Biomedical subjects
T Ogihara
Publications and source records attributed to T Ogihara.
[Progressive autonomic failure].
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Overexpression of prostacyclin synthase inhibits growth of vascular smooth muscle cells.
To define the local effects of prostacyclin (PGI2) on the growth of vascular smooth muscle cells (VSMC), we transfected VSMC with an expression vector harboring the cDNA for PGI2 synthase (PGIS), which catalyzes the rearrangement of prostaglandin H2 to PGI2. Transfection of the human PGIS cDNA into rat VSMC did not affect DNA synthesis under serum-free basal conditions, but it increased PGI2 synthesis and decreased DNA synthesis under serum-stimulated conditions (in the presence of 1 or 5% fetal calf serum). These results demonstrated that locally synthesized PGI2 can exert autocrine and/or paracrine inhibitory effects on VSMC growth. It was also suggested that in vivo transfer of PGIS gene may be useful for the gene therapy for vascular disease such as neointimal hyperplasia.
Participation of a proton-cotransporter, MCT1, in the intestinal transport of monocarboxylic acids.
A molecular mechanism for the intestinal monocarboxylic acid transport was characterized by using a proton/monocarboxylate transporter, MCT1, in Chinese hamster ovary (CHO) cells, first found by Garcia et al. (Cell, 76, 865-873, 1994). Northern blotting analysis showed that MCT1-isomers exist in the rat and rabbit intestinal enterocytes and Caco-2 cells. The expression of [14C]lactic acid uptake by Xenopus laevis oocytes injected with rabbit intestinal mRNA was reduced by hybridizing the mRNA with a MCT1 cDNA of CHO cells before microinjection used as the antisense DNA. [14C]Lactic acid uptake by CHO cells was pH dependent, saturable, stereospecific, and reduced in the presence of acetic acid, benzoic acid, S- and R-ibuprofen, S- and R-mandelic acid, nicotinic acid, pravastatin, propionic acid and valproic acid. In addition, several monocarboxylic acids were transported in pH-dependent and saturable manners. These results suggest that the intestinal MCT1-related protein contributes to a carrier-mediated absorption for organic weak acid compounds.
Angiotensin-converting-enzyme genotype and ischemic heart disease.
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Genetic association between chromosome 8 microsatellite (MS8-134) and Werner syndrome (WRN): chromosome microdissection and homozygosity mapping.
Werner syndrome (WRN) is an autosomal recessive disorder characterized by premature aging that has been mapped to the short arm of chromosome 8, 8p11.2-p12. To refine the genetic map around the WRN region, we have isolated eight microsatellites for this region from a microdissection library. We typed members of Japanese families with WRN on the basis of homozygosity mapping analysis. There was no obligate recombination between the WRN locus and microsatellite clone, MS8-134 (D8S1055). The maximum lod score was 20.28 at theta = 0.00. Alleles for MS8-134 showed association with WRN in a case-control study (OR = 3.55, 95% CI 1.56-8.07, P < 0.01). Such microsatellites from a microdissection library of the definite chromosome region may be useful for positional cloning of the WRN gene.
[Genetic aspects of hypertension--etiology of essential hypertension].
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Hepatic damage induced by perfusion of radical generating azo compound and its inhibition by vitamin E.
The damaging effect of perfusion of hydrophilic radical generating azo compound on the liver of normal and vitamin E-deficient rats and its inhibition by antioxidants were studied in order to increase understanding of the action of free radicals on biological tissues. The hepatic damage was evaluated from the release of cytosolic enzymes such as glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, mitochondrial oxidation metabolism and morphological change. Two kinds of hydrophilic azo compounds were used, one which decomposes spontaneously at a uniform rate to generate free radicals and the other which does not. The former induced hepatic damage in a dose-dependent manner, while the latter did not exert any damage. Both endogenous vitamin E in the membranes and a water soluble vitamin E analogue added simultaneously with a radical initiator suppressed the hepatic damage. These results show that the hepatic damage induced by perfusion of radical generating azo compound is caused not by the azo compound itself but by free radicals.
A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo.
The application of DNA technology to regulate the transcription of disease-related genes in vivo has important therapeutic potentials. The transcription factor E2F plays a pivotal role in the coordinated transactivation of cell cycle-regulatory genes such as c-myc, cdc2, and the gene encoding proliferating-cell nuclear antigen (PCNA) that are involved in lesion formation after vascular injury. We hypothesized that double-stranded DNA with high affinity for E2F may be introduced in vivo as a decoy to bind E2F and block the activation of genes mediating cell cycle progression and intimal hyperplasia after vascular injury. Gel mobility-shift assays showed complete competition for E2F binding protein by the E2F decoy. Transfection with E2F decoy inhibited expression of c-myc, cdc2, and the PCNA gene as well as vascular smooth muscle cell proliferation both in vitro and in the in vivo model of rat carotid injury. Furthermore, 2 weeks after in vivo transfection, neointimal formation was significantly prevented by the E2F decoy, and this inhibition continued up to 8 weeks after a single transfection in a dose-dependent manner. Transfer of an E2F decoy can therefore modulate gene expression and inhibit smooth muscle proliferation and vascular lesion formation in vivo.
Antioxidant, probucol, can inhibit the generation of hydrogen peroxide in islet cells induced by macrophages and prevent islet cell destruction in NOD mice.
The preventive effect of antioxidant on the islet cell destruction in NOD mice was studied. Oral administration of probucol prevented the development of cyclophosphamide induced diabetes in NOD mice (incidence of diabetes; probucol: 0/10 vs. control: 9/15) As in vitro studies, the effects of probucol on the generation of H2O2 in the content of H2O2 in islet cells co-cultured with M phi were measured. Intracellular oxidation of 2'7'-dichlorofluorescin by H2O2 was analyzed by flow cytometry. The probucol-treated M phi induced less marked increase of H2O2 in islet cells in comparison with control M phi. The generation of H2O2 by M phi in islet cells from treated mice was less marked than that in controls. These results suggest that the preventive effect of probucol may be mediated by protection of islet cells from oxidative stress induced by M phi.
A novel isoform of syntaxin-binding protein homologous to yeast Sec1 expressed ubiquitously in mammalian cells.
munc-18/n-Sec1/rbSec1, a brain homologue of the yeast Sec1p protein, is thought to participate in regulating the docking and fusion of synaptic vesicles. We have screened the mouse cDNA library of an MIN6 cell line, derived from pancreatic beta cells, for its novel isoform and have identified a cDNA encoding a 593-amino acid protein having 63, 53, and 30% identity with munc-18/n-Sec1/rbSec1, Caenorhabditis elegans unc18, and Saccharomyces cerevisiae Sec1p, respectively. While munc-18/n-Sec1/rbSec1 expression has been reported to be neural-specific, RNA blot analysis has revealed that the novel isoform, which we refer to as muSec1 (mammalian ubiquitous Sec1), is expressed ubiquitously. We have also identified mouse munc-18/n-Sec1/rbSec1 from the MIN6 cDNA library, indicating that different isoforms of a protein participating in vesicular transport exist in a single cell. muSec1 bound to glutathione S-transferase-syntaxin 1A and, although with lower affinity, to glutathione S-transferase-syntaxin 4 fusion protein. These findings suggest that muSec1 is, via its binding to the syntaxin family, involved in the protein trafficking from the Golgi apparatus to the plasma membrane and that the fundamental mechanisms of protein trafficking have been conserved from yeast through virtually all mammalian cells.
[Treatment of hypertension in the elderly].
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Lysophosphatidylcholine causes Ca2+ influx, enhanced DNA synthesis and cytotoxicity in cultured vascular smooth muscle cells.
The effects of lysophosphatidylcholine (LPC), a vasoactive phospholipid, on intracellular free calcium concentration ([Ca2+]i), DNA synthesis and cytotoxicity of vascular smooth muscle cells (VSMC) were studied. LPC from 10(-7) to 10(-5) mol/l dose-dependently induced a sustained increase in [Ca2+]i. In contrast to the response of [Ca2+]i induced by angiotensin II, that induced by LPC was totally abolished when extracellular Ca2+ was removed, was not affected by pretreatment of the cells with islet-activating protein, and was not desensitized by repeated addition. 8-(N,N-Diethylamino)octyl 3,4,5-trimethoxybenzoic acid (TMB-8), an inhibitor of Ca2+ release from intracellular Ca2+ stores, 1-(5-isoquinolinesulfonyl)-2-methylpiperadine dihydrochloride (H-7), an inhibitor of protein kinase C, KT5823, an inhibitor of protein kinase G, and Ca2+ channel blockers failed to suppress the LPC-induced increase in [Ca2+]i. LPC at 10(-5) mol/l caused significant stimulation of [3H]thymidine incorporation into VSMC, and at concentrations of 10(-5) mol/l and higher dose-dependently stimulated release of lactate dehydrogenase in cell culture supernatants. Moreover, digitonin mimicked the effects of LPC on [Ca2+]i, and also caused similar effects to those of LPC on DNA synthesis and cytotoxicity in VSMC. These observations suggest that LPC causes both cell growth and cell injury of VSMC, at least partly, through its detergent action, causing membrane leakiness and resultant [Ca2+]i overload in vitro, thus indicating the possible participation of LPC in atherosclerosis and/or injury of the vascular wall.
Rapid typing of variable number of tandem repeat locus in the human apolipoprotein B gene for DNA diagnosis of heart disease by polymerase chain reaction and capillary electrophoresis.
The apolipoprotein B (apoB) variable number of tandem repeat (VNTR) alleles containing larger repeat units is a risk factor for coronary heart disease. Capillary electrophoresis (CE) in entangled polymer solution was applied to the analysis of polymerase chain reaction (PCR) amplified apoB VNTR locus for DNA diagnosis of heart disease. The CE separation gives an excellent resolution of two alleles differing by one or two 16 bp repeat units in the DNA size range up to 600 bp with high speed. The apoB alleles differing in length by 2 or 4 repeat units are readily distinguishable by CE in the DNA size range from 600 to 1000 bp. The plate number achieved was 1 million plates per meter. CE combining with PCR provides an excellent technique for accurate determination of the number of repeat units of apoB VNTR alleles and differentiation of heterozygous from homozygous individuals. Using the CE technique, the apoB VNTR loci from some individuals in genotyping were examined towards precise DNA diagnosis for coronary heart disease.
Alterations of bone mineral density of the femurs in hemiplegia.
We evaluated the bone mineral density (BMD) of the bilateral femurs in 112 patients with hemiplegia using dual-energy X-ray absorptiometry in order to elucidate the effect of disuse and immobilization. BMD of the paretic side was significantly reduced compared with that of the non-paretic side in hemiplegic patients (femoral neck 0.582 +/- 0.014 g/cm2 versus 0.623 +/- 0.014 g/cm2 and total femur 0.645 +/- 0.02 g/cm2 versus 0.702 +/- 0.017 g/cm2; mean +/- SEM, P < 0.01, respectively). Femoral BMD in both the paretic and nonparetic limb had significantly (P < 0.01) lower values than in age- and sex-matched controls, but the paretic side had a more significant reduction of BMD; femoral neck -20% versus -14% and total femur -24% versus -18%. In addition, patients with impaired activities of daily living (ADL), evaluated by a mobility score, had significantly decreased BMD ratios of paretic/nonparetic side than patients with improved ADL (femoral neck 91% versus 97%, P < 0.01 and total femur 89% versus 94%, P < 0.05). Our results indicated that BMD of both femurs of patients with hemiplegia was reduced, although the paretic side showed a greater BMD decrease. This decrease might be prevented or reduced by improvement of ADL.
A mutation in the glucagon receptor gene (Gly40Ser): heterogeneity in the association with diabetes mellitus.
A possible pathogenic mutation in the glucagon receptor gene causing a Gly to Ser change at codon 40 (Gly40Ser) was reported to be associated and linked with non-insulin-dependent diabetes mellitus (NIDDM), in France and Sardinia. Since the frequency of the mutation (Gly40Ser), about 5% in the French population of familial NIDDM and 8% in randomly chosen diabetic patients in Sardinia, was much higher than that of any of the previously reported mutations in candidate genes, it is important to clarify whether the contribution of this mutation to NIDDM is universal. In this study, we investigated the association of this mutation with diabetes mellitus in a large number of Japanese diabetic patients (383 NIDDM and 53 insulin-dependent diabetic patients) by polymerase chain reaction-restriction fragment length polymorphism analysis. None of the Japanese diabetic patients showed Gly40Ser mutation and the association of this mutation with NIDDM was significantly different (p < 4.10(-5) vs French, p < 3.10(-6) vs Sardinian by Fisher's exact test). The results not only indicate that the mutation plays little, if any, role in susceptibility to diabetes in Japan, but also indicate the genetic heterogeneity in NIDDM and further emphasize the importance of studies on genetic susceptibility to NIDDM and other complex traits in different ethnic groups.
Class I HLA is associated with age-at-onset of IDDM, while class II HLA confers susceptibility to IDDM.
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Two dinucleotide repeat polymorphisms at the D8S1442 and D8S1443 loci.
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