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Biomedical subjects

H Ishihara

Publications and source records attributed to H Ishihara.

At least 19 recordsLinked to original sources
PubMed1995

Unusual long target duplication by insertion of intracisternal A-particle element in radiation-induced acute myeloid leukemia cells in mouse.

Retrotransmission into the IL-3/GM-CSF gene locus by the retrotransposon intracisternal A-particle (IAP) had been observed in distinct tumor cell lines. We analyzed the locus in genomes from 7 different myeloid leukemia cell strains which were originally generated by whole-body X-irradiation of the inbred C3H/He mice at a dose of 3 Gy and maintained by in vivo passage. In one leukemia cell strain out of 7 such cases, RFLP of an allele of the interleukin-3 gene was found. Sequence analysis after cloning from the genomic library showed that a type I delta 2 IAP element was inserted in the region upstream of the IL-3 gene in the head-to-head orientation. This suggests that the locus in myeloid cells is sensitive for integration of IAP elements. Additionally, an unusual long target duplication of 82 bp, 14-fold larger than normal one, was found at the junction of the element. This suggests the possibility of a radiation-induced integration mechanism which is distinct from normal retrotransmission.

Animals↗
PubMed1995

Specific detection of the precursor of ras p21 with a mouse monoclonal anti-C-terminal peptide antibody, SARA-K1.

In an attempt to clarify the post-translational modifications of ras oncogene product p21, we have established a mouse monoclonal antibody specific for the precursor of p21. The C-terminal peptide (156-188) of K(4A)-ras oncogene product p21 (p21K(4A), termed K(4A)-peptide, was used as the immunogen. In Western blotting, monoclonal antibodies were examined for their differential reactivity between two types of p21K(4A) expressed in Escherichia coli (esh-p21K(4A)) and mammalian cell (mam-p21K(4A)). One monoclonal antibody, designated SARA-K1, reacted selectively with esh-p21K(4A). The epitope for SARA-K1 was defined on tryptic peptide (177-184), containing Cys180, of the K(4A)-peptide. Pulse-chase experiments of mam-p21K(4A) synthesis at 24 degrees C revealed that SARA-K1 precipitated a 21 kDa protein within a 7 min chase but not after a 10 min chase, indicating that SARA-K1 recognizes the precursor of mam-p21K(4A). Furthermore, in Triton X-114 partitioning experiments using mammalian cells pre-treated with Mevalotin, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, SARA-K1 precipitated [35S]methionine-labeled, [3H]mevalonic acid-unlabeled mam-p21K(4A) in the aqueous phase, but did not precipitate [3H]mevalonic acid-labeled mam-p21K(4A) in either aqueous or detergent phase. The data presented clearly show that the SARA-K1 specifically recognizes the primary translational product pro-p21K(4A).

Amino Acid Sequence↗
PubMed1995

Inhibition of pancreatic beta-cell glucokinase by antisense RNA expression in transgenic mice: mouse strain-dependent alteration of glucose tolerance.

We have generated transgenic mice, in either C57BL/6 or C3H background, expressing antisense glucokinase mRNA in beta-cells. The glucose phosphorylating activity at 60 mM glucose in transgenic islets was significantly lower than that in controls, and the insulin secretory response to glucose was lower in transgenic islets than in those of controls in both strains. Following i.p. glucose challenge, higher blood glucose levels were observed in transgenic mice than in controls in the C57BL/6 but not the C3H background. These data suggest that a beta-cell secretory defect, in combination with other undefined genetic factors, causes impaired glucose homeostasis in mice.

Animals↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

Human naive CD4 T cells produce interleukin-4 at priming and acquire a Th2 phenotype upon repetitive stimulations in neutral conditions.

The maturation of naive CD4 T cells into interleukin (IL)-4-producing effectors was shown to require the presence of IL-4 at priming, the cellular origin of which remains unclear. We demonstrate here that naive T cells themselves release IL-4 at very low levels that are nevertheless sufficient to promote their development into Th2-like cells. This conclusion is based on three observations: (1) highly purified human naive CD4 T cells, of neonatal or adult origin, develop into Th2 effectors upon repetitive cycles of stimulation with anti-CD3 monoclonal antibody (mAb) cross-linked to CD32-B7 transfected L fibroblasts followed by IL-2 expansion; (2) IL-4 protein is readily detectable in the concentrated supernatant fluids of priming cultures performed in the presence of anti-IL-4 receptor mAb; and (3) addition of anti-IL-4 or anti-IL-4 receptor mAb at priming markedly inhibits the acquisition of IL-4- and IL-5-producing capacity while enhancing that of interferon-gamma.

Adult↗
PubMed1995

Mitochondrial diabetes mellitus--glucose-induced signaling defects and beta-cell loss.

Japanese diabetic patients whose mothers were also diabetic were screened, using peripheral leukocytes, for an A to G transition at nucleotide pair 3243 of the mitochondrial gene, a tRNA(Leu)(UUR) mutation. This mutation was identified in four pedigrees from among 300 unrelated patients. Diabetes mellitus cosegretated with the mutation, except in 1 young subject, and was maternally inherited. Long-term follow-up revealed that the underlying disorder in affected members is a progressive impairment of insulin secretion. In accord with this finding, this mutation was found to be highly prevalent in a subset of diabetes mellitus called slowly progressive IDDM; the mutation was identified in 3 of 27 Japanese patients enrolled in the prospective study of islet cell antibody (ICA)-positive, initially non-insulin-dependent diabetic patients, who are very likely to become insulin dependent in several years. The histologic characteristics of slowly progressive IDDM include loss, though incomplete, of pancreatic beta-cells. Mitochondrial gene defects in beta-cells could therefore cause glucose-induced signaling defects as well as beta-cell loss, which explains the wide range of diabetic phenotypes, from NIDDM phenotype to IDDM, in patients with this mitochondrial gene mutation.

Adolescent↗
PubMed1995

Haemodynamic changes during induced hypotension--comparison of trimethaphan with prostaglandin E1 assessed using transoesophageal echocardiography.

Haemodynamic changes during induced hypotension depend upon the hypotensive agent used. We investigated if, using transoesophageal echocardiography (TEE), we could identify the haemodynamic differences between trimethaphan and prostaglandin E1. Twenty-nine patients undergoing total hip replacement were selected for study. Hypotension was induced to a mean arterial pressure of 8.0-9.3 kPa with either trimethaphan (5-20 micrograms.kg-1.min-1) or prostaglandin E1 (0.5-2.0 micrograms.kg-1.min-1). The left atrial dimension, cardiac output, fractional shortening, pulmonary venous flow and mitral valve flow were evaluated using TEE. During induced hypotension, left atrial dimension decreased in both trimethaphan and prostaglandin E1 groups (P < 0.05). In the trimethaphan-treated patients systolic velocity in pulmonary venous flow decreased from 41.9 +/- 4.8 cm.sec-1 before induced hypotension to 27.8 +/- 4.2 cm.sec-1 by 30 min after stable hypotension had been established (P < 0.01). The late/early ratio of peak velocity in mitral blood flow decreased in prostaglandin E1 treated patients. Cardiac output increased from 4.2 +/- 0.5 L.min-1 to 5.3 +/- 0.4 L.min-1 during 30 min hypotension with prostaglandin E1 administration (P < 0.05), but cardiac output decreased from 5.0 +/- 0.5 to 3.5 +/- 0.4 L.min-1 with trimethaphan (P < 0.01). The differences in haemodynamic variables could be attributed to the venule dilatation effect of trimethaphan. We conclude that it was possible to detect the haemodynamic differences between trimethephan and prostaglandin E1 using TEE.

Adult↗
PubMed1995

Does the initial distribution volume of glucose reflect plasma volume after haemorrhage in dogs?

To test the hypothesis that the initial distribution volume of glucose (IDVG) reflects plasma volume, the relationship between the IDVG and indocyanine green (ICG) assessments of plasma volume (Vd-ICG) were evaluated simultaneously both before and after induced haemorrhage (30 ml.kg-1) in eight mongrel dogs. The IDVG and the Vd-ICG were calculated with a one-compartment model from repeated measurements of plasma glucose three to seven minutes, and of plasma ICG three to nine minutes after simultaneous infusions of both glucose 100 mg.kg-1 and ICG 0.5 mg.kg-1. The IDVG calculated with a one-compartment model (IDVG-OCM) was also compared with a two-compartment model within 15 min (IDVG-TCM) on nine occasions among a total of 12 determinations. Using Bland and Altman analysis to compare the two analytical models, the IDVG-OCM tends to overestimate the IDVG-TCM by an average of 0.04 L. Although the IDVG-OCM was two to three times larger than the Vd-ICG at each corresponding point, a correlation was obtained between the IDVG-OCM and the Vd-ICG before and after induced haemorrhage (r = 0.85, n = 16, P < 0.001). We conclude that the IDVG reflects plasma volume in normal and hypovolaemic dogs, although the IDVG cannot be used directly to estimate plasma volume.

Animals↗
PubMed1995

Platinum concentrations in uterus and serum after internal iliac arterial infusion of platinum compounds in rabbits.

OBJECTIVE: To compare three platinum compounds, and study the pharmacokinetics of these compounds after systemic and intra-arterial infusion. METHODS: Adult female rabbits received infusions of 1.7 mg/kg cisplatin, 10 mg/kg carboplatin, or 6 mg/kg cisdiammine(glycolato)platinum (254-S) via the internal iliac artery or jugular vein. The doses were equitoxic. Platinum tissue concentration in uterus and platinum serum levels were measured after internal iliac arterial or intravenous (i.v.) infusion with these platinum compounds. RESULTS: Platinum uterine concentration after intra-arterial infusion was significantly higher than that after i.v. infusion for each drug (P < .05). The ratios of platinum uterine concentration after intra-arterial infusion to those after i.v. infusion were 2.24 for cisplatin, 1.83 for carboplatin, and 1.67 for 254-S measured 20 minutes after drug administration. The area under the curve of filtrated platinum (micrograms/mL.hours) was significantly lower for cisplatin compared with carboplatin and 254-S in both infusion methods (1.5 for cisplatin, 32.1 for carboplatin, and 16.0 for 254-S after i.v. administration, and 0.8, 30.9, and 15.2 after intra-arterial administration, respectively). CONCLUSION: Cisplatin produced the highest ratio of uterine to serum concentration of platinum after intra-arterial infusion.

Animals↗
PubMed1995

Influence of left ventricular hypertrophy on left ventricular function during dynamic exercise in the presence or absence of coronary artery disease.

OBJECTIVES: We investigated the influence of left ventricular hypertrophy in the presence or absence of coronary artery disease on hemodynamic characteristics during exercise in subjects without previous myocardial infarction. BACKGROUND: Left ventricular hypertrophy has been found to increase the vulnerability of the myocardium to the development of ischemia. However, the independent influences of left ventricular hypertrophy and coronary artery disease have not been assessed in humans. METHODS: Symptom-limited supine leg exercise tests were performed by 78 patients. They were classified into the following subgroups: no coronary artery disease or left ventricular hypertrophy (group I, n = 30), left ventricular hypertrophy only (group II, n = 12), coronary artery disease only (group III, n = 20) and both left ventricular hypertrophy and coronary artery disease (group IV, n = 16). Mean left ventricular mass index was 105, 158, 109 and 159 g/m2 in groups I to IV, respectively. RESULTS: Pulmonary artery wedge pressure increased from 6 +/- 3 (mean +/- SD) mm Hg at rest to 10 +/- 5 mm Hg at peak exercise in group I, from 8 +/- 2 to 18 +/- 8 mm Hg in group II (p < 0.05 vs. group I), from 6 +/- 3 to 23 +/- 6 mm Hg in group III (p < 0.01 vs. group I) and from 8 +/- 4 to 30 +/- 7 mm Hg in group IV (p < 0.01 vs. group I; p < 0.01 vs. group II; p < 0.05 vs. group III). Multiple regression analysis showed that the number of diseased coronary vessels and left ventricular mass index were independent predictors of peak pulmonary artery wedge pressure (F = 59.2 and 19.1, respectively; multiple correlation coefficient r = 0.74, p < 0.0001). CONCLUSIONS: Left ventricular hypertrophy and coronary artery disease independently increased left ventricular filling pressure during supine leg exercise. Severe left ventricular dysfunction was induced by exercise when both conditions were present.

Adult↗
PubMed1995

Structure and regulation of the chicken GATA-3 gene.

To elucidate the mechanisms underlying the tissue-restricted expression of GATA factor transcription, we have isolated and analyzed the genomic chicken GATA-3 (cGATA-3) locus. Structural analysis of the clones showed that the cGATA-3 gene consists of six exons which span more than 19 kb. Two trans-activating domains and two Zn finger domains of cGATA-3 were found to be encoded separately by exons 2/3 and 4/5, respectively, indicating that each functional domain of GATA-3 is encoded by a discrete exon. We have determined 1.7 kb of upstream promoter sequence and found a number of sequence motifs which match those of known transcription factor binding sites. Activities of presumptive regulatory regions of this gene were assessed by transfecting chimeric constructs into a chicken T cell line MSB-1. The results showed three features of cGATA-3 gene regulation. The basal promoter activity of the cGATA-3 gene is determined by sequences lying between -104 and -29 bp of the promoter region. The upstream region containing the GATA and CACCC elements in close proximity (-1280 to -1152) appeared to act as a negative transcriptional regulator, whereas the region -1151 to -850 acts as a positive regulator. Thus, the expression of cGATA-3 gene is under complex regulation and the mode of regulation of cGATA-3 gene expression is suggested to be different from that of GATA-1 genes.

Animals↗
PubMed1995

Human GLUT-2 overexpression does not affect glucose-stimulated insulin secretion in MIN6 cells.

Accumulated evidence suggests that GLUT-2, in addition to its role in glucose transport, may also have other functions in glucose-stimulated insulin secretion. As a first step in addressing this possibility, we have engineered MIN6 cells overexpressing human GLUT-2 by transfection with human GLUT-2 cDNA. Stable transformants harboring human GLUT-2 cDNA exhibited an approximately twofold increase in 3-O-methyl-D-glucose uptake at 0.5 and 15 mM. Glucokinase activity or glucose utilization measured by conversion of [5-3H]glucose to [3H]H2O was not, however, altered in the MIN6 cells overexpressing human GLUT-2. Furthermore, glucose-stimulated insulin secretion was not affected by over-expression of human GLUT-2. An abundance of GLUT-2, therefore, does not correlate with the glucose responsiveness of cells in which glycolysis is regulated at the glucose phosphorylating step. These data suggest that GLUT-2 by itself does not have significant functions other than its role in glucose transport in glucose sensing by MIN6 cells.

3-O-Methylglucose↗
PubMed1995

Quantitative analysis of endometrial stromal cells including behavioral changes related to the menstrual cycle in smear specimens.

We aimed to obtain quantitative data regarding certain characteristics of stromal cells in smear specimens of normal endometrium, including the cytological changes associated with progression through the menstrual cycle. Eleven histological and 25 cytological specimens of normal endometrium were examined immunochemically using monoclonal antibodies to vimentin and keratin by streptavidin-biotin method. As a result, we decided to use the antibody to vimentin for the differential staining of endometrial smears: cells showing positive staining to vimentin were considered stromal cells. The quantitative nuclear measurements in the smear specimens revealed that the nuclei of endometrial stromal cells were more elongated and had a more dispersed distribution than the glandular cells in the proliferative phase. Although a part of the nuclei of stromal cells became rounder in shape in the secretory phase, they were larger in size than those of glandular cells.

Adolescent↗
PubMed1995

Characterization of rat GLUT5 and functional analysis of chimeric proteins of GLUT1 glucose transporter and GLUT5 fructose transporter.

To investigate the biological and biochemical properties of GLUT5, rat GLUT5 complementary DNA was transfected into Chinese hamster ovary cells. Rat GLUT5 was exclusively targeted to the plasma membrane and exhibited a transport activity, not for glucose, but for fructose. The affinity for fructose (Km = 11.6mM) was much higher than that of GLUT2, the other glucose transporter with fructose transport activity. Interestingly, rat GLUT5 was not photolabeled with 0.5 microM cytochalasin B, whereas a similar amount of GLUT1 was adequately photolabeled under the same experimental conditions. Next, to investigate the domains required for transport of glucose/fructose in GLUT1 and/or GLUT5, several chimeric GLUT1/GLUT5 proteins were expressed, and their glucose and/or fructose transport activities were studied. The intracellular middle loop and the region encompassing the membrane spanning domains 7-12 were observed to have crucial roles in GLUT1 glucose transport, whereas replacement of the N-terminal half or the intracellular C-terminal region with the corresponding region of GLUT5 produced no marked effects on glucose transport activity. In contrast, both the N-terminal half encompassing the region from the N-terminus through the 6th membrane spanning domain and the intracellular C-terminal region were mandatory for GLUT5 fructose transport. In conclusion, GLUT5 is a transporter exclusively for fructose and the structural requirements for fructose transport are more stringent than those for glucose transport among hexose transporter proteins.

Affinity Labels↗
PubMed1995

Inhibitory effect of clopidogrel, vapiprost and argatroban on the middle cerebral artery thrombosis in the rat.

This study investigated the roles of thromboxane A2 (TXA2), ADP and thrombin in middle cerebral artery (MCA) thrombosis in the rat. The rat MCA was occluded by a thrombus induced by the photochemical reaction of rose bengal by green light that causes endothelial damage followed by platelet adhesion, aggregation and formation of a platelet and fibrin-rich thrombus at the site of the photochemical reaction. Vapiprost, a specific TXA2-receptor antagonist; clopidogrel, which has the thienopyridine structure of ticlopidine and is a more potent inhibitor of ADP-induced platelet aggregation than ticlopidine; argatroban, a specific thrombin inhibitor; or heparin was administered intravenously before rose bengal injection. The MCA local blood flow was monitored by a laser Doppler flowmeter. The MCA was occluded by thrombus about 5 min after the initiation of the photochemical reaction. Vapiprost, clopidogrel and argatroban all significantly prolonged the time taken for the thrombotic occlusion of the MCA, but in this respect, heparin was ineffective. Our observations suggest that vapiprost and clopidogrel are useful antithrombotic agents against platelet and fibrin-rich thrombi. The effect of argatroban is attributable to inhibition of thrombin-induced platelet activation and fibrin generation. The thrombosis model described in this study is useful for understanding the mechanism(s) of thrombogenesis in the rat MCA and may be applied to other mammalian species.

Animals↗