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K Kariya

Publications and source records attributed to K Kariya.

16 recordsLinked to original sources
PubMed1992

Identification of a receptor binding site in the carboxyl terminus of human interleukin-6.

To identify a receptor binding site of human interleukin-6 (IL-6), we created a library of IL-6 variants with single amino acid substitutions in the last 15 residues (171-185) in the COOH terminus of IL-6. Twenty-seven IL-6 variants were tested for biological activity on a human hepatoma and a mouse hybridoma cell line. Most variants were additionally tested in a receptor binding assay using a human myeloma cell line. Several single amino acid substitutions in the COOH terminus of IL-6 were found to decrease biological activity significantly. This is especially seen in variants with amino acid substitutions that alter the postulated amphipathical alpha-helix structure between residues 178 and 183. The two highly conserved Arg residues at positions 180 and 183 seem to play a very important role in biological activity. The loss of biological activity in all inactive variants is completely paralleled by a decrease of IL-6 receptor binding, as determined by competition binding experiments. One mutant (Leu171) displayed a higher activity on human cells and a higher binding affinity to the receptor and can be considered an IL-6 agonist. It is concluded that the amphipathical alpha-helix structure in the COOH terminus of IL-6 is critical for ligand receptor interaction. Furthermore, the region between residues Ser178 and Arg183 (Ser-Leu-Arg-Ala-X-Arg) is identified as a receptor binding site in the COOH terminus of human IL-6.

Amino Acid Sequence↗
PubMed1992

Sympathetic modulation of the cardiac myocyte phenotype: studies with a cell-culture model of myocardial hypertrophy.

Myocardial hypertrophy is the common endpoint of many cardiovascular stimuli such as hypertension, myocardial infarction, valvular disease, and congestive failure. Catecholamines have long been implicated in the pathogenesis of myocardial hypertrophy, however, it is very difficult to sort out catecholamine mechanisms in vivo. We have developed a cell-culture model which excludes hemodynamic effects and allows the assignment of receptor specificity to catecholamine effects. Utilizing this system, we have shown that stimulation of the alpha 1 adrenergic receptor leads to the development of myocardial hypertrophy and results in the selective up-regulation of the fetal/neonatal mRNAs encoding skeletal alpha-actin and beta-MHC, a pattern similar to that seen with hypertrophy in-vivo. Utilizing a co-transfection assay, we have also obtained data that suggest that the beta-PKC isozyme is in a pathway regulating transcription of the beta-MHC isogene. Beta adrenergic stimulation of the cultured cardiac myocytes also results in a modest degree of hypertrophy, however, this effect may be dependent upon myocyte contractile activity and may involve, at least in part, the non-muscle cells present in the culture system.

Animals↗
PubMed1992

Increased monoamine turnover in the subfornical organ area following body fluid depletion.

To clarify whether monoaminergic inputs to the subfornical organ (SFO) area participate in fluid regulatory systems, we examined the effects of body fluid depletion on monoamine turnover in the region of the SFO using microdialysis techniques in rats. An iso-osmotic reduction of fluid volume following subcutaneous treatment with polyethylene glycol (PEG) significantly increased dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) concentrations in the SFO area. 5-hydroxytryptamine (5-HT) in the SFO area could be detected after the PEG treatment, while 5-HT was undetectable before the treatment. The data imply that both dopaminergic and serotonergic systems in the SFO area may be involved in controlling body fluid balance.

Animals↗
PubMed1992

[Effects of sodium hyaluronate on the nociceptive response of rats with experimentally induced arthritis].

Antinociceptive effects of sodium hyaluronate (Na-HA) were studied on the basis of improvement in the graded abnormal gait elicited by arthritis induced by intra-articular administration of monosodium urate crystal (MSU) to rats. One hour before MSU injection, intra-articular administration of a 1.0% solution of Na-HA with different molecular weights, ranging from 4.70 x 10(5) to 2.02 x 10(6) (HA-200), improved the score of abnormal gait in a molecular weight-dependent manner in the experimental arthritis model. Similarly, administrations of HA-200 at concentrations ranging from 0.1 to 1.0% prior to MSU treatment resulted in improvement of the score in abnormal gait in a dose-dependent manner. To elucidate the antinociceptive mechanisms of Na-HA, effects of pretreatment with Na-HA (1.0%) of different molecular weights on prostaglandin E2 (PGE2) and bradykinin (BK) releases in synovial fluid 3 hr after MSU injection were studied. Increases in PGE2 and BK concentration in the synovial fluid were depressed in a molecular weight-dependent manner by Na-HA (1.0%) pretreatment. These results indicate that Na-HA attenuates the nociceptive responses inflicted by the MSU-induced arthritis. Such an antinociceptive effect may be due to the inhibition of PGE2 and BK synthesis in the synovial joint of rats.

Analgesics↗
PubMed1992

Stereoselective conjugation of a uricosuric diuretic with glutathione by glutathione transferase 3-3.

1. The activity of glutathione (GSH) transferases in rat liver cytosol was inhibited by the (-) enantiomer of a uricosuric diuretic (6,7-dichloro-5[N,N-dimethylsulphamoyl]-2,3-dihydrobenzofuran-2-ca rboxylic acid, DBCA) in a concentration-dependent manner. Although the DBCA (+) enantiomer inhibited the activity of liver cytosol GSH transferases, it was less effective. 2. Among four purified GSH transferase isozymes obtained from rat liver cytosol, isozyme 3-3 showed stereoselective interactions with the enantiomers of DBCA. This isozyme most actively and preferentially catalysed the transfer of GSH to DBCA (-) enantiomer.

Animals↗
PubMed1992

[Bleeding time].

Bleeding time indicates the interaction of the platelets with the damaged vessel wall and the subsequent formation of the primary hemostatic plug. Bleeding time has been widely used in the diagnosis of bleeding disorders, especially thrombocytopenia, abnormalities in platelet function, vascular disorders, and von Willebrand's disease. There are a number of methods to perform the bleeding time test, but there are significant problems concerning sensitivity, specificity, and reproducibility. To study the inhibitory effects of monoclonal antibodies (anti-vWF, anti-GPIb, and anti-GPIIb/IIIa) on primary hemostasis, these antibodies were infused to normal pigs. Anti-vWF and anti-GPIb antibodies markedly prolonged the bleeding time and inhibited hemostatic plug formation. The anti-GPIIb/IIIa antibody completely inhibited ADP-and collagen-induced platelet aggregation, but no or only mild prolongation of bleeding time was observed. The quantitative bleeding time which measures both the time and the amount of blood loss is useful in the diagnosis of hemorrhagic disorders and in judging the efficacy of the treatment. It will provide important information to understand the mechanism of primary hemostasis.

Animals↗
PubMed1992

Mimicking of superoxide dismutase activity by protein-bound polysaccharide of Coriolus versicolor QUEL, and oxidative stress relief for cancer patients.

The protein-bound polysaccharide of Coriolus versicolor QUEL (PS-K) has been found to express antioxidant activity as an "ion-radical scavenger" in diamine oxidation reactions. The mode of this expression was examined to determine whether the drug functioned as a simple radical scavenger or mimicked the action of superoxide dismutase (SOD). The latter was confirmed in both enzymatic and nonenzymatic superoxide anion radical (O2-.) producing systems in vitro. The SOD mimetic activity of PS-K was demonstrated by quantitative analysis of hydrogen peroxide as the end product of O2-., its formation being assisted catalytically by SOD or PS-K. Analysis by electron spin resonance also confirmed the SOD mimetic activity of PS-K in a xanthine-xanthine oxidase reaction. Relative SOD activity with PS-K was approximately 1/8,000 in a KO2-O2-.-producing system. The SOD mimetic activity of PS-K resisted treatment by 0.7N HCl, 0.7N NaOH, boiling for 30 minutes in a double water bath, and digestion by pronase. Fractionation according to differences in molecular mass caused no significant increase in relative SOD activity within a certain range of molecular mass, indicating that there is no definite molecule expressing SOD mimetic activity. Tumor-bearing rats and human patients with digestive tract cancer who suffered from oxidative stress were relieved by a single intraperitoneal administration of PS-K or a 1-day peroral prescription.

Animals↗
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

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

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

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↗
PubMedDate not supplied

Adrenergic hormones and control of cardiac myocyte growth.

The molecular mechanisms of cardiac myocyte growth are relevant to important problems in cardiovascular disease. A cell culture model has been developed to explore the role of adrenergic hormones in cardiac myocyte growth and gene expression. Activation of a cardiac myocyte alpha 1-adrenergic receptor by catecholamines induces hypertrophic growth of neonatal rat cardiac myocytes and initiates selective increases in contractile protein gene transcription. These effects on growth and gene expression do not depend on contractile activity. The cardiac myocytes contain at least two subtypes of alpha 1-adrenergic receptors and at least three isoforms of protein kinase C (PKC). A distinct alpha 1 receptor subtype may mediate hypertrophy and gene transcription. Different isoforms of PKC are translocated to different intracellular sites on activation, and there is evidence that the beta-PKC isoform may be an element in the signal transduction pathway from an alpha 1 receptor at the surface to the cardiac myocyte nucleus. Growth regulation through a beta-adrenergic receptor can also be demonstrated in the culture model. The growth response mediated through a beta-adrenergic receptor differs in several respects from that transduced through an alpha 1-adrenergic receptor.

Catecholamines↗