-treated mouse, Fos expression was observed in brain structures, which were divided into two groups. The structures in group 1 showed Fos expression between 1.5 h and 2 days postdosings, and in those in group 2 expression remained for up to 30 days postdosing. As most of these structures, especially in group 2, were identical to some Fos-positive structures observed after unilabyrinthectomy, the present results indicate that each nitrile induces Fos expression by causing a change in the peripheral vestibular system, resulting in behavioral abnormalities.

Composition of halophilic bacteria survived in bioaerosol

Teruya Maki, Shinzi Susuki, Fumihisa Kobayashi, Makiko Kakikawa, Maromu Yamada, Tomomi Higashi, Chunsang Hong, Yutaka Tobo, Hiroshi Hasegawa, Kazumasa Ueda and Yasunobu Iwasaka 1 Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, 920-1192, Japan. 2 Institute of Nature and Environmental Technology, Kanazawa University, Kakuma, Kanazawa, 920-1192, Japan. 3 Faculty of Environmental and Symbiotic Science, Prefectural University of Kumamoto.3-1-100 Tsukide, Kumamoto 862-8502, Japan 4 Hygiene, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920-8640, Japan 5 Frontier Science Organization, Kanazawa University, Kakuma, Kanazawa, 920-1192, Japan.