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Biomedical subjects

H Hagiwara

Publications and source records attributed to H Hagiwara.

At least 73 records · Page 4Linked to original sources
PubMed1998

Laminar shear stress-induced GRO mRNA and protein expression in endothelial cells.

BACKGROUND: The shear stress induced by blood flow may play a pivotal role in the induction or prevention of atherosclerosis by changing endothelial functions. To disclose the mechanisms of this change, we prepared an endothelial cell (EC) cDNA library to select specific clones expressed in response to shear stress. METHODS AND RESULTS: The mRNA of cultured confluent bovine aortic ECs (BAECs) subjected to steady laminar shear stress (30 dyne/cm2) for 4 hours was separated, and a cDNA library was prepared. Nine clones whose expressions were specifically enhanced by the shear stress were selected by use of a differential hybridization method. One clone had 94% homology at the nucleotide sequence level to Oryctolagus cuniculus gro (GRO) mRNA and 79% homology at the amino acid sequence level to human GRO-beta. The GRO mRNA expression was increased in both BAECs and human umbilical vein ECs (HUVECs) after the ECs were subjected to high (30 dyne/cm2) and low (5 dyne/cm2) laminar shear stress. GRO-alpha and/or -beta protein expression also increased after the HUVECs and BAECs were subjected to shear stress. Because GRO protein has been shown to function as an adhesion factor of monocytes on the surface of ECs, we studied whether shear stress-induced monocyte adhesion was caused by GRO protein expression on ECs. The 4-hour shear stress enhanced monocyte adhesion to ECs by 2.5-fold over control levels, and this enhancement was inhibited by 53% by anti-GRO-alpha antibody. CONCLUSIONS: The present study is the first report that shear stress induced the expression of GRO mRNA and protein in ECs and enhanced the monocyte adhesion on ECs via GRO protein. Further investigations of the functions and participation in atherogenesis of this selected clone may clarify the significance of shear stress on atherogenesis.

Animals↗
PubMed1998

Effects of nitric oxide from exogenous nitric oxide donors on osteoblastic metabolism.

We examined the effects of nitric oxide (NO) on the differentiation and mineralization of newborn rat calvarial osteoblastic cells (ROB cells) using exogenous NO donors, sodium nitroprusside, 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamin e (NOC-7) and 2,2'-(hydroxynitrosoydrazino)bis-ethanamine (NOC-18). Sodium nitroprusside and NOC-7 dose-dependently enhanced the rate of production of intracellular cGMP in ROB cells and the rat clonal osteogenic cell line ROB-C26. We used NOC (NOC is the trade name for NO complex manufactured by Dojindo, Kumamoto, Japan) as an NO donor in our experiments because sodium nitroprusside exhibited a marked cytotoxicity. Northern blot analysis revealed that the level of mRNA for osteocalcin, one of the osteoblastic differentiation markers, was enhanced in the ROB cells, which was continuously treated by NOC-18. NOC-18, however, did not affect the level of mRNA for alkaline phosphatase and the activity of alkaline phosphatase. Both the number and the total area of mineralized nodules that are a model of in vitro bone formation were shown to be increased by 10(-5) M NOC-18. Our data suggest that NO might act as a local regulator of the metabolism of osteoblastic cells.

Animals↗
PubMed1998

Enantioselective Total Syntheses of (-)-7betaH-Eudesmane-4alpha,11-diol and (+)-ent-7betaH-Eudesmane-4alpha,11-diol

The syntheses of (-)-7betaH-eudesmane-4alpha,11-diol (2) and (+)-ent-7betaH-eudesmane-4alpha,11-diol (ent-2) were carried out starting from (-)- and (+)-dihydrocarvones. As a result, the structure, including absolute configuration, of the naturally occurring eudesmane-4,11-diol isolated from Pluchea arguta was determined to be (+)-ent-7betaH-eudesmane-4alpha,11-diol (ent-2).

Journal Article↗
PubMed1998

Caveolae: from a morphological point of view.

Caveolae in the plasma membrane have been a focus of intensive research during the past several years. There has been confusion concerning caveolae and caveola-like membrane domains, but it is now generally thought that the latter is a region distinct from caveolae. However, due to similar buoyancy of caveolae and caveola-like membranes, whether caveolae in situ are enriched with a given molecule is often difficult to be concluded by biochemical techniques alone. Furthermore, relatively shallow caveolae may be detected by some techniques, but not by others. Thus whether a molecule is enriched in caveolae should be confirmed by methods based on different principles. Among many putative caveolar molecules, those related to Ca2+ influx and extrusion were shown to be concentrated in caveolae by both immunocytochemical and biochemical techniques. In conjunction with other characteristics, the result implies that caveolae may function as a mobile compartment for Ca2+ signalling.

Animals↗
PubMed1998

Endothelins inhibit mineralization of rat calvarial osteoblast-like cells.

We examined the effects of members of the endothelin (ET) family on mineralization of rat calvarial osteoblast-like cells. The accumulation of calcium in cells and cell layers was attenuated by ETs with the rank order of potency ET-1 = ET-2 > ET-3. We stained the mineralized nodules by von Kossa staining and measured the number and area of mineralized nodules. The inhibitory effects of ET-1 and ET-2 on the formation of mineralized nodules were stronger than those of ET-3. Our data suggest that ET-1 may inhibit the mineralization process of osteoblastic cells through the ETA receptor.

Animals↗
PubMed1998

A new mutation in the POU3F4 gene in a Japanese family with X-linked mixed deafness (DFN3).

OBJECTIVE: The molecular defect in patients with X-linked mixed deafness showing a perilymphatic gusher at stapedectomy (DFN3) has been attributed to mutations in the POU3F4 gene. This study aimed to clarify an allelic variant of this gene. STUDY DESIGN: This was a genetic study of a single Japanese family with DFN3. METHODS: Products of a polymerase chain reaction (PCR) were subjected to single-strand conformation polymorphism (SSCP) analysis. Direct sequencing of PCR products from patients and carriers showing SSCP variants was performed using the fluorescent dideoxy termination method and a sequencer. RESULTS: Sequencing of the PCR product revealed a 6-base deletion (TTCAAA) at nucleotides 601 to 606, resulting in a two-amino-acid deletion in the POU3F4 protein, (phenylalanine and lysine at amino acid residues 201 and 202). The deletion was adjacent to the site of a nonsense mutation previously described. CONCLUSION: Microdeletions at a previously undescribed location account for some clinically important POU3F4 mutations.

Asian People↗
PubMed1998

Endothelins inhibit the mineralization of osteoblastic MC3T3-E1 cells through the A-type endothelin receptor.

We examined the effects of various endothelins on the mineralization of mouse clonal preosteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed mRNAs for endothelin (ET)-1 and the A-type receptor for ET (ETA). A pharmacological study also demonstrated the predominant expression of the ETA receptor. Northern blotting analysis revealed that ETs decreased the expression of mRNA for osteocalcin, which is a marker protein for the maturation of osteoblastic cells. ET-1 also decreased in the deposition of calcium by MC3T3-E1 cells in a dose-dependent manner and it had an inhibitory effect even at 10(-11) M. The rank order of potency of ETs was ET-1 = ET-2 > ET-3. Brief treatment with 10(-7) M ET-1 on days 6-8 alone suppressed mineralization. ET-1 enhanced the rate of production of inositol 1,4, 5-trisphosphate (IP3) in MC3T3-E1 cells, but it had no effect on the rate of production of cAMP. Taken together, our data indicate that ET-1 might inhibit the mineralization of osteoblastic cells via an interaction with the ETA receptor, with generation of IP3 as the intracellular signal.

Animals↗
PubMed1998

Deceleration by angiotensin II of the differentiation and bone formation of rat calvarial osteoblastic cells.

We examined the effects of angiotensin II (Ang II) on the differentiation of rat calvarial osteoblastic cells and on the formation of bone by these cells. Northern blotting analysis revealed that Ang II inhibited the expression of mRNA for osteocalcin, which is a protein that is specifically expressed during maturation of osteoblastic cells. Ang II decreased the activity of alkaline phosphatase, a marker of osteoblastic differentiation, in the cells, acting via the type 1 (AT1) receptor. We used von Kossa staining to examine the formation of mineralized nodules by osteoblastic cells. Both the number and the total area of mineralized nodules were quantified and shown to be decreased by 10(-7) M Ang II. The accumulation of calcium in cells and the matrix layer was also decreased by Ang II. Binding analysis with subtype-specific antagonists revealed the presence of AT1 receptors for Ang II in this culture system. Ang II caused a marked increase in the rate of production of intracellular cAMP in this system. Our data suggest that Ang II might be intimately involved in osteoblastic metabolism through its interaction with the AT1 receptor.

Alkaline Phosphatase↗
PubMed1998

Langerhans cells in the human oviduct mucosa.

Langerhans cells (LCs) are the predominant antigen-presenting cells in epithelial tissues. They have been known to be present in the vagina and uterine cervix. In the present study, localization of LCs in the oviduct was investigated by electron microscopy and immunohistochemistry using an anti-CD1a (CD; cluster of differentiation) antibody. Although the cell density was variable, CD1a-positive LCs were detected in the oviduct epithelium. Their occurrence was most common in women at the age of 40 to 59. LCs extended cell processes along the base of the epithelium and were ultrastructurally characterized by rod-shaped Birbeck granules and a well-developed Golgi apparatus. LCs, together with lymphocytes and macrophages, are considered to engage in the mucosal immune system of the oviduct.

Adult↗
PubMed1997

Peculiar distribution of fodrin in fat-storing cells.

Fat-storing cells (FSCs) show unique morphology containing many lipid droplets in the cytoplasm. In this study, we found that a membrane skeletal protein, fodrin, shows peculiar distribution in FSCs of rat liver. By immunofluorescence microscopy of FSCs in culture, intense labeling for fodrin was seen as coarse filaments in the cytoplasm. Especially in FSCs isolated from vitamin A-treated rats, the labeling was often seen as many small rings in the cytoplasm. In contrast, labeling for fodrin in human fibroblasts or rat adipocytes in culture was seen diffusely in the cell cortex. Distribution of actin, tubulin, vimentin, and desmin in FSCs was also examined, but none of them appeared correlated with fodrin. By immunoelectron microscopy using nanogold labeling with silver enhancement, positive labeling for fodrin was seen around some lipid droplets in FSCs in vivo. We assume that the peculiar distribution of fodrin may be related to the morphological characteristics of FSCs.

Adipocytes↗
PubMed1997

Interrelation between nitric oxide synthase and heme oxygenase in rat endothelial cells.

The gene expression and interrelation of the constitutive type nitric oxide (NO) synthase-III as a NO-forming enzyme and heme oxygenase-2 as a carbon monoxide-forming enzyme were studied in cultured rat aortic endothelial cells. Both NO synthase-III and heme oxygenase-2 mRNAs were demonstrated in the endothelial cells by RNAase protection analysis. NO synthase-III mRNA was upregulated in the presence of the heme oxygenase inhibitor, zinc protoporphyrin IX, but not in the presence of the NO synthase inhibitor, N(G)-nitro-L-arginine. Although heme oxygenase-2 mRNA was significantly upregulated in the presence of both NO synthase inhibitor and heme oxygenase inhibitor, the increase was greater with the NO synthase inhibitor. These results provide the first evidence for the concomitant gene expression of NO synthase-III and heme oxygenase-2, and their compensatory interrelation in endothelial cells.

Animals↗
PubMed1997

The insulin-like growth factor II receptor gene is mutated in genetically unstable cancers of the endometrium, stomach, and colorectum.

Disruption of the DNA mismatch repair system, characterized by microsatellite instability (MI), plays an important role in the course of human carcinogenesis. Repetitive sequences constitute targets for mutation in MI+ cells, and frequent mutations have indeed been reported in such regions within the transforming growth factor beta receptor II (RII) gene in genetically unstable colorectal and gastric cancers. However, other genes that are targets for mutations in MI+ cells during the course of carcinogenesis have proven elusive. Because the insulin-like growth factor II receptor (IGFIIR) gene contains several repetitive sequences within its coding region, we examined mutations of this gene in MI+ cancers occurring at various primary sites. We found frameshift mutations in the poly(G)8 tract of IGFIIR in eight tumors, all of which were MI+: 4 of 26 (15%) MI+ endometrial cancers, 3 of 12 (25%) MI+ gastric cancers, and 1 of 18 (6%) MI+ colorectal cancers. In contrast, no mutation was found in 51 pancreatic cancers, 7 of which (14%) were MI+. These results implicate abnormal IGFIIR-mediated growth control in carcinogenesis involving the endometrium, stomach, and colorectum but not the pancreas.

Breast Neoplasms↗
PubMed1997

Angiotensin II stimulates the proliferation of osteoblast-rich populations of cells from rat calvariae.

We examined the effects of angiotensin II (Ang II) on the proliferation of osteoblast-rich populations of cells obtained from calvariae of newborn rat. Addition of Ang II to the culture medium caused dose-dependent increases in the rate of DNA synthesis. Such increases were completely inhibited by the addition of DuP753, an antagonist of AT1 receptor. Ang II potentiated the production of inositol 1,4,5-trisphosphate (IP3) in the culture. Ang II also stimulated the activities of mitogen-activated protein kinases (MAPKs) upon binding to the AT1 receptor. These results suggest that Ang II might be intimately involved in the proliferation of the cells in calvariae through the AT1 receptor.

Angiotensin II↗
PubMed1997

Expression profiles of transcripts from 126 open reading frames in the entire chromosome VI of Saccharomyces cerevisiae by systematic northern analyses.

Chromosome VI of Saccharomyces cerevisiae contains 126 open reading frames (ORFs), and the functions of proteins encoded by 80 ORFs are still unknown. In this report, we have systematically examined the expression profiles of all 126 ORFs on chromosome VI under five kinds of growth conditions by quantitative Northern hybridization. A series of Northern analyses and reverse transcription polymerase chain reactions have revealed that more than 64 novel ORFs are transcribed. Two ORFs (YFL059w and YFR011c) are specifically expressed in the presence of galactose. Two ORFs (YFL012w and YFR032c) are specifically transcribed in sporulation. Six ORFs (YFL049w, YFL035c, YFL010c, YFR006w, YFR010w and YFR017c) are abundantly expressed in many growth conditions.

Blotting, Northern↗
PubMed1997

Partial cloning of the hormone-binding domain of the cortisol receptor in tilapia, Oreochromis mossambicus, and changes in the mRNA level during embryonic development.

Cortisol is one of the central hormones in osmoregulation in fish, especially in seawater adaptation. A cDNA of 453 bp was cloned from liver mRNA of freshwater-reared tilapia (Oreochromis mossambicus), by reverse transcription polymerase chain reaction (RT-PCR) with primers designed for the hormone-binding domain of glucocorticoid receptors (GRs) in mammals and rainbow trout. The sequence of PCR product has 83% homology to the trout GR at the nucleotide level and 92% at the amino acid level. The PCR product of tilapia showed highest homology (74% at the amino acid level) to GR among human steroid hormone receptors, including mineralocorticoid receptor. The length of the receptor mRNA of tilapia was about 6.5 kb as determined by Northern blot hybridization. The mRNA concentration in the gills was relatively higher among various organs, the highest concentration being observed in blood cells. Signal intensity of the receptor message in the gills was stronger in fish reared in freshwater than in those reared in seawater or in concentrated (160%) seawater. During early development of tilapia, the highest concentration of receptor mRNA in the total RNA extracted from the whole egg was found just after fertilization, and its concentration decreased steadily toward hatching. The absolute amount of receptor mRNA per egg increased gradually before the initiation of cortisol production by the embryo. When embryos were transferred from fresh water to seawater 2 days before hatching, no difference was observed in the signal intensity of the receptor mRNA among embryos after 1, 2 (the day of hatching), 4, and 7 days.

Animals↗
PubMed1997

Development of striated rootlets during ciliogenesis in the human oviduct epithelium.

Striated rootlets in ciliated cells are conical banded structures composed of longitudinally aligned filaments. The formation of striated rootlets during ciliognesis in the human oviduct epithelium was studied by electron microscopy. Primitive rootlets appeared at the proximal side of basal bodies before or at the same time as ciliary budding. After the formation of several striations, the tip of the rootlets extended deeply toward the interior of the cell and became differentiated into two distinct parts, viz., the proximal conical part connected to the basal body and the distal fibrillar part. The periodicity of the striations in the fibrillar part was 68. 5+/-2.95 nm, about 5 nm longer than that of the conical part (63. 9+/-2.25 nm). The dark band in the striation was thicker in the fibrillar part than in the conical part. Since the fibrillar part was not observed in the mature cilium, this part was considered as being either degraded or changed into the conical part during ciliogenesis.

Adult↗