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Biomedical subjects

N Ohishi

Publications and source records attributed to N Ohishi.

At least 37 records · Page 2Linked to original sources
PubMed1998

Dissipation of mitochondrial membrane potential by exogenous phospholipid monohydroperoxide and protection against this effect by transfection of cells with phospholipid hydroperoxide glutathione peroxidase gene.

Two hours after its addition to cultures of a guinea pig cell line, 104C1, dilinoleoyl phosphatidylcholine monohydroperoxide (PCOOH) at concentrations of 5-160 microM induced a dissipation of the mitochondrial inner membrane potential (delta psi m), without any apparent morphological changes, in the cells. The PCOOH-induced loss of delta psi m was restored 4 hr after the replacement of the medium with PCOOH-free fresh medium. In contrast, 104C1/O4C cells, a stable clone from 104C1 cells transfected with the human phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene encoding a sequence including a signal peptide towards mitochondria, were resistant to the loss of delta psi m after a 2-hr exposure to PCOOH at concentrations up to 160 microM. Even after an 8-hr exposure to 80 microM PCOOH, the transfected cells retained their delta psi m intact, though the parent cells were killed by the same treatment. The present results strongly suggest that the expression of PHGPx protected the host cells from PCOOH-mediated injury at least by protecting their mitochondria from lipid hydroperoxide-induced loss of delta psi m.

Animals↗
PubMed1998

Arsenic induces apoptosis in B-cell leukaemic cell lines in vitro: activation of caspases and down-regulation of Bcl-2 protein.

We showed that arsenic inhibited the cell growth of four B-cell leukaemia cell lines of 11 various cell lines in vitro. In two of these four lines, KOCL44 and LyH7, apoptosis was identified by morphological and nucleosomal DNA fragmentation studies. Three of the four B-cell lines that were growth inhibited were acute infantile leukaemia with t(11;19)(q23;p13) translocations involving the MLL gene that encodes the transcriptional factor Drosophila trithorax. The arsenic-induced apoptosis in KOCL44 and LyH7 cells was found to be linked to caspases by Western blot and enzymological analyses. The amount of Bcl-2 was reduced during apoptosis in LyH7 as judged by Western blot analysis. We concluded that combined activation of the caspases and down-regulation of Bcl-2 could determine the fate of B-cell leukaemic cells in response to arsenic.

Animals↗
PubMed1998

Effect on colon cancer cells of human interferon-beta gene entrapped in cationic multilamellar liposomes.

When cultured cells of human colon cancer cell line SW480 were transfected with human interferon-beta (hIFN-beta) gene by means of cationic multilamellar liposomes, the endogenously produced hIFN-beta exhibited a remarkable anti-proliferative effect on the cells, which was more effective than that of exogenously added hIFN-beta. This effect lasted for several days, and was blocked completely by the addition of sufficient amounts of anti-hIFN-beta antibody. From experiments using a transwell plate and an infusion pump, we found that endogenously produced hIFN-beta acted effectively on the cells around the transfectants and that the growth-inhibitory effect was totally retained upon continuous dilution of the medium. These data indicate that hIFN-beta expressed endogenously by transfer of its gene acted on these cancer cells mainly in a paracrine manner. Although the transfection with hIFN-gamma gene also revealed a definite growth-inhibitory effect on the same tumor cells, the extent was less than that of hIFN-beta gene.

Antibodies↗
PubMed1998

Human large luteal cells in the menstrual cycle and early pregnancy express leukotriene A4 hydrolase.

Leukotriene (LT) A4 hydrolase (EC3.3.2.6) converts LTA4 to LTB4 which shows a chemotactic activity to leukocytes. To investigate the involvement of LTB4 in human corpus luteum (CL) function, the localization of LTA4 hydrolase in human ovarian tissues was examined by immunohistochemistry with a rabbit polyclonal antibody against LTA4 hydrolase. The enzyme was weakly expressed on granulosa cells of the follicles. After ovulation, the intensity of LTA4 hydrolase on large luteal cells increased and was highest in the midluteal phase. High expression was also observed in the CL of early pregnancy. In theca interna and small luteal cells, LTA4 hydrolase was weakly detected in all developmental stages. Immunoblot analysis demonstrated that the molecular mass of LTA4 hydrolase expressed in CL was 62 kDa, and confirmed that LTA4 hydrolase expression increased during CL formation and remained high in early pregnancy. The sequence encoding mRNA of LTA4 hydrolase, which was isolated from CL and amplified by polymerase chain reaction, was shown to be identical to the previously reported one. Immunocytochemistry showed that LTA4 hydrolase expression in cultured granulosa cells increased over 4 days in vitro and was enhanced by human chorionic gonadotrophin treatment. These expression profiles of LTA4 hydrolase suggest the involvement of LTB4 in luteal cell function during CL formation and early pregnancy.

Adolescent↗
PubMed1998

Contribution of HLA genes to genetic predisposition in diffuse panbronchiolitis.

Diffuse panbronchiolitis (DPB) in East Asia is a distinctive chronic obstructive pulmonary disease of unknown etiology. We hypothesize that the disease susceptibility is due to genetic predisposition unique to Asians. Association between human leukocyte antigen (HLA)-Bw54 and the disease was previously reported. In the present study, using newly developed polymerase chain reaction (PCR)- based methods, we directly analyzed HLA class I and II alleles in 76 Japanese patients. HLA-A, -B, and -C antigens were screened by the conventional typing method, and then B22-group alleles including HLA-B54 were genotyped by single-strand conformation polymorphism analysis. Alleles of HLA-DRB1 gene were fully determined by the microtiter plate hybridization method. Thirty-seven percent of the patients possessed HLA-B*5401 allele conserved predominantly in East Asians, as compared with 15% of 110 healthy volunteers (chi2 = 12.4, p = 0.0004). In addition, 4% of the patients possessed B*5504 also unique to Asians but a rare allele which was not found in normal control subjects in this study. Typing of HLA-DRB1 class II gene did not demonstrate strong positive association with the disease. A33, B44, and DRB1*1302 showed negative association with the disease. We conclude that distinctive molecular structure of HLA-B alleles or a closely linked gene in the HLA region contributes to genetic predisposition in diffuse panbronchiolitis. This may partly explain why this disorder is found primarily in Asians.

Alleles↗
PubMed1998

Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress collagen accumulation in experimental glomerulonephritis.

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that has been shown to play a major role during the biosynthesis and secretion of procollagen molecules. The expression of HSP47 has been reported to increase in parallel with the expression of collagens during the progression of various fibrosis models. However, it remains unclear whether an inhibition of HSP47 overexpression would suppress collagen accumulation and thus reduce the progression of fibrotic diseases. In this study, we attempted to attenuate glomerular collagen accumulation by inhibiting the overexpression of HSP47 with antisense oligodeoxynucleotides in an experimental glomerulonephritis model induced by anti-Thy-1 antibodies. The administration of antisense oligodeoxynucleotides against HSP47 at the induction of the glomerular disease markedly suppressed the increased production of collagens and attenuated the histologic manifestations of the disease. These results provide direct evidence of a pivotal role for HSP47 in the pathogenesis of glomerulonephritis.

Animals↗
PubMed1997

Adenovirus-mediated transduction with human glial cell line-derived neurotrophic factor gene prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopamine depletion in striatum of mouse brain.

As a novel trial of neuroprotective therapy of neurodegenerative diseases, we have constructed a recombinant adenovirus vector (rAdv) bearing a neurotrophic factor gene to deliver the factor to rescue neurons in vivo. In the present study, human glial cell line-derived neurotrophic factor (hGDNF) was chosen to examine the applicability of our strategy to a mouse model of Parkinson's disease. During the construction of the rAdv, we found that the strong constitutive hGDNF expression unit somehow inhibited the appearance of the rAdv. Therefore we adopted a self-contained tetracycline-regulated expression system to acquire an rAdv expressing hGDNF. By analyzing the condition medium of SH-SY5Y cells infected with our constructed virus vector, we confirmed that biologically active GDNF was successfully expressed in vitro. For an animal study, we delivered this virus vector directly to the C57 black mouse brain and then exposed the animal to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to injure the nigrostriatal dopaminergic neurons. One week after the MPTP exposure, the neuroprotective effect of the virus vector was estimated by measurement of the dopamine content in the striatum of the mouse brain. The mice that had received our constructed virus had significantly higher dopamine levels in their striatum, demonstrating that our rAdv expressing hGDNF has therapeutic potential to protect the nigrostriatal dopaminergic neurons in vivo.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1997

Isolation and expression of rat interferon beta gene and growth-inhibitory effect of its expression on rat glioma cells.

Rat interferon beta (IFN beta) was cloned from a part of rat genomic DNA by the polymerase chain reaction (PCR) with reference to the nucleotide sequence data of a mouse IFN beta. The sequence result showed that the clone had 555 bp of open reading frame (ORF) which encoded a 184-amino acid polypeptide. Comparison of our data with those of mouse and human IFN beta revealed 86.7 and 67.6% homology at the nucleotide level, and 76.2 and 48.9% homology at the amino acid level, respectively. This ORF was inserted into an expression vector, pCAGGS, and COS-1 cells were then transfected with the plasmid encapsulated in cationic multilamellar liposomes. From the results of reverse transcription-PCR and cytopathic activity assay, this ORF expressed IFN beta in COS-1 cells. When the cultured cells of rat glioma cell line T9 were transfected with the plasmid, their growth was markedly suppressed due to the expression of rat IFN beta.

Amino Acid Sequence↗
PubMed1997

Growth suppression and induction of heat-shock protein-70 by 9-cis beta-carotene in cervical dysplasia-derived cells.

The aim of the present study was to determine to what extent 9-cis beta-carotene, one of the most abundant naturally-occurring cis-isomers of beta-carotene, can inhibit the growth of cervical dysplasia-derived cells in comparison with all-trans beta-carotene. We found that 9-cis beta-carotene was dose-dependently more effective than all-trans beta-carotene. Both carotenes induced the intracellular accumulation of heat-shock protein-70 (HSP70), and the treated cells showed morphological changes indicative of apoptosis. The results of the present study strongly suggest that the induction of HSP70 by beta-carotene might be involved in beta-carotene-mediated suppression of the cell growth through apoptosis.

Cell Division↗
PubMed1997

Bronchiolitis obliterans organizing pneumonia after syngeneic bone marrow transplantation for acute lymphoblastic leukemia.

We report a patient who developed bronchiolitis obliterans organizing pneumonia (BOOP) after syngeneic BMT for ALL. The patient complained of persistent low-grade fever and non-productive cough after engraftment. Chest CT scan showed patchy infiltration bilaterally in the lower lung fields. Antibiotics were ineffective. Cultures, serological studies and polymerase chain reaction detected no infectious pathogens. We finally made a diagnosis of BOOP by thoracoscopical lung biopsy. The lung lesion disappeared in a month with corticosteroid therapy. While BOOP following allogeneic BMT has been reported, this is the first report after syngeneic transplantation.

Adult↗
PubMed1997

Effect of selenium on human phospholipid hydroperoxide glutathione peroxidase expression and host cell susceptibility to lipid hydroperoxide-mediated injury.

We recently isolated stable transfectants expressing human phospholipid hydroperoxide glutathione peroxidase (PHGPx) from the cells of guinea pig cell line 104C1 (Biochem. Biophys. Res. Commun. 219, 486-491, 1996). Among them, one transfectant, designated 104C1/O4C, expressed high glutathione peroxidase activity toward dilinoleoyl phosphatidylcholine hydroperoxide (PCOOH); and another one, 104C1/O2D, moderate activity. In the present study, we investigated the effect of selenium on the PHGPx activity and on the lipid hydroperoxide-mediated cell injury in the transfectants to clarify further the action of PHGPx in preventing oxidative injury of the cells. When transfectant 104C1/O2D cells were cultured in the medium added with 250 nM selenium, glutathione peroxidase activity toward PCOOH increased 8-fold. Western blot analysis also revealed an increase in the amount of protein immunoreactive against anti-rat PHGPx antibody in this transfectant. Lipid hydroperoxide-mediated cell injury to the transfectant 104C1/O2D was significantly suppressed in accordance with the increase in the enzyme activity when the cells were cultured in the medium added with selenium. On the contrary, neither glutathione peroxidase activity toward PCOOH nor susceptibility to the injury was affected by selenium addition to the medium of the parental 104C1 cells, which have no selenium-dependent glutathione peroxidase. These results clearly support our previous conclusion that expression of PHGPx is responsible for the protection of host cells from lipid hydroperoxide-mediated injury.

Animals↗
PubMed1997

Long-term effects of LTB4 antagonist on lipid induced renal injury.

Glomerular infiltration of macrophages is a characteristic alteration of renal histopathology in hyperlipidemic renal injury. Each macrophage has two key enzymes to synthesize LTB4:5-lipoxygenase and LTA4 hydrolase. In this study we examined the long-term effects of LTB4 antagonist on real function and histopathology of spontaneously hypercholesterolemic (SHC) rat, which is model of hyperlipidemic renal injury, to see if the LTB4 antagonist could reduce the progression of renal damage. Spontaneously hypercholesterolemic rats fed a normal diet (C) developed end-stage renal failure in 26 weeks, while those fed a diet supplemented with LTB4 antagonist (E) showed normal renal function, and mild histopathological alterations (SCr: C, 1.4 +/- 0.3; E, 0.6 +/- 0.1 mg/dl, P < 0.03) without statistical differences in serum total cholesterol, body weight and blood pressure between two groups. These results suggest that LTB4 plays an important role in progression of hyperlipidemic renal injury.

Animals↗
PubMed1996

Expression of human phospholipid hydroperoxide glutathione peroxidase gene for protection of host cells from lipid hydroperoxide-mediated injury.

A cDNA encoding human phospholipid hydroperoxide glutathione peroxidase (PHGPx) was obtained by PCR amplification from human testis cDNA and was inserted into the plasmid pRc/CMV to construct an expression vector for human PHGPx. Guinea pig cell line 104C1 cells were transfected with the expression vector. One of the transfectants, designated 104Cl/O4C, expressed high glutathione peroxidase activity toward dilinoleoyl phosphatidylcholine hydroperoxide and linoleic acid hydroperoxide. Western blot analysis revealed a large amount of protein immunoreactive against anti-PHGPx antibody in the transfectant. When the cells were incubated with these hydroperoxides, the parental cells suffered from serious cell injury, whereas the transfectant was extremely resistant against lipid hydroperoxide-mediated injury.

Animals↗
PubMed1996

A simple procedure for large-scale purification of 9-cis beta-carotene from Dunaliella bardawil.

For experiments designed to obtain reliable data on the metabolic pathway and biological function of 9-cis beta-carotene, sufficient quantity of this substance in pure form is needed. For this purpose, we decided to purify 9-cis beta-carotene from the dry powder of the alga Dunaliella bardawil. By use of both silica gel and ODS open column chromatography and high-performance liquid chromatography (HPLC) on an ODS column and by treatment of the sample with ethanol, we could obtain this carotenoid as fine needle-shaped orange crystals. They were found to be highly pure as judged by analytical HPLC, absorption spectrum, and nuclear magnetic resonance analysis.

Chlorophyta↗
PubMed1996

Alpha-class isozymes of glutathione S-transferase in rat liver cytosol possess glutathione peroxidase activity toward phospholipid hydroperoxide.

Selenium-independent enzymes, found in the liver cytosol of selenium deficient rats, that are capable of reducing dilinoleoyl phosphatidylcholine hydroperoxide in the presence of reduced glutathione [Guan et al., (1995) Biochem. Mol. Biol. Int., 37, 1103-1110] were purified to homogeneity by use of successive chromatography on glutathione affinity and Mono P columns. The molecular weight of the purified protein was estimated by gel filtration to be approximately 50 kDa. Upon isoelectric focusing, the purified preparation showed two protein bands having pI values of 8.6 and 8.8. Both proteins had reactivity against both 1-chloro-2,4-dinitrobenzene and dilinoleoyl phosphatidylcholine hydroperoxide in the presence of reduced glutathione. Each of them consisted of two subunits having molecular weights of 24.3 kDa and 26 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit was identified as rat glutathione S-transferase (GST) 2 (Yc subunit) based on the amino-terminal amino acid sequence analysis. The small subunit was considered to be most probably rat GST 1 (Ya subunit). From these results, we conclude that the basic alpha-class isozymes of GST in rat liver cytosol possess glutathione peroxidase activity toward phospholipid hydroperoxide.

Animals↗