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Biomedical subjects

K Kariya

Publications and source records attributed to K Kariya.

At least 91 records · Page 5Linked to original sources
PubMed1991

Expression of a constitutively activated mutant of the beta-isozyme of protein kinase C in cardiac myocytes stimulates the promoter of the beta-myosin heavy chain isogene.

Cultured neonatal rat cardiac myocytes express at least three isozymes of protein kinase C (PKC), and two PKC isozymes are translocated to different intracellular sites on activation with alpha 1-adrenergic agonists or phorbol myristate acetate. Differential intracellular localization upon activation was compatible with differential function, and we therefore asked whether PKC isozymes had distinct roles in regulating transcription of the cardiac myosin heavy chain (MHC) genes. Cardiac myocytes were transfected with chloramphenicol acetyltransferase reporter plasmids containing the promoters of the beta-MHC or alpha-MHC isogenes. An alpha 1-adrenergic agonist stimulated the beta-MHC promoter by 3-fold but had no effect on the alpha-MHC promoter. This pattern of MHC promoter regulation by an alpha 1 agonist was the same as that found previously for the endogenous MHC mRNAs in this model system. Myocytes were then co-transfected with the beta- or alpha-MHC-chloramphenicol acetyltransferase plasmids and expression plasmids encoding wild-type or constitutively activated mutants of the alpha- and beta-isozymes of PKC. Co-transfection with wild-type alpha-PKC or wild-type beta-PKC did not stimulate the beta-MHC promoter, and none of the expressed PKCs affected the alpha-MHC promoter. However, the constitutively activated mutant of beta-PKC stimulated the beta-MHC promoter by 8-fold, whereas stimulation by the activated alpha-PKC mutant was only 40% as great (3-fold). In contrast, the constitutively activated alpha-PKC and beta-PKC mutants were equally potent in stimulating a reporter plasmid containing AP-1 recognition sequences. All transfected PKCs were expressed equally in the myocytes, as judged by immunofluorescence. These data indicate that transcription of the beta-MHC isogene is stimulated preferentially by beta-PKC in cardiac myocytes and provide direct evidence for differential functions of alpa-PKC and beta-PKC in transcriptional regulation.

Animals↗
PubMed1991

Stimulation of phagocytosis in rat polymorphonuclear leukocytes by A23187 is accompanied by activation of myeloperoxidase.

The incubation of polymorphonuclear leukocytes from rats with A23187 resulted in stimulation of phagocytosis with a concomitant increase in the activity of myeloperoxidase. Activation of the enzyme by A23187 reached the maximum at 2.5 min of incubation and preceded the maximum stimulation of phagocytosis. Propylthiouracil, an inhibitor of myeloperoxidase, and Ca2+ chelators prevented activation of myeloperoxidase and phagocytosis by A23187. These results indicate that A23187 increases intracellular Ca2+ level, followed by activation of myeloperoxidase involved in stimulation of phagocytosis.

Affinity Labels↗
PubMed1991

Effects of site-specific mutations on biologic activities of recombinant human IL-6.

To examine structure-activity relationships of human IL-6, we have determined the effects of specific mutations on the biologic activity of a human rIL-6 expressed in bacteria. Three types of mutants were examined: 1) a variant that contains serines in place of the four naturally occurring cysteines; 2) a series of cysteine-containing deletion mutants, each having a single internal 20 amino acid deletion; and 3) a cysteine-free variant containing a single 20 amino acid deletion. The mutants of the second type constitute a set of nonoverlapping, adjacent deletions spanning amino acids 4 through 183 of the 184 amino acids in natural human IL-6. All of the mutants were expressed, along with the full length, cysteine-containing analogue, in Escherichia coli as fusion proteins, joined to beta-galactosidase through a collagen linker. This system allows microgram quantities of the rIL-6 variants to be partially purified from small bacterial cultures without chromatographic or refolding steps. Each of the rIL-6 variants was released from the beta-galactosidase fusion protein with collagenase, and the recovered rIL-6 was quantitated by laser densitometry of Coomassie-stained, SDS polyacrylamide gels. The sp. ac. of each of the rIL-6 variants was determined using four assays: induction of IgM secretion from an EBV transformed human B cell line, induction of fibrinogen secretion from a human hepatoma cell line, induction of fibrinogen secretion from a rat hepatoma cell line, and induction of proliferation of a murine hybridoma cell line. Replacement of cysteines with serines reduced activity relative to cysteine-containing rIL-6 to about 20% in the rat hepatoma assay and about 3% in the mouse hybridoma assay, whereas activity in both of the human cell lines was reduced to less than 0.1%. These data suggest that the murine and rat cell lines are less selective than the human cell lines in their requirements for recognition of biologically active IL-6. Each of the deletions, except that of amino acids 4 through 23, resulted in loss of activity in all four assays. These results suggest that the information necessary for activity is not contained within any one portion of the IL-6 molecule, but rather that multiple segments of the protein are required for each of the biologic activities that we tested.

Amino Acid Sequence↗
PubMed1991

Selective release of glutathione transferase subunits from primary cultured rat hepatocytes by carbon tetrachloride and deoxycholic acid.

The change in the activity of glutathione (GSH) transferases by carbon tetrachloride or deoxycholic acid, which induced hepatotoxicity, was studied using primary cultured rat hepatocytes. The activity of GSH transferases in the hepatocytes was decreased after the treatment with carbon tetrachloride or deoxycholic acid in their concentration- and incubation time-dependent manners. On the other hand, these compounds elicited the release of the activity of GSH transferases into the medium. Glycyrrhizin, an antihepatotoxic agent, inhibited the release of both aspartate transaminase (AST) and GSH transferases induced by carbon tetrachloride or deoxycholic acid. All subunits comprised of GSH transferases could not be released by these compounds. The main subunits of GSH transferases released by hepatotoxicity were identified as 3 and 4. These results indicate that hepatotoxicity is accompanied by the selective release of GSH transferase isozymes (class mu) following the loss of the enzymes activity in the cells.

Animals↗
PubMed1991

Subunit selective alteration of hepatic glutathione transferases in spontaneously hypertensive rats.

1. The activities of glutathione (GSH) transferases in male, spontaneously hypertensive rats (SHR) and stroke-prone rats (SHR-SP) were different from those of normotensive male Wistar Kyoto rats (WKY). 2. These alterations of the enzyme activities were partly due to the changes in the levels of subunits 2 and 4. 3. Subunit selective alterations were observed in pathophysiological conditions, namely spontaneous hypertension. 4. The sex-related difference of GSH transferases in these animals was also discussed.

Animals↗
PubMed1991

Blue rubber bleb nevus syndrome with disseminated intravascular coagulation and thrombocytopenia: successful treatment with high-dose intravenous gammaglobulin.

A 63-year-old woman was diagnosed as having blue rubber bleb nevus syndrome (BRBNS) with disseminated intravascular coagulation (DIC). Hematological data showed typical DIC: PT 13.2 sec, activated PTT 55.3 sec, fibrinogen 20 mg/100 ml, FDP-E 928 ng/ml, D-dimer 3,477 ng/ml, platelet count 25 x 10(3)/microliters. Although hypofibrinogenemia was successfully controlled by the continuous infusion of heparin, 10,000 units/day, thrombocytopenia has continued. Based on shortened platelet life span, high level of platelet associated IgG, and increased number of megakaryocyte in the bone marrow, the thrombocytopenia was thought to be due to antiplatelet antibody. Her platelet count returned to normal after intravenous infusion of high-dose gamma globulin (IVIg, Sandoz) at the dose of 400 mg/kg for 2-5 days, while corticosteroid, Gabexate mesilate, synthetic thrombin inhibitor MD-805, urinastatin and warfarin had no effect. Thus, DIC or thrombocytopenia may become a serious complication in some patients with BRBNS and IVIg may be useful for correcting thrombocytopenia in the patient.

Disseminated Intravascular Coagulation↗
PubMed1991

Sympathetic activity: modulator of myocardial hypertrophy.

The mechanisms regulating myocardial hypertrophy are largely unknown. Furthermore, the hypertrophic phenotype can be associated with either normal or abnormal function. To study the molecular mechanisms involved in myocardial hypertrophy, we have established a cell culture system in which stimulation of the alpha 1-adrenergic receptor leads to the development of myocardial cell hypertrophy. In addition to producing a generalized twofold increase in both cell size, total protein, and total RNA, activation of the alpha 1-receptor produces specific alterations in gene expression that are reflected by changes at both the mRNA and protein levels. In particular, alpha 1 stimulation leads to an increase in the expression of the c-myc oncogene as well as a selective increase in skeletal alpha-actin and beta-myosin heavy-chain isogene expression, isoforms normally found only in fetal/neonatal hearts. Similar changes in gene expression are seen in pressure-load hypertrophy in vivo. Skeletal alpha-actin gene expression is induced preferentially to that of the cardiac actin isogene resulting from a specific preferential increase in gene transcription. Work with subtype-specific inhibitors indicates that it is a particular alpha 1-receptor subtype that is responsible for the development of hypertrophy in culture. The finding that alpha 1 stimulation leads to an increase in protein kinase C activity is suggestive of a potential second messenger involving the phosphorylation of a transcriptional factor or factors.

Animals↗
PubMed1990

Mechanism of inactivation of myeloperoxidase by propylthiouracil.

The mechanism of inactivation of myeloperoxidase purified from rat bone marrow by propylthiouracil (PTU) was studied. PTU inhibited not only the peroxidase activity but also the chlorinating activity of myeloperoxidase in a concentration dependent manner. When myeloperoxidase was treated with PTU and hydrogen peroxide (5 microM), inactivation of the enzyme was still observed after the excess reagents were removed by a column of Sephadex G-25. The treatment of the enzyme with PTU in the absence of hydrogen peroxide caused a slight inhibition of the enzyme activity. In addition, [14C]PTU became bound to myeloperoxidase in the presence of hydrogen peroxide. Difference spectrum of myeloperoxidase incubated with the small (0.1 mM) and large (2 mM) amounts of hydrogen peroxide revealed the formation of compounds II and III, respectively. Difference spectrum of myeloperoxidase treated with PTU in the presence of a low concentration of hydrogen peroxide (5 microM) was similar to that of compound II. Therefore, these results indicate that PTU inactivates myeloperoxidase through binding to the enzyme and the conversion to a compound II-like form in the presence of hydrogen peroxide.

Animals↗
PubMed1990

Stimulation of platelet-derived growth factor-induced DNA synthesis by angiotensin II in rabbit vascular smooth muscle cells.

In cultured rabbit vascular smooth muscle cells (VSMCs), angiotensin II by itself had little mitogenic effect even in the presence of cell-free plasma-derived serum (PDS), but markedly stimulated the platelet-derived growth factor (PDGF)-induced DNA synthesis in the presence of PDS. The maximal extent of DNA synthesis induced by PDGF plus angiotensin II was about twice that induced by PDGF alone. The stimulatory effect of angiotensin II was dose-dependent with the maximal response seen at 1 microM and was inhibited by the specific angiotensin II receptor antagonist, [Sar1, Ile8]angiotensin II. In VSMCs, both PDGF and angiotensin II induced expression of the c-fos gene in dose-dependent manners. In contrast to the synergistic effect of angiotensin II and PDGF on DNA synthesis, they induced expression of the c-fos gene in an additive manner. These results suggest that angiotensin II may act as a growth regulator for VSMCs in addition to acting as a vasoconstrictor.

Angiotensin II↗
PubMed1990

Bone marrow peroxidases of spontaneously hypertensive rats.

The activity of peroxidases and the level of myeloperoxidase in the bone marrow of spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHR-SP) were determined in comparison with normotensive Wistar Kyoto rats (WKY). In the cetyltrimethylammonium bromide extract of bone marrow, the peroxidase activities using guaiacol or Kl as the electron donor of male and female WKY were different from those of SHR and SHR-SP. The peroxidase activity was also separately determined as myeloperoxidase and eosinophil peroxidase by the use of ion-exchange high pressure liquid chromatography. In males, SHR and SHR-SP contained a low activity of eosinophil peroxidase compared with WKY. Bone marrows of female SHR and SHR-SP contained a lower activity of myeloperoxidase, while SHR and SHR-SP possessed a higher activity of eosinophil peroxidase compared with WKY. No change of the level of myeloperoxidase in the bone marrow was observed among male animals. A significant decrease in the level of myeloperoxidase was observed in female SHR and SHR-SP. Therefore, these results indicate that the change in the activity of the peroxidases in the bone marrow is accompanied by the spontaneously hypertensive state.

Animals↗
PubMed1990

Trophic factors for cardiac myocytes.

During early neonatal myocardial growth, cardiac myocytes undergo a terminal differentiation stage after which cellular proliferation no longer occurs. Subsequent growth occurs by means of enlargement/hypertrophy of the existing cells. In an effort to dissect the molecular mechanisms underlying myocardial hypertrophy, a cell culture model of cardiac hypertrophy was developed in our laboratory. In this model, neonatal rat heart muscle cells respond to alpha 1-adrenergic receptor stimulation with an increase in cell size, total protein and the cellular contents of several messenger (m)RNA encoding fetal/neonatal contractile protein isoforms, and also that encoding the proto-oncogene c-myc. Similar changes in gene expression are seen in pressure-load hypertrophy in vivo. Recent observations on the production of peptide growth factors by the myocardium suggest an additional role of cell-cell interaction in cardiac growth and development. Some of the growth factors which have been found in normal myocardium include platelet-derived growth factor B chain, fibroblast growth factor (both acidic and basic), transforming growth factor beta, insulin-like growth factor-1 and insulin-like growth factor-2, and nerve growth factor. We recently identified a heparin-binding growth factor produced by cardiac non-myocytes in culture, which acts in a dose-dependent fashion to produce hypertrophy of cardiac muscle cells in culture. Preliminary work suggests that this is dissimilar from previously identified growth factors. Furthermore, the quantitative response of the cardiac myocytes to our growth factor exceeds that seen for other factors. Further work is necessary to ascertain how these factors and the alpha 1-adrenergic system interact to produce the different clinical forms of myocardial hypertrophy.

Animals↗
PubMed1990

[Bleeding time and volume in vitro by THROMBOSTAT].

We tested an in vitro system simulating bleeding time reported by Kratzer et al. Primary hemostasis was studied perfusing an artificial vessel with citrated blood under a constant pressure of 40 mmHg, measuring the blood volume perfused (bleeding volume) and the time until blood flow stopped (bleeding time). The artificial vessel consists of a glass capillary simulating arteriole and a filter covered with collagen type I to provide a surface for the adhesion of platelets. The bleeding volume (mean +/- SD microliters) was 317.7 +/- 93.8 in controls (n = 19), 487.3 +/- 242.1 in idiopathic thrombocytopenic purpura (n = 9), 666.8 +/- 224.1 in aplastic anemia and paroxysmal nocturnal hemoglobinuria (n = 4), greater than 820 in von Willebrand's disease (n = 3), 231.0 +/- 74.5 in hemophilia A (n = 3), 499.0 +/- 269.4 in liver cirrhosis (n = 6), and 457.7 +/- 229.0 in myeloproliferative disorders (n = 11). When citrated blood was applied to this system after incubation with monoclonal antibodies (MoAb) to von Willebrand factor or platelet membrane glycoprotein Ib (GPIb), bleeding volume was significantly increased while no effects were observed after incubation with MoAb to GPIIb/IIIa, factor VIII: CAg and factor XIIIa. These data suggest that in vitro model of primary hemostasis could be used for not only diagnosing bleeding disorders although 'time' is not reliable, but also investigating the mechanisms of hemostasis.

Antibodies, Monoclonal↗
PubMed1989

Two types of protein kinase C with different functions in cultured rabbit aortic smooth muscle cells.

Incubation of cultured rabbit aortic smooth muscle cells (SMC) with phorbol-12, 13-dibutyrate (PDBu) for 48 h caused the down-regulation of protein kinase C (PKC) to the level of 30-40% of that in the control cells. The proliferative and antiproliferative actions of PKC were abolished in parallel with the loss of the down-regulation-sensitive component of PKC, but the inhibitory actions in the whole blood serum (WBS)-induced phospholipase C (PLC) reactions and intracellular Ca2+ mobilization were not affected. Immunoblot analysis with specific monoclonal antibodies against three PKC isozymes (type I, II and III) revealed that only the type III isozyme was detected in rabbit aortic SMC and that this isozyme completely disappeared after the incubation with PDBu. These results indicate that the type III isozyme is responsible for the proliferative and antiproliferative actions and suggest that the unidentified isozyme(s) is involved in the inhibitory actions in the WBS-induced PLC reactions and intracellular Ca2+ mobilization in rabbit aortic SMC.

Animals↗
PubMed1989

Endothelin-induced biphasic formation of 1,2-diacylglycerol in cultured rabbit vascular smooth muscle cells--mass analysis with a radioenzymatic assay.

Mass analysis of 1,2-diacylglycerol (DG) with a radioenzymatic assay revealed that endothelin induced a biphasic formation of DG with an early transient phase peaking at 30 sec and a late sustained phase peaking at 5 min in cultured rabbit vascular smooth muscle cells (VSMCs). The amounts of DG after the 30-sec and 5-min incubation with endothelin were 0.74 +/- 0.08 (mean +/- SE) nmol and 0.87 +/- 0.10 nmol/100 nmol of lipid phosphorus, representing 2.6- and 3.1-fold increases of the resting level, respectively. The EC50 values of endothelin for the early and late phases of DG formation were about 1 nM and 40 nM, respectively. In the [3H] inositol-labeled VSMCs, endothelin induced a rapid transient formation of inositol tris- and bisphosphates which peaked at 30 sec and a sustained formation of inositol monophosphate which peaked at 5 min. The EC50 values for the formation of these inositol phosphates were the same and about 1 nM. These results suggest that the early transient phase of DG is derived from the hydrolysis of polyphosphoinositides, while a large part of the late sustained phase of DG is from the reaction(s) other than the hydrolysis of phosphoinositides but its sources remain to be clarified.

Animals↗
PubMed1989

Stimulation of phospholipase C-mediated hydrolysis of phosphoinositides by endothelin in cultured rabbit aortic smooth muscle cells.

Incubation of the [3H] inositol-labeled cultured rabbit vascular smooth muscle cells (VSMCs) with either endothelin or angiotensin II caused a rapid formation of inositol mono-, bis- and trisphosphates (IP1, IP2 and IP3, respectively). Time courses of the endothelin- and angiotensin II-induced formation of these inositol phosphates were similar. The maximal levels of IP1, IP2 and IP3 formation induced by endothelin were about 50%, 25% and 40%, respectively, of those induced by angiotensin II. The doses of endothelin necessary for the half maximal and maximal extents of the formation of IP1 were about 1 nM and 100 nM, respectively. Protein kinase C-activating 12-Q-tetradecanoylphorbol-13-acetate (TPA) inhibited the endothelin-induced formation of IP1 with the half maximal extent of inhibition seen at 3 nM. The inhibitory action of TPA was mimicked by another protein kinase C-activating phorbol ester, phorbol-12,13-dibutyrate, but not by 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme. These results indicate that endothelin causes the phospholipase C-mediated hydrolysis of phosphoinositides, though to a lesser extent than angiotensin II, in cultured VSMCs and suggest that protein kinase C modulates the signaling mechanism of endothelin to the phospholipase C.

Angiotensin II↗
PubMed1989

Antiproliferative action of cyclic GMP-elevating vasodilators in cultured rabbit aortic smooth muscle cells.

In cultured rabbit aortic smooth muscle cells (SMCs), sodium nitroprusside (SNP) (10(-7) to 10(-4) M), atrial natriuretic peptide (ANP) (10(-9) to 10(-6) M) and 8-bromo-cyclic GMP (10(-6) to 10(-3) M) inhibited the whole blood serum (WBS)-induced DNA synthesis by about 30%. The doses of SNP and ANP necessary for the inhibition of the WBS-induced DNA synthesis were similar to those necessary for the formation of cellular cyclic GMP (cGMP). These agents were effective even when added 6 h after stimulation of the cells with WBS. These results suggest that cGMP inhibits the proliferation of rabbit aortic SMCs by inhibiting the progression from the G1 into S phase of the cell cycle and raise the possibility that cGMP-elevating vasodilators may suppress the atherogenic process by inhibiting vascular SMC proliferation.

Animals↗