Biomedical subjects

N Ishii

Publications and source records attributed to N Ishii.

At least 37 records · Page 2Linked to original sources

PubMed2002

Perimesencephalic nonaneurysmal subarachnoid haemorrhage and variations in the veins.

Perimesencephalic nonaneurysmal subarachnoid haemorrhage (PN-SAH) is characterised by accumulation of blood around the midbrain, normal angiographic findings and an excellent prognosis. The etiology of PN-SAH has not yet been determined. Therefore we decided to compare the venograms of patients with PN-SAH with those of patients with aneurysmal SAH (A-SAH) in order to examine the relationship between PN-SAH and venous structures. We retrospectively studied 6 patients with PN-SAH and 102 cases of angiographically evaluated A-SAH during the past 12 years by reviewing their venograms for possible abnormalities in venous structures, particularly in the basal vein of Rosenthal (BVR). More abnormalities in venous structures were found in the patients with PN-SAH than in those with A-SAH. Most of the BVR in the patients with PN-SAH appeared to drain into various dural sinuses instead of the galenic system. The relationship between PN-SAH and abnormalities in venous structures was determined.

Aged↗

PubMed2002

Immunomodulation by apoptosis-inducing caspases for an influenza DNA vaccine delivered by gene gun.

Apoptosis-inducing caspases have been tested for immunomodulatory effect on a gene gun-delivered DNA vaccine which expresses influenza hemagglutinin. Attenuated murine caspase 2 and a chimera of murine caspase 2 prodomain and human caspase 3 strongly enhanced humoral and cell-mediated immune response to hemagglutinin when they were co-administered with an immunogen DNA. In contrast, wild-type caspases did not enhance the DNA-raised immune response. Caspase dose-dependent antibody response curve revealed that the antibody level was in inverse relation to the amount of administered caspase. These findings indicate that bland apoptosis of antigen-harboring cells can elicit enhanced immune responses. Extensive apoptosis interferes with the generation of immune response. Gene gun delivery involving caspases elicited type-2 immune responses that characterized with dominant IL-4 and IgG1 production. ELISPOT assays showed that CD4 T cells were preferentially activated, while CD8 T cell response remained at marginal level. Using attenuated caspases for gene gun DNA vaccination is a useful approach to amplify type-2 immune responses.

Adjuvants, Immunologic↗

PubMed2002

Roles of DNA-dependent protein kinase and ATM in cell-cycle-dependent radiation sensitivity in human cells.

PURPOSE: The roles of DNA-dependent protein kinase (DNA-PK) and ATM in the cell-cycle-dependent radiosensitivity in human cells were investigated. METHODS AND MATERIALS: A DNA-PK activity-deficient human glioblastoma cell line M059J, ataxia telangiectasia cell lines AT3BISV and AT5BIVA, and control cell lines were used. Wortmannin inhibited DNA-PK and ATM activities. Cells were synchronized by hydroxyurea. Progression through the cell cycle was analysed by flow cytometry. RESULTS: M059J exhibited hyper-radiosensitivity throughout the cell cycle, with extreme hyper-radiosensitivity in G to early S-phase compared with the control cell line M059K. AT3BISV and AT5BIVA exhibited hyper-radiosensitivity throughout the cell cycle but showed a similar pattern of cell-cycle-dependent radiosensitivity to that observed in LM217 or HeLa cells. In AT3BISV and AT5BIVA, radiosensitization by wortmannin was observed throughout the cell cycle and was most prominent in G1 to early S-phase. Wortmannin did not sensitize M059J to ionizing radiation in any cell-cycle phase. DNA-PK activities were not different throughout the cell cycle. CONCLUSION: The results suggest that (1) non-homologous endjoining plays a dominant role in G1 to early S-phase and a minor role in late S to G2-phase in repairing DNA double-strand breaks, (2) the role of ATM in repairing double-strand breaks may be almost cell-cycle-independent and (3) the dominant role of non-homologous end-joining during G1 to early S-phase is not due to cell-cycle-dependent fluctuations in DNA-PK activity.

Androstadienes↗

PubMed2002

Gamma-irradiation effect: variation of photosynthetic activity of Euglena.

OBJECTIVE: To study the effects of gamma-ray irradiation on carbon fixation (Specific production rate: SPR), CO2 utilization efficiency (CUE) and electron transfer rate (ETR) in the photosynthetic flagellate Euglena gracilis strain Z in a dose-response dependent manner. METHODS: Euglena cells were cultured in an inorganic nutrient medium containing ammonium chloride or proteose peptone. Cells were exposed to gamma-ray at 5 doses (0, 100, 250, 350, 500 Gy for water). Five days after irradiation, three photosynthetic activities were measured. SPR, which is a carbon uptake rate per unit carbon mass, was determined by 13C tracer methodology. CUE was evaluated using a relation of carbon isotope fractionation in Calvin cycle. ETR in photosystem II (PS II) was measured by a chlorophyll fluorescence analysis. RESULTS: Even at a dose of 500 Gy, 80% of ETR of the non-irradiated control (0 Gy) was sustained, while SPR and CUE were about half the level in the non-irradiated control at 500 Gy. Furthermore, the dose response of ETR was considerably different from the others. CONCLUSION: Our findings suggest that not only PS II but also the Calvin cycle in the photosynthetic system is affected by gamma ray irradiation.

Animals↗

PubMed2001

Topical application of HIV DNA vaccine with cytokine-expression plasmids induces strong antigen-specific immune responses.

The topical application of DNA vaccine to the skin is a useful method of immunization because of its simplicity, painlessness and economy. But the immune responses that it elicits are relatively low. In this study, we administered human immunodeficiency virus type-1 (HIV-1) DNA vaccine with cytokine-expressing plasmids to the skin of mice by a new topical application technique involving prior elimination of keratinocytes using fast-acting adhesive. Our results revealed that the topical application of HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. Co-administration of the DNA vaccine with cytokine expression plasmids of IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by this new method raised the levels of both the HIV-specific cytotoxic T lymphocyte (CTL) response and delayed-type hypersensitivity (DTH) and facilitated the induction of substantial immune responses by DNA vaccine. Skin biopsy sections, thus, immunized showed significant increases of S-100 protein-positive dendritic cells (DCs). These results suggest that the topical application method described here is an efficient route of DNA vaccine administration and that the immune response may be induced by DNA plasmids taken in by DCs, Langerhans cells (LCs), or others such as antigen-presenting cells. This new topical application is likely to be of benefit in clinical use.

AIDS Vaccines↗

PubMed2001

Retrovirus-mediated gene therapy for hepatocellular carcinoma with reversely oriented therapeutic gene expression regulated by alpha-fetoprotein enhancer/promoter.

In the present study, to achieve more selective and efficient therapeutic gene expression in hepatoma cells, we compared the therapeutic efficacies of the retroviral vectors expressing the herpes simplex virus thymidine kinase (HSV-tk) gene by the alpha-fetoprotein (AFP) enhancer/promoter in the forward (LNAFE0.3TK) and reverse (LN[AFE0.3TK]R) orientation to the vector long terminal repeats. By Northern blotting, the level of the HSV-tk mRNA in LN[AFE0.3TK]R-infected HepG2 human hepatoma cells was much higher than that in LNAFE0.3TK-infected cells. Consistent with this, LN[AFE0.3TK]R infection into HepG2 cells caused a greater cytotoxicity by ganciclovir exposure together with a stronger bystander effect than LNAFE0.3TK infection. In an animal model, intratumorous injection of LN[AFE0.3TK]R with ganciclovir treatment resulted in pronounced growth inhibition of HepG2 tumor. Thus, the reversely oriented therapeutic gene expression under the control of AFP enhancer/promoter is a possible candidate for the retrovirus-mediated gene therapy for hepatocellular carcinoma.

Animals↗

PubMed2001

Restoration of endogenous wild-type p53 activity in a glioblastoma cell line with intrinsic temperature-sensitive p53 induces growth arrest but not apoptosis.

p53 protein is a transcription factor involved in multiple tumor-suppressor activities including cell cycle control and apoptosis. TP53 gene is frequently mutated in glioblastoma, suggesting the importance of inactivation of this gene product in gliomagenesis. Restoration of p53 function in glioblastoma cell lines deficient for p53 has shown that p53 induces growth arrest or apoptosis depending on the cell line and vector used to transduce wild-type TP53 alleles. Considering that astrocytes grow and express p53, it is not clear whether these results reflect physiologic responses or the result of p53 overexpression in combination with cellular responses to viral vector infection. Here, we reassessed this issue using a glioblastoma cell line (LN382) that expresses an endogenous temperature-sensitive mutant p53. This cell line expresses TP53 alleles (100% as determined by a p53 transcriptional assay in yeast) mutated at codon 197 GTG (Val) > CTG (Leu). We found that the p53 protein in these cells acted as an inactive mutant at 37 degrees C and as a functional wild-type p53 below 34 degrees C as demonstrated by several lines of evidence, including (i) restoration of transactivating ability in yeast, (ii) induction of p53-modulated genes such as CDKN1(p21) and transforming growth factor-alpha, (iii) disappearance of accumulated p53 protein in the nucleus and (iv) decrease in steady state p53 protein levels. This temperature switch allowed p53 levels, which were close to physiological levels to dramatically reduce LN382 cell proliferation by inducing a G(1)/S cell cycle block, but not to induce apoptosis. The lack of apoptosis was considered to be a result of the low level p53 expression, because increasing wild-type p53 levels by adenoviral-mediated gene transfer caused apoptosis in these cells. The LN382 cell line will be extremely useful for investigations into the roles of p53 in cellular responses to a variety of stimuli or damages.

Apoptosis↗

PubMed2001

Oxidative stress causes abnormal accumulation of familial amyotrophic lateral sclerosis-related mutant SOD1 in transgenic Caenorhabditis elegans.

Mutations in the Cu/Zn superoxide dismutase (SOD1) genes are present in approximately 20% of families suffering from familial amyotrophic lateral sclerosis (FALS). Results from several transgenic studies in which FALS-related SOD1 mutations have been expressed have suggested that mutant SOD1 proteins induce cytotoxicity through a toxic gain of function, although the specific mechanism of this has not been fully clarified. To investigate the mechanism of toxicity induced by the mutant SOD1 associated with FALS, we generated transgenic Caenorhabditis elegans strains that contain wild-type and mutant human A4V, G37R and G93A SOD1 recombinant plasmids. The transgenic strains expressing mutant human SOD1 showed greater vulnerability to oxidative stress induced by 0.2 mM paraquat than a control that contained the wild-type human SOD1. In the absence of oxidative stress, mutant human SOD1 proteins were degraded more rapidly than the wild-type human SOD1 protein in C.elegans. In the presence of oxidative stress, however, this rapid degradation was inhibited, and the transgenic C.elegans co-expressing mutant human SOD1 and green fluorescent proteins (GFPs) in muscle tissues demonstrated discrete aggregates in the adult stage. These results suggest that oxidative damage inhibits the degradation of FALS-related mutant human SOD1 proteins, resulting in an aberrant accumulation of mutant proteins that might contribute to the cytotoxicity.

Amyotrophic Lateral Sclerosis↗

PubMed2001

Wobble modification defect in tRNA disturbs codon-anticodon interaction in a mitochondrial disease.

We previously showed that in mitochondrial tRNA(Lys) with an A8344G mutation responsible for myoclonus epilepsy associated with ragged-red fibers (MERRF), a subgroup of mitochondrial encephalomyopathic diseases, the normally modified wobble base (a 2-thiouridine derivative) remains unmodified. Since wobble base modifications are essential for translational efficiency and accuracy, we used mitochondrial components to estimate the translational activity in vitro of purified tRNA(Lys) carrying the mutation and found no mistranslation of non-cognate codons by the mutant tRNA, but almost complete loss of translational activity for cognate codons. This defective translation was not explained by a decline in aminoacylation or lowered affinity toward elongation factor Tu. However, when direct interaction of the codon with the mutant tRNA(Lys) defective anticodon was examined by ribosomal binding analysis, the wild-type but not the mutant tRNA(Lys) bound to an mRNA- ribosome complex. We therefore concluded that the anticodon base modification defect, which is forced by the pathogenic point mutation, disturbs codon- anticodon pairing in the mutant tRNA(Lys), leading to a severe reduction in mitochondrial translation that eventually could result in the onset of MERRF.

Animals↗

PubMed2001

Critical involvement of OX40 ligand signals in the T cell priming events during experimental autoimmune encephalomyelitis.

OX40 ligand (OX40L) expressed on APCs, and its receptor, OX40 present on activated T cells, are members of the TNF/TNFR family, respectively, and have been located at the sites of inflammatory conditions. We have observed in OX40L-deficient mice (OX40L(-/-)) an impaired APC capacity and in our recently constructed transgenic mice expressing OX40L (OX40L-Tg), a markedly enhanced T cell response to protein Ags. Using these mice, we demonstrate here the critical involvement of the OX40L-OX40 interaction during the T cell priming events in the occurrence of experimental autoimmune encephalomyelitis (EAE). In OX40L(-/-) mice, abortive T cell priming greatly reduced the clinical manifestations of actively induced EAE, coupled with a reduction in IFN-gamma, IL-2, and IL-6 production in vitro. Adoptive transfer experiments however revealed an efficient transfer of disease to OX40L(-/-) mice using wild-type donor T cells, indicating an intact capacity of OX40L(-/-) mice to initiate effector responses. On the other hand, OX40L(-/-) donor T cells failed to transfer disease to wild-type recipient mice. Furthermore, OX40L-Tg mice developed a greater severity of EAE despite a delayed onset, while both OX40L-Tg/CD28(-/-) and OX40L-Tg/CD40(-/-) mice failed to develop EAE demonstrating a requisite for these molecules. These findings indicate a pivotal role played by OX40L in the pathogenesis of EAE.

Adoptive Transfer↗

PubMed2001

An in vitro temporomandibular joint-nerve preparation for pain study in rats.

A novel in vitro TMJ-nerve preparation was developed to quantitatively study peripheral sensory mechanisms of temporomandibular joint (TMJ). The TMJ region on one side (including mandibular head, disc, retrodiscal tissue and mandibular fossa) of adult Wistar albino rats was excised together with the auriculo-temporal nerve. The block was preserved in a modified Krebs-Henseleit solution saturated with O(2)/CO(2) (95/5%) gas mixture. Using a calibrated von Frey type apparatus, mechanical noxious stimulation was applied directly to various sites within the TMJ region. In addition, thermal and chemical noxious stimuli were also attempted. Stable recordings of single unit activities from the auriculo-temporal nerve could be obtained for as long as 5 h, which was sufficient to analyze the response properties of the TMJ units to various stimuli. This new preparation would be useful for investigating TMJ peripheral sensory mechanisms, especially pain, and potentially makes it possible to reveal neural mechanisms of temporomandibular arthralgia, a syndrome that has recently shown an increased incidence in clinical dentistry.

Action Potentials↗

PubMed2001

A defect in the cytochrome b large subunit in complex II causes both superoxide anion overproduction and abnormal energy metabolism in Caenorhabditis elegans.

A mev-1(kn1) mutant of the nematode Caenorhabditis elegans is defective in the cytochrome b large subunit (Cyt-1/ceSDHC) in complex II of the mitochondrial electron transport chain. We have previously shown that a mutation in mev-1 causes shortened life span and rapid accumulation of aging markers such as fluorescent materials and protein carbonyls in an oxygen-dependent fashion. However, it remains unclear as to whether this hypersensitivity is caused by direct toxicity of the exogenous oxygen or by the damage of endogenous reactive oxygen species derived from mitochondria. Here we report important biochemical changes in mev-1 animals that serve to explain their abnormalities under normoxic conditions: (i) an overproduction of superoxide anion from mitochondria; and (ii) a reciprocal reduction in glutathione content even under atmospheric oxygen. In addition, unlike wild type, the levels of superoxide anion production from mev-1 mitochondria were significantly elevated under hyperoxia. Under normal circumstances, it is well known that superoxide anion is produced at complexes I and III in the electron transport system. Our data suggest that the mev-1(kn1) mutation increases superoxide anion production at complex II itself rather than at complexes I and III. The mev-1 mutant also had a lactate level 2-fold higher than wild type, indicative of lactic acidosis, a hallmark of human mitochondrial diseases. These data indicate that Cyt-1/ceSDHC plays an important role not only in energy metabolism but also in superoxide anion production that is critically involved in sensitivity to atmospheric oxygen.

Adenosine Triphosphate↗

PubMed2001

Cutting edge: the common gamma-chain is an indispensable subunit of the IL-21 receptor complex.

The common gamma-chain (gamma(c)) is an indispensable subunit of the functional receptor complexes for IL-4, IL-7, IL-9, and IL-15 as well as IL-2. Here we show that the gamma(c) is also shared with the IL-21R complex. Although IL-21 binds to the IL-21R expressed on gamma(c)-deficient ED40515(-) cells, IL-21 is unable to transduce any intracytoplasmic signals. However, in EDgamma-16 cells, a gamma(c)-transfected ED40515(-) cell line, IL-21 binds to the IL-21R and can activate Janus kinase (JAK)1, JAK3, STAT1, and STAT3. The chemical cross-linking study reveals the direct binding of IL-21 to the gamma(c). These data clearly demonstrate that the gamma(c) is an indispensable subunit of the functional IL-21R complex.

Cell Line↗

PubMed2001

Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene.

DNA vaccination is characterized by its preferential induction of the cytotoxic T cell lymphocyte (CTL) response and is expected to be a useful means of protection against viral infection. We examined the protective effect of an expression plasmid (pME18S-M) containing M1 and M2 genes of influenza A/PR/8/34. We detected the CTL activity by introducing these plasmids into BALB/c mice by either the intramuscular or the intranasal route. The influenza-specific antibody response was also induced, although its neutralizing effect against influenza virus was not observed. From 70 to 80% protection was observed in the mice immunized with the pME18S-M plasmid followed by lethal infection with influenza viruses of the A/WSN/33 and A/PR/8/34 strains, whereas all mice without the plasmid vaccination failed to survive. This protective activity was significantly weakened when the CD8(+) cells of these immunized mice were eliminated by several injections of anti-CD8 antibody. The protective activity was also weakened when anti-CD4 antibody was injected in the early phase of DNA vaccination. These data suggest that the pME18S-M plasmid is useful as a DNA vaccine for overcoming highly mutational influenza viruses.

Amino Acid Sequence↗

PubMed2001

Decreased expression of c-myc family genes in thymuses from myasthenia gravis patients.

The thymus is a critical organ for the elimination of autoreactive T cells by apoptosis. We studied the expression of apoptosis-associated genes, bcl-xL, bad, caspase-3, and c-myc family genes in myasthenia gravis (MG) thymuses. We observed that the mRNA levels of myc family genes, c-myc and max, were markedly reduced in MG thymuses. These results indicate that c-myc-mediated signaling is abnormal in MG thymuses. The levels of molecules whose expressions are associated with myc, such as STAM, prothymosin-alpha, and NFkappaB, were also analyzed.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Geranylgeranylacetone induces antiviral gene expression in human hepatoma cells.

Geranylgeranylacetone (GGA), an isoprenoid compound, is used clinically as an anti-ulcer drug. Since some isoprenoids including retinoids have anti-tumor and anti-viral activities in a variety of cell types, we investigated whether GGA could induce anti-viral proteins in human hepatoma cells. The HuH-7 and HepG2 cells were treated with GGA, and expression of anti-viral proteins such as 2'5'-oligoadenylate synthetase (2'5'-OAS) and double-stranded RNA-dependent protein kinase (PKR) in these cells was analyzed. GGA stimulated 2'5'-OAS and PKR gene expression at the transcriptional level through the formation of interferon-stimulated gene factor 3 (ISGF3), which regulates both gene transcription. By Western blotting, GGA induced expression of signal transducers and activators of transcription 1, 2 (STAT1, STAT2) and p48 proteins, components of ISGF3, together with the phosphorylation of STAT1. These results suggest that GGA acts as a potent inducer of anti-viral gene expression by stimulating the ISGF3 formation in human hepatoma cells.

2',5'-Oligoadenylate Synthetase↗

PubMed2001

Altered quinone biosynthesis in the long-lived clk-1 mutants of Caenorhabditis elegans.

Mutations in the clk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ(9)) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ(9). Instead, the UQ(9) biosynthesis intermediate, demethoxyubiquinone (DMQ(9)), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ(9) in C. elegans. Interestingly, the activity levels of NADH-cytochrome c reductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ(9) can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ(2) was chemically synthesized. We find that DMQ(2) can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ(2), cannot. The accumulation of DMQ(9) and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.

Animals↗

PubMed2001

CpG methylation of promoter region inactivates E-cadherin gene in renal cell carcinoma.

CpG methylation in the promoter region has been shown to be important in the regulation of genes implicated in malignant transformation. The present study was designed to test the hypothesis that CpG methylation of the promoter region of the E-cadherin gene may inactivate its expression in renal cell carcinoma. To test this hypothesis, five kidney cancer cell lines and 34 microdissected renal cell carcinoma samples were analyzed for gene and protein expression by reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. CpG methylation in the promoter regions of the E-cadherin gene was analyzed by the sodium bisulfite genome sequencing technique. Our results show that all normal renal tissue expressed the E-cadherin gene and protein. Of the renal cancer tissues analyzed, 67% (23 of 34) lacked E-cadherin expression, with an associated increase in methylation, compared with normal tissue. E-cadherin gene promoter was methylated in all renal cancer cell lines and was accompanied by a loss of E-cadherin gene and protein expression. The treatment of renal cancer cell lines with the demethylating agent 5-aza-2'-deoxycytidine restored E-cadherin mRNA expression in all renal cancer cell lines. This is the first report that shows inactivation of the E-cadherin gene and protein in renal cell carcinoma through CpG hypermethylation in the promoter region of this gene. The results of these experiments may contribute to an understanding of the role of E-cadherin inactivation in renal cell carcinoma.

Azacitidine↗