The mouse U2af1-rs1(SP2) gene, which was cloned by a two-dimensional genome scanning method, is expressed exclusively from the paternally inherited chromosome. This gene has significant similarity to U2AF and located in chromosome 11, of which maternal duplication/paternal deficiency results in a small body. In this report, we cloned genomic U2af1-rs1(SP2) and found its promoter was methylated in a maternal-allele-specific manner. This allelic methylation was not established in parental gametes, but established between 1.5 d.p.c. and 12.5 d.p.c. on the contrary, the allele-specific expression occurred in the two-cell stage when transcription initiates. Absence of the methylation of the upstream region in this stage indicates that methylation is not necessary for inactivation of the expression.

Alleles↗

PubMed1995

Response of mucosal mast cells to intestinal ischemia-reperfusion injury in the rat.

The goals of this study were to investigate the in vivo effects of intestinal ischemia-reperfusion on mucosal mast cells, and to evaluate the morphological changes induced by standardized arterial occlusion in anesthetized rats. Complete segmental ileal ischemia was maintained for 15, 30, or 60 min, and was followed by a 30 min reperfusion period. Intestinal biopsies taken at the end of ischemia and in the 30th min of reperfusion were evaluated by image analysis, and the rate of release of type II rat mast cell protease, a marker of mast cell exocytosis, was determined from the venous effluent of the segment. Electron microscopy revealed cytoplasmic vacuolization of the mast cells of the villi after the 15 min ischemia. Ischemia induced a continuous diminution of the mucosal thickness and a significant fall in the number of mast cells in the villi; with immunoperoxidase staining with a monoclonal antibody that recognizes the AD1 mast cell surface antigen, the decrease was 57, 49, and 66% in the 15, 30, and 60 min ischemia groups, respectively. In these groups, the mucosal type II mast cell protease concentration increased to 2.4-, 2.5-, and 3.6-fold, respectively, and a significant increase in plasma protease levels was observed on reperfusion. These results lead us to conclude that mucosal mast cells are very sensitive to intestinal ischemia, with the majority of mast cells in the ileal villi already involved in the response to ischemia after a short period of arterial occlusion.

Animals↗

PubMed1995

Expression of PDGF in relation to cell division in atherosclerotic intima of human carotid arteries.

The platelet derived growth factor (PDGF) plays an important role for development of atherosclerosis. We therefore immunostained carotid atheroma specimens for PDGF. We also detected dividing cell species of the atheroma with in vitro labeling of bromodeoxyuridine (BUdR). Thirty specimens of carotid atheroma were obtained by endarterectomy and they were incubated for 3 hours with Dulbecco's modified Eagle medium/20% fetal calf serum culture medium containing BUdR/fluorodeoxyuridine (FUdR). They were ethanol-fixed, thin-sliced, and immunostained for BUdR, PDGF, smooth muscle actin and macrophage. The PDGF immunoreactivity was mainly detected in the macrophages of the subendothelial area, where BUdR-positive cells were present. Percentage of BUdR-positive cells in the atheroma specimens ranged from 3% to 15%. The BUdR-labeled small cells were mainly located in the subendothelial area, and they were identified as non-foamy macrophages by double immunostaining with anti-macrophage antibody. The results indicate that nonfoamy macrophages have potentials for cell division and they might play an important role for the development and growth of atheroma by secreting PDGF.

Aged↗

PubMed1994

Position-independent, high-level, and correct regional expression of the rat aldolase C gene in the central nervous system of transgenic mice.

Aldolase C is mainly expressed in the central nervous system (CNS). To clarify the regulatory mechanisms for the CNS-specific expression, transgenic mice were created using two constructs of the rat aldolase C gene. A fusion gene comprising the 5' regulatory region of the aldolase C gene was expressed in a CNS-specific manner. However, the expression levels and the cellular localization of the gene varied among transgenic mice. The other construct, including both 5' and 3' regulatory regions of the gene, showed position-independent and high-level expression as well as the correct regional distribution in the CNS. These results indicate that the 13-kb sequence of the rat aldolase C gene contains sufficient information for faithful expression of the gene.

Animals↗

PubMed1994

Gene expression of monocyte chemoattractant protein-1 in human monocytes is regulated by cell density through protein tyrosine kinase and protein kinase C.

The present study investigated the signal transduction pathways leading to the gene expression for monocyte chemoattractant protein-1 (MCP-1) in human monocytes. By Northern blot analysis, MCP-1 mRNA was undetectable in freshly isolated monocytes, but was induced and reached a maximal level at 4 h during culture. The level of accumulated mRNA altered with cell density of the monocytes and was highest at a density of 1 x 10(6) cells/ml. Nuclear run-on assay demonstrated that this cell density-dependent expression of MCP-1 mRNA was regulated at the transcriptional level, and protein tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, completely abrogated this gene transcription. Immunoblot analysis for phosphotyrosine in whole cell lysates demonstrated gradual increases in tyrosine phosphorylation of 55-, 60-, and 70-kDa proteins during culture. Cell density regulated tyrosine phosphorylation of 70-kDa protein in parallel with alterations in MCP-1 mRNA expression. The protein kinase C (PKC) inhibitor H-7 also abrogated the gene transcription and suppressed tyrosine phosphorylation of 70-kDa protein, whereas HA1004, a structural analogue of H-7, did not. These results suggest that MCP-1 gene expression in cultured monocytes is regulated by the cell density at the transcriptional level and that the signaling pathways leading to the gene transcription are mediated through PTK and PKC. It is also suggested that PKC activity plays a critical role in tyrosine phosphorylation of 70-kDa protein, which may mediate signals regulating the cell density-dependent expression of the MCP-1 gene.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1994

N2O emissions at solid waste disposal sites in Osaka City.

Nitrous oxide (N2O) is a trace gas contributing to stratospheric ozone depletion and global warming. Although a large quantity of information exists about N2O emissions from various ecosystems, this study was initiated to demonstrate the features of N2O emissions from sea-based waste disposal sites in Osaka City in relation to CH4 emissions. Average N2O emissions at an active landfill (S-Site) were several times higher than those at a closed landfill (N-Site). Average CH4 emissions were also much greater at the S-Site. Regarding the nature of N2O emissions, remarkable emissions often were observed with aerobic waste layers at the N-Site, suggesting almost inversely related N2O emissions with CH4 production at the N-Site. However, at the S-Site a few exceptionally high N2O emissions were noted in cases of high CH4 emissions.

Air Pollutants↗

PubMed1994

Mechanisms of cerebral artery thrombosis: a histopathological analysis on eight necropsy cases.

The mechanisms of thrombus formation in atherosclerotic cerebral arteries are still controversial, although intraplaque haemorrhage and rupture of the atheromatous plaques have been proposed. A histopathological analysis of segments of the thrombosed large intracranial arteries was carried out on eight patients who died within 28 days after brain infarction. The study revealed occlusive thrombi in six and mural thrombi in two, developing mostly at the site of greatest stenosis or just distal to it. The histological characteristics of the thrombosed arteries were plaque rupture in three, intramural haemorrhage in one, ulceration in one, and thrombosis in the absence of plaque rupture or intramural haemorrhage in three. Occlusive emboli distal to the site of cerebral artery thrombosis (intracranial artery-to-artery thromboembolism) were observed in two. The following conclusions were drawn from the study: 1) plaque rupture or intraplaque haemorrhage is not a sine qua non for cerebral artery thrombosis; 2) occlusions occur at the site of greatest luminal compromise or just distal to it, and 3) non-occlusive mural thrombosis can occur in the absence of plaque rupture and eventually lead to local occlusion or distal embolisation.

Aged↗

PubMed1994

Neointima formation after vascular stent implantation. Spatial and chronological distribution of smooth muscle cell proliferation and phenotypic modulation.

Intravascular stents have proved useful as angioplasty devices, but intimal hyperplasia after stent implantation remains an unsolved problem. In the present study, we analyzed the spatial and chronological distribution of proliferation and phenotypes of smooth muscle cells (SMCs) in rabbit aortas during the process of neointima formation after stent implantation (Gianturco's Z type) by immunohistochemistry for proliferating cell nuclear antigen (PCNA) and myosin heavy chain isoforms (SM1, SM2, and SMemb). Stent implantation induced regional injury in the arterial wall. Medial SMCs then began to proliferate adjacent to the injured SMCs, maximally on day 4 (PCNA index in the media: 3.9 +/- 3.4% [mean +/- SD]), and were modulated to the embryonic phenotype (SMemb-positive and SM2-negative). They migrated into the intima and proliferated most frequently on day 7 (PCNA index in the intima: 20.3 +/- 5.5%) and subsequently led to fibrocellular neointima formation at 2 weeks and later. At 1 month after implantation and later, SMC proliferation was rare, and the phenotype of intimal SMCs was gradually returning to the adult type (SMemb-negative and SM2-positive). Thus, this stent implantation model demonstrates that the regional effect on arterial wall by stenting leads to neointima formation through transient and regional proliferation and migration of SMCs and their phenotypic modulations.

Animals↗

PubMed1994

Atheromatous embolism in the brain: a clinicopathologic analysis of 15 autopsy cases.

We report 15 autopsy cases with cerebral atheromatous embolism (14 men and one woman, 57 to 76 years of age) and analyze their pathologic features. Cardiovascular surgery or catheterization triggered the atheromatous embolism in the brain in six cases (aortocoronary bypass, two; emergency aortocoronary bypass after percutaneous transluminal coronary angioplasty, one; graft implantation for thoracic aortic aneurysm, two; coronary angiography, one). The events that had triggered embolism were not clear in the remaining nine cases. Pathologic examination of the brains revealed that nine cases had single or multiple cortical hemorrhagic infarcts corresponding to the border zones between two main cerebral arterial territories. Many of the leptomeningeal arteries located in the subarachnoid spaces of cortical sulci and surfaces adjacent to the infarcts were occluded by atheromatous emboli composed mostly of cholesterol crystals. The internal diameters of the occluded arteries ranged from 50 to 300 microns. Arterial territorial infarcts were present in six cases, three of which had thromboemboli containing various amounts of cholesterol crystals occluding the major arteries or their large branches supplying the infarcted areas, which were pale in two cases and hemorrhagic in one. The other three cases had hemorrhagic infarcts in which atheromatous emboli were present only in the small leptomeningeal arteries and were composed mostly of cholesterol crystals. Atheromatous embolism in the brain frequently causes border-zone infarcts by occlusion of the terminal cortical branches, and sometimes causes arterial territorial infarcts if the emboli are associated with fibrin and sufficiently large to occlude the larger arteries.

Aged↗

PubMed1994

Bidirectional regulation of smooth muscle cell proliferation by IFN-gamma.

We studied the effects of interferon gamma (IFN-gamma), a T-cell lymphokine, on the proliferation and chemotaxis of vascular smooth muscle cells (SMC). Recombinant human IFN-gamma dose-dependently inhibited the proliferation of SMC cultured in the presence of 20% fetal calf-serum. It also inhibited PDGF-induced chemotaxis of SMC. Similar concentrations of IFN-gamma induced DNA-synthesis of SMC cultured in mitogen-depleted medium for 5 days. The inhibition and the stimulation of SMC proliferation were accompanied by concomitant decrease and increase in the number of PDGF receptors. Our study indicated that IFN-gamma is a bidirectional regulator of SMC proliferation.

Cell Division↗

PubMed1994

Angiotensin II receptor antagonist, TCV-116, prevents myocardial hypertrophy in spontaneously hypertensive rats.

Recently, it has been suggested that angiotensin II (AII) might be associated with cardiac hypertrophy and fibrosis. We investigated the preventive effect of an AII receptor antagonist, TCV-116, on the development of cardiac hypertrophy and fibrosis in spontaneously hypertensive rats (SHR) at 24 weeks of age through histopathological study and an AII receptor assay. Treatment with TCV-116, enalapril (an angiotensin-converting enzyme inhibitor, ACEI), and hydralazine for 20 weeks lowered systolic blood pressure (SBP) significantly (-39 mmHg, -45 mmHg, and -45 mmHg, respectively). The heart weight/body weight ratio, cardiac myocyte diameter, and percent cardiac fibrosis were significantly reduced by treatment with TCV-116 and enalapril as compared with hydralazine treatment or no treatment. The AII receptor density was significantly increased by treatment with TCV-116 and enalapril as compared with hydralazine treatment or no treatment. The results of this study suggest that AII receptors are involved in the development of cardiac hypertrophy and fibrosis in SHR. It was demonstrated that the AII receptor antagonist, TCV-116, was comparable to the ACEI, enalapril, in inhibiting the progression of cardiac hypertrophy and fibrosis via the AII receptor.

Angiotensin II↗

PubMed1994

[Vaporizer malfunction--a cause of high concentration halothane vapor delivery].

Excessively high concentration of halothane vapor was delivered to the inspiratory gas mixture due to the vaporizer malfunction (Fluotec 4). After disassembling the vaporizer, corrosion was observed on the internal components. This was considered as the reason for the high halothane output. Proper regular check up, use of appropriate monitoring devices, and a preuse equipment checklist may help improve the quality of patient care and decrease the morbidity and mortality associated with anesthesia equipment. Anesthesia machines should receive regular check up at least once a year and anesthesia providers should routinely use an equipment checklist. It is also important to realize that anesthesiologist's senses of sight, hearing, touch, and smell are exceedingly reliable monitors for decreasing the incidence of anesthesia mishaps.

Anesthesia, Inhalation↗

PubMed1993

Protein kinase C-mediated inhibition of cyclin A expression in human vascular endothelial cells.

Proliferation of cultured human vascular endothelial cells may be negatively regulated by the protein kinase C (PKC) pathway, because phorbol 12-myristate, 13-acetate (PMA) inhibits DNA synthesis and cell population doubling in PKC-retaining cells, but not in cells depleted of PKC by a long-term exposure to PMA. We investigated the mechanism through which PKC arrests the cell cycle with regard to cyclin A, which has been reported to play a key role in G1/S progression activating CDK2. Cyclin A mRNA was elevated from late G1 in accordance with the protein expression, which reached the maximal level during the S phase. PMA added at late G1 potently reduced the levels of cyclin A mRNA and the protein in concentration-dependent manners parallel to its effect on the proliferation. However, it failed to inhibit the expression in PKC-depleted cells. The mRNA reduction by PMA was due to inhibition of the transcription. The PMA effects were mimicked by multiple doses of 1,2-dioctanoylglycerol. These findings suggest that PKC inhibits G1/S progression through suppression of cyclin A gene transcription in endothelial cells.

Blotting, Western↗

PubMed1993

Protein kinase C-mediated inhibition of vascular smooth muscle cell proliferation: the isoforms that may mediate G1/S inhibition.

The role of protein kinase C (PKC) in the regulation of vascular smooth muscle cell proliferation was studied using not only phorbol ester but also diacylglycerol, with regard to the molecular species of PKC. Phorbol 12,13-dibutyrate (PDBu) and 1,2-dioctanoylglycerol (DOG) both potently inhibited serum-stimulated DNA synthesis and cell population doubling. The PDBu effect on DNA synthesis was maximal when applied at late G1. Neither PDBu nor DOG inhibited DNA synthesis in cells incubated with phorbol 12-myristate 13-acetate (PMA) for 24 h, which down-regulates PKC. Moreover, long exposure to PMA shortened the G1 period and the cell population doubling time. Therefore, a PKC isoform(s) that can be activated by phorbol ester and down-regulated by long exposure to PMA should be involved in the G1/S inhibition. A PKC enzyme assay of the soluble proteins extracted from late G1 cells and fractionated by anion exchange and hydroxylapatite chromatography showed that the activity eluting with PKC-alpha predominated, whereas that eluting with PKC-zeta was detectable. The former was dependent on Ca2+ and phorbol ester but the latter was not. PKC-zeta appeared to be expressed as two subspecies of M(r) 70 and 80 kDa. In cells incubated with PMA for 24 h, the activity eluting with PKC-alpha was completely abolished, whereas the significant activity eluting with PKC-zeta (70 kDa) remained. On the other hand, a relatively low, Ca(2+)-independent activity eluted with PKC-epsilon from the particulate fraction. This was reduced by long exposure to PMA, although not completely. Therefore, PKC-alpha and -epsilon may be the most probable mediators of the G1/S inhibition.

Animals↗