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Biomedical subjects

T Ohnishi

Publications and source records attributed to T Ohnishi.

At least 91 records · Page 5Linked to original sources

Analysis of deletion mutations of the rpsL gene in the yeast Saccharomyces cerevisiae detected after long-term flight on the Russian space station Mir.

Using the yeast Saccharomyces cerevisiae on board the Russian space station Mir, we studied the effects of long-term space flight on mutation of the bacterial ribosomal protein L gene (rpsL) cloned in a yeast-Escherichia coli shuttle vector. The mutation frequencies of the cloned rpsL gene on the Mir and the ground (control) yeast samples were estimated by transformation of E. coli with the plasmid DNAs recovered from yeast and by assessment of the conversion of the rpsL wild-type phenotype (Sm(S)) to its mutant phenotype (Sm(R)). After a 40-day space flight, some part of space samples gave mutation frequencies two to three times higher than those of the ground samples. Nucleotide sequence analysis showed no apparent difference in point mutation rates between the space and the ground mutant samples. However, the greater part of the Mir mutant samples were found to have a total or large deletion in the rpsL sequence, suggesting that space radiation containing high-linear energy transfer (LET) might have caused deletion-type mutations.

Base Sequence↗

Development of highly sensitive enzyme-linked immunosorbent assays for hepatocyte growth factor/scatter factor (HGF/SF): determination of HGF/SF in serum and urine from normal human subjects.

A sandwich enzyme-linked immunosorbent assay (ELISA) using rabbit anti-hepatocyte growth factor (HGF) IgG for human HGF, also known as the scatter factor, has previously been developed for determining increases in serum HGF levels in various liver diseases. The sensitivity limit of the ELISA is, however, approximately 0.2 ng/ml sample, and HGF concentrations in about 50% of normal subjects are not accurately measurable by this method, because the mean level of HGF in normal serum is close to the sensitivity limit. In the present study, chicken Fab' from egg yolk anti-HGF immunoglobulin Y and rabbit Fab' from rabbit anti-HGF IgG were conjugated with beta-D-galactosidase. With these conjugates as the second antibodies, we developed two sandwich ELISAs for human HGF and found that the sensitivities were about 20 pg/ml with the former conjugate and 2 pg/ml with the latter. The HGF concentration in sera from 138 normal subjects determined by the ELISA with the rabbit conjugate was 244+/-65 (SD) pg/ml serum, and it correlated very well with the number of leukocytes. Moreover, the ELISA with the rabbit conjugate permitted the determination of HGF levels in urine from normal subjects without first concentrating the sample. The determination of HGF in various biological fluids other than blood and urine by these ELISAs may aid the diagnosis and prognosis of various diseases.

Adult↗

Characterization of the complex I-associated ubisemiquinone species: toward the understanding of their functional roles in the electron/proton transfer reaction.

NADH-ubiquinone oxidoreductase (called complex I for mitochondrial enzyme and NDH-1 for bacterial counterparts) is an energy transducer, which utilizes the redox energy derived from the oxidation of NADH with ubiquinone to generate an electrochemical proton gradient (Deltamu(H(+))) across the membrane. The complex I/NDH-1 contain one non-covalently bound flavin mononucleotide and as many as eight iron-sulfur clusters as electron transfer components in common. In addition, electron paramagnetic resonance (EPR) spectroscopic studies have revealed that three ubisemiquinone (SQ) species with distinct spectroscopic and thermodynamic properties are detectable in complex I and function as electron/proton translocators. Thus, the understanding of molecular properties of the individual quinone species is prerequisite to elucidate the energy-coupling mechanism of complex I. We have investigated these SQ species using EPR spectroscopy and found that the three SQ species have strikingly different properties. We will report characteristics of these SQ species and discuss possible functional roles of individual quinone species in the electron/proton transfer reaction of complex I/NDH-1.

Animals↗

Fetuin in the developing neocortex of the rat: distribution and origin.

Immunocytochemical distribution of the fetal protein fetuin in the neocortex of developing rat brain and the presence of its mRNA, as detected by using reverse transcriptase-polymerase chain reaction analysis, was studied in fetuses at embryonic day 15 (E15) through E22, in neonates at postnatal day 0 (P0) through P20, and in adults. Quantitative estimates of fetuin in cerebrospinal fluid (CSF) and plasma were obtained over the same period. Exogenous (bovine) fetuin injected intraperitoneally into fetal and postnatal rats was used to study the uptake of fetuin into CSF and brain and its distribution compared with endogenous fetuin; bovine albumin was used as a control. Fetuin was identified immunocytochemically in the cortical plate and subplate cells of the developing neocortex. In the rat fetus, fetuin first was apparent at E17, mainly in cell processes, but a few subplate cells also were positive. By E18, there was strong staining in subplate neurons and in inner cells of the cortical plate. At E21, these inner cells of the cortical plate were beginning to differentiate into layer VI neurons, many of which were positive for fetuin. By P0-P1, more layer VI neurons and some layer V neurons had become positive for fetuin. Fetuin immunoreactivity generally was weaker at P1, and, by P2-P3, it had disappeared from all of the layers of the developing neocortex. Bovine fetuin (but not albumin), probably taken up through CSF over the neocortical dorsal surface, had a cytoplasmic distribution; endogenous rat fetuin was both cytoplasmic and membrane bound. Thus, much of this fetuin can be accounted for by uptake, although the presence of fetuin mRNA indicates that in situ synthesis may also contribute.

Animals↗

Precise measurement of dp elastic scattering at 270 MeV and three-nucleon force effects

The cross section, the deuteron vector A(d)(y) and tensor analyzing powers A(ij), the polarization transfer coefficients K(y('))(ij), and the induced polarization P(y(')) were measured for the dp elastic scattering at 270 MeV. The cross section and A(d)(y) are well reproduced by Faddeev calculations with modern data-equivalent nucleon-nucleon forces plus the Tucson-Melbourne three-nucleon force. In contrast, A(ij), K(y('))(ij), or P(y(')) are not described by such calculations. These facts indicate the deficiencies in the spin dependence of the Tucson-Melbourne force and call for extended three-nucleon force models.

Journal Article↗

Induction of aquaporin-4 water channel mRNA after focal cerebral ischemia in rat.

Aquaporin-4 (AQP4) is a member of a water-selective channel aquaporin-family and mainly expressed in the several structures of the brain and in the collecting duct of the kidney. Here we show its functional involvement in the water homeostasis of the ischemic brain. The expression of AQP4-mRNA is increased in the peri-infarcted cortex during the observation period ( approximately 7 days) after MCA-occlusion, maximally on day 3. The change corresponds to the generation and resolution of brain edema monitored by MRI. The signals for the mRNA are predominantly observed in glial cells in the molecular and outer granular layer of the peri-infarcted cortex. These results indicate that AQP4 plays a role in post-ischemic edema formation.

Animals↗

Transfection with mutant p53 gene inhibits heat-induced apoptosis in a head and neck cell line of human squamous cell carcinoma.

PURPOSE: To confirm that human cancer cells show p53-dependent heat sensitivity through an apoptosis-related mechanism, we examined the heat sensitivity and Bax-mediated apoptosis after heating in a human squamous cell carcinoma cell line, SAS, with identical genetic backgrounds except for the p53 status. MATERIALS AND METHODS: We performed colony formation assay, Western blotting and analyses of apoptosis, using the SAS cells transfected with pC53-248 vector with mutant p53 gene (SAS/Trp248 cells) or the cells transfected with pCMV-Neo-Bam vector (SAS/neo cells) as a control. RESULTS: SAS/Trp248 cells showed heat resistance due to the dominant negative nature of mp53, compared with SAS/neo cells. The incidence of DNA ladders and apoptotic bodies increased markedly after heating in SAS/neo cells, but increased very little in SAS/Trp248 cells. CONCLUSION: These results suggest that heat resistance brought by mp53-transfection is p53-dependent and closely correlates with the induction of apoptosis in human squamous cell carcinomas.

Apoptosis↗

The dependence of p53 on the radiation enhancement of thermosensitivity at different let.

PURPOSE: The aim of this study is to investigate the dependence of p53-gene status on the radiation enhancement of thermosensitivity at different levels of linear energy transfer (LET). METHODS AND MATERIALS: We used two kinds of human glioblastoma transfectants of A-172 cells bearing the wild-type p53 gene, A-172/neo cells with control vector containing the neo gene and A-172/mp53 cells with both the dominant negative mutated p53 gene and neo gene. We exposed these cells to X-rays and accelerated carbon-ion (C-) beams (13-200 KeV/microm) followed by heating at 44 degrees C. Cellular sensitivities were determined using clonogenic assay. RESULTS: The radiation enhancement of thermosensitivity was LET-dependent for the A-172/neo cells, but this was not clearly demonstrated in the A-172/mp53 cells. The supraadditive radiation enhancement of thermosensitivity was observed in A-172/neo cells at the LET range of 13 to 70 KeV/microm, though only an additive effect was observed at higher LET. In A-172/mp53 cells, only an additive effect was observed through all the LET examined. CONCLUSION: These results indicate that the radiation enhancement of thermosensitivity is p53- and LET-dependent. Our results suggest that the combined use of high-LET radiation and hyperthermia brings useful application for cancer therapeutic purposes.

Cell Survival↗

Quantitative analysis of glioma cell invasion by confocal laser scanning microscopy in a novel brain slice model.

To quantitatively analyze the spatial extent of glioma cell migration in an organotypic brain slice culture, we developed a new invasion model with the aid of confocal laser scanning microscopy (CLSM). CLSM allowed not only for three-dimensional visualization of the invasive pattern of human T98G glioma cells in the living brain slice but also for serial analysis of the invasive process over several weeks. Twenty-four hours after the T98G glioma spheroid was initiated to coculture with a brain slice, the glioma cells detached themselves from the spheroid and spontaneously continued to migrate on the surface of the brain slice, while they diffusely invaded into the slice by migrating to a deeper site. Immunohistochemical analysis revealed that these migrating glioma cells much more strongly immunostained for matrix metalloproteinase (MMP)-2 and -9 than the tumor spheroid which remained at the implanted site. Treatment of the T98G glioma spheroid with 1,10-phenanthroline, a specific inhibitor of MMPs, significantly inhibited not only the cell migration on the surface of the brain slice but also the invasion of the glioma cells into the slice. The present version of the glioma invasion model using CLSM makes it possible to spatially and serially analyze the extent of glioma cell invasion in the living brain slice for several weeks, making it a very useful tool for investigating the cellular and molecular mechanisms of glioma invasion under conditions most analogous to those of normal brains in vivo.

Animals↗

Bax and Bcl-2 protein expression following radiation therapy versus radiation plus thermoradiotherapy in stage IIIB cervical carcinoma.

BACKGROUND: The relative amounts of Bcl-2 and Bax proteins determine cell survival or death following an apoptotic stimulus. To clarify the molecular mechanism of cell death after radiotherapy or thermoradiotherapy and its relation to the response of AJCC/UICC Stage IIIB cervical carcinomas, the expression of Bax and Bcl-2 proteins was investigated both before and in the course of treatment given during this study. METHODS: Thirty-seven patients with Stage IIIB carcinoma of the uterine cervix were treated with external beam irradiation to the pelvis combined with iridium-192 high-dose-rate intracavitary brachytherapy. All patients were randomized to one of the following two groups: the radiotherapy (RT) group of 19 patients who were given radiotherapy alone, and the thermoradiotherapy (TRT) group of 18 patients who were given 3 sessions of hyperthermia in addition to RT. Specimens of the cervical tumors were obtained by punch biopsy both before and in the course of the treatment (after a total dose of 10.8 grays ¿Gy for the RT group or after 10.8 Gy plus 1 session of hyperthermia for the TRT group). The tumor sections were stained with anti-Bax and anti-Bcl-2 monoclonal antibody. On the basis of the percentage of immunopositive cells, both pretreatment and posttreatment samples were scored. Furthermore, relative changes in protein expression were determined by comparing the pretreatment scores with those in the course of treatment. In addition, treatment response was evaluated. RESULTS: A complete response was achieved in 52.6% (10 of 19) of the RT group versus 83. 3% (15 of 18) of the TRT group (P = 0.049). Better tumor control was accompanied by increased Bax expression, i.e., 10.5% (2 of 19) of the RT group versus 44.4% (8 of 18) of the TRT group (P = 0.02). The respective number of patients who partially responded (PR) or did not respond to treatment (NC) was 26.3% (5 of 19) and 21.1% (4 of 19) of the RT group versus 11.1% (2 of 18) and 5.6% (1 of 18) of the TRT group (P = 0.2 for both the PR and NC subgroups). CONCLUSIONS: TRT was found to result in better treatment responses than RT for patients with Stage IIIB cervical carcinoma. An additive or synergistic antitumor effect of TRT is likely to occur through induction of apoptosis involving one of the bax pathways.

Aged↗

Loss of regulation of circulating 1,25-dihydroxyvitamin D with paradoxically decreased serum phosphate levels in individuals with recurrent kidney stones.

A low serum phosphate concentration is characteristic in individuals in whom kidney stones form, this being related to serum 1,25-dihydroxyvitamin D, parathyroid hormone and urinary phosphate excretion. In order to determine whether these parameters are related to recurrence of stone formation, they were analyzed in single and recurrent stone formers as well as controls. An inverse correlation between serum levels of phosphate and 1,25-dihydroxyvitamin D was observed in control subjects, indicating that a drop in serum phosphate results in upregulated circulating 1,25-dihydroxyvitamin D level in controls. While the circulating low phosphate level upregulated the 1,25-dihydroxyvitamin D level in single stone formers, the elevation was less than expected from the drop in serum phosphate in recurrent stone formers. The results thus suggest that loss of upregulation of 1,25-dihydroxyvitamin D by serum levels of phosphate might be important for stone formation. The possibility of deregulation of 1,25-dihydroxyvitamin D to maintain physiological requirements in stone formers and prevent further nephrolithiasis therefore warrants attention.

Adult↗

P53 status plays no role in radiosensitizing effects of SN-38, a camptothecin derivative.

PURPOSE: Topoisomerase inhibitors including camptothecin are being studied as potential radiosensitizers. CPT-11 is a derivative of camptothecin and is clinically available. In this study, we investigated the effects of SN-38 (an active metabolite of CPT-11) on four nonirradiated and irradiated murine fibroblast cell lines with different p53 statuses to clarify the role of p53 in the radiosensitizing activity of SN-38. MATERIALS AND METHODS: Four fibroblast cell lines, MT158, MT158/neo, MT158/wtp53 and MT158/mp53 with the same genetic background but with different p53 statuses, were used. Exponentially growing cells were treated with SN-38 (200 nM) and incubated with the drug for 30 min. Cells were then irradiated (0 to 12 Gy) and further incubated with the drug for 2 h. The cell survival rate was determined using a conventional clonogenic assay. The effects of the treatments on the cell cycle were analyzed with a flow cytometric assay. Apoptosis after these treatments was also detected by an annexin V assay. RESULTS: There were no significant differences in sensitivity to radiation or SN-38 treatment among these cell lines. The combined treatment of irradiation and SN-38 showed supraadditive effects in all four cell lines independent of their p53 status. Transient arrest in G2 with a decreased percentage of cells in both the S and G1 phases was observed 8 h after treatment with either SN-38 alone, radiation or their combination, regardless of the p53 status. No significant differences in frequency of apoptosis were observed between treatment and control groups in two cell lines with or without wild-type p53. CONCLUSION: The combination of irradiation and SN-38 treatment showed supraadditive effects in all four cell lines tested, and the p53 status did not play a role in the combination effect.

Antineoplastic Agents, Phytogenic↗

Intracavernous sinus ectopic adrenocorticotropin-secreting tumours causing therapeutic failure in transsphenoidal surgery for Cushing's disease.

BACKGROUND: The major cause of surgical failure in the treatment of Cushing's disease lies in unsuccessful identification of adenomas. In the present study, we investigated the pathogenesis of negative exploration in transsphenoidal surgery for Cushing's disease by analyzing neuro-imaging studies, endocrinological examination and selective venous sampling. METHODS: Thirty patients with ACTH-dependent Cushing's syndrome that met the endocrinological criteria for Cushing's disease were treated by transsphenoidal microsurgery. Depending on positive or negative identification of adenomas during the surgery, selective adenomectomy, partial hypophysectomy or subtotal hypophysectomy was performed. FINDINGS: All nine patients who underwent selective adenomectomy showed endocrinological remission and did not need any hormone replacement therapy. Of the 12 patients who underwent partial hypophysectomy, 11 showed normalization of hypercortisolism but seven needed permanent replacement of hydrocortisone. Four patients underwent subtotal hypophysectomy because no adenoma could be identified in spite of detailed exploration, and three of them showed remission but with permanent requirements of hydrocortisone and other pituitary hormones. Five patients, whose hypercortisolism persisted after initial surgery, underwent total hypophysectomy as secondary transsphenoidal surgery, and only one of them showed endocrinological remission. Of the six patients with surgical failure, four were thought to be rare cases in whom the ACTH-secreting tumours may have occurred ectopically in the cavernous sinus without direct contact with the pituitary gland. Endocrinologically, these four patients showed a low or no response to corticotropin releasing hormone (CRH) stimulation, and for three of them, radiation therapy to the sellar region including the cavernous sinus was effective for persistent hypercortisolemia. INTERPRETATION: Recognition of an ectopic intracavernous sinus ACTH-secreting tumour as a pathological entity for Cushing's disease may not only enhance the diagnostic accuracy but also be important for determining the optimal surgical mode for persistent Cushing's disease.

Adenoma↗

Accumulation of tumor suppressor p53 in rat muscle after a space flight.

Tumor suppressor p53 functions as a cell cycle checkpoint under stressful conditions. Early studies have shown that genotoxic stress activates p53 pathway. Recently, many kinds of non-genotoxic stress such as heat shock, cold shock, and low pH also have been found to activate p53 pathway. The effects on living organism remains to be explored. Here, we show that an 18-day space flight induced a 3.6 fold accumulation of p53 in rat skeletal muscle. This results suggests that the p53 pathway plays a role in safeguarding genomic stability against the stressful space environments and supports our previous observation of p53 accumulation in rat skin after a space flight.

Animals↗

Sickle cell anemia: a potential nutritional approach for a molecular disease.

A certain population of red blood cells in patients with sickle cell anemia has an elevated density and possesses an abnormal membrane. These "dense cells" have a tendency to adhere to neutrophils, platelets, and vascular endothelial cells, and, thus, they could trigger vasoocclusion and the subsequent painful crisis from which these patients suffer. We developed a laboratory method of preparing such dense cells and found that nutritional antioxidant supplements, hydroxyl radical scavengers, and iron-binding agents could inhibit the formation of dense cells in vitro. The concentrations at which effective nutritional supplements could inhibit dense cell formation by 50% were 4.0 mg/mL for aged garlic extract, 0.38 mg/mL for black tea extract, 0.13 mg/mL for green tea extract, 0.07 mg/mL for Pycnogenol, 930 microM for alpha-lipoic acid, 270 microM for vitamin E, 45 microM for coenzyme Q(10), and 32 microM for beta-carotene. Both an ex vivo study and a pilot clinical trial demonstrated that a cocktail consisting of daily doses of 6 g of aged garlic extract, 4-6 g of vitamin C, and 800 to 1200 IU of vitamin E may indeed be beneficial to the patients.

Adolescent↗

Detection of microsatellite alterations in nipple discharge accompanied by breast cancer.

Nipple discharge in breast cancer cases was examined loss of heterozygosity (LOH). DNA samples were extracted from both supernatant and cell pellet components of the discharge, and examined for LOH at microsatellite markers, D11S1818, D11S2000, D16S402, D16S504, D16S518, D17S520, and D17S786. At least one LOH was found in either the supernatant or cell pellet in seven out of 10 patients (70%). Five of seven samples, which were cytologically negative, were LOH positive, and only one case, which was cytologically positive, showed no LOH on the markers examined. All three samples, which were judged 'negative' by CEA measurement (<400 ng/ml), were LOH positive. This method could be a useful novel diagnostic modality for nonpalpable breast cancer with nipple discharge.

Adult↗