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Biomedical subjects

T M Chiang

Publications and source records attributed to T M Chiang.

At least 55 records · Page 3Linked to original sources
PubMed1988

Isolation and characterization of a receptor for type 1 fimbriae of Escherichia coli from guinea pig erythrocytes.

The adhesion of Escherichia coli to eukaryotic cells is mediated by proteinaceous surface appendages called fimbriae and complementary receptors on host cells. Although type 1 fimbriae, which contain a D-mannose-reactive lectin, have been well studied little is known about the binding mechanism of isolated fimbriae to individual cell receptors. This report describes the isolation and purification of a guinea pig erythrocyte receptor for type 1 fimbriae. Erythrocyte membranes were dissolved in 0.5% Triton X-100 and the receptor isolated and purified by affinity chromatography using type 1 fimbriae immobilized on Sepharose. The 65-kDa receptor, which inhibits the agglutination of guinea pig erythrocytes by type 1 fimbriated E. coli, has a pI of 8.5-8.7, and binds concanavalin A and type 1 fimbriae in a dose-dependent and saturable manner. The fimbrial binding activity of the receptor was reduced when treated with sodium metaperiodate, endoglycosidase H, trypsin, and V8 protease, suggesting the isolated receptor is a glycoprotein with N-linked carbohydrate units. Isolated type 1 fimbriae inhibited the binding of fimbriated E. coli to purified receptor in a dose- and time-related fashion. The calculated binding affinity was 6 X 10(6) M-1, a value consistent with the low binding affinity expected from previous studies of the agglutination of guinea pig erythrocytes by isolated type 1 fimbriae.

Animals↗
PubMed1988

Prostaglandins in type II collagen-induced ear diseases.

This study examines the role of prostaglandins in type II collagen-induced inner ear disease. Hartley guinea pigs were immunized with type II collagen in complete Freund's adjuvant. Auditory brain stem response was measured initially and at 2, 3, 4, and 6 months after immunization. The presence of PGE2 and 6-keto-PGF1 alpha in the perilymph was determined by radioimmunoassay. Perilymph from immunized animals contained about five times more PGE2 and about three times more 6-keto-PGF1 alpha than the perilymph from control animals. However, the two groups showed no difference in the level of either PGE2 or 6-keto-PGF1 alpha in cerebrospinal fluid and serum. Immunized guinea pigs also showed evidence of hearing loss. The temporal bones showed the following changes: spiral ganglion degeneration; mild to moderate degeneration in the organ of Corti; and infrequent and very mild endolymphatic hydrops. Thus, prostaglandins might play an important role in the pathogenesis of type II collagen-induced inner ear disease.

Animals↗
PubMed1987

Platelet-collagen interaction. Inhibition by a monoclonal antibody raised against collagen receptor.

Monoclonal antibodies to the purified platelet type I collagen receptor were produced to study platelet receptor function. The antibody specifically reacted with the platelet receptor in immunoblot experiments. The IgG purified from the monoclonal antibodies and isolated Fab' fragments inhibited the binding of radiolabeled alpha 1(I) chain to washed platelets competitively. Soluble and fibrillar type I collagen-induced platelet aggregations were inhibited by purified IgG suggesting that soluble and fibrillar collagens shared a common receptor. The adhesion of platelets to an artificial collagen matrix was also inhibited by the monoclonal antibody. However, adenosine diphosphate-induced platelet aggregation was not inhibited by the same amount of IgG that inhibited collagen-induced platelet aggregation. The results suggest that collagen-induced platelet aggregation is mediated through the interaction of collagen with the platelet receptor.

Adenosine Diphosphate↗
PubMed1987

Identification of a 42K phosphoprotein of platelets modulated by collagen: the alpha-subunit of pyruvate dehydrogenase.

Platelets exposed to collagen sufficient to stimulate the release reaction show an increase in labeling of two intracellular proteins with molecular weights of 20,000 and 42,000. The 20,000 Mr protein has already been identified as the light chain of myosin whereas the identity of the 42,000 Mr protein had not been established. By use of biochemical and immunological techniques, the identify of the 42,000 Mr component of prelabeled platelets found in the 100,000g supernatant of freeze-thawed or sonicated cells appears to be one of the subunits of pyruvate dehydrogenase complex which is translocated from the mitochondria to the 100,000g supernatant during the preparative procedure. Increased phosphorylation of the 42,000 Mr protein occurred after collagen stimulation and was accompanied by diminished pyruvate dehydrogenase activity.

Adenosine Diphosphate↗
PubMed1987

Borderline immunodeficiency in male homosexuals: is life-style contributory?

Results of our study suggest that white Southern male homosexuals without clinical evidence of AIDS who patronize "gay bars" may have significant zinc deficiency and moderately depressed T-helper/T-suppressor cell ratios. No single causative factor could be identified to explain the significantly low zinc and elevated copper levels measured in whole blood, as well as the depressed OKT4/OKT8 cell ratios. Seventy-four percent of the homosexual male subjects were "recreational" drug abusers, 81% used inhalant nitrites routinely, and 41% routinely treated themselves with antibiotics. Eighty-one percent practiced active and/or passive penile-oral insertion, and 55.5% practiced both active and passive anal intercourse. Of the latter, 19% reported anal bleeding. Clinically inapparent, though statistically significant, borderline immunodeficiency and aberrant zinc and copper levels may be a consequence of multiple factors comprising the gay bar life-style.

Acquired Immunodeficiency Syndrome↗
PubMed1986

The effect of platelet surface protein phosphorylation on collagen-platelet interaction.

Phosphorylation of the surface proteins of human platelets by treatment with human plasma kinase and exogenous ATP enhances their aggregation response to collagen. This effect can be reversed by treatment of the phosphorylated platelets with acid phosphatase. Moreover, acid phosphatase treatment of normal human platelets results in a decreased aggregation response to collagen. The increased responsiveness of phosphorylated platelets to collagen is associated with a concomitant increase in Thromboxane A2 formation. These results suggest that the platelet surface protein phosphorylation/dephosphorylation plays an important role in modulating platelet function.

Acid Phosphatase↗
PubMed1986

The effect of Fe2+ on the activity of human plasma protein kinase.

A Fe2+ stimulatory protein kinase in human plasma is demonstrated. This enzyme was partial purified from the 100,000 X g supernatant of human plasma by using (NH4)2SO4 fractionation. The activity of this protein kinase existed in 30-50% fraction. The effect of various compounds on the activity of this partial purified protein kinase was studied. The Fe2+ stimulates 5 to 10-fold of the protein kinase activity. The maximum concentration of Fe2+ to stimulate the activity of this plasma protein kinase was 2 mM. Double reciprocal plot for the stimulation showed that Fe2+ increased the Vm but not the Km for ATP. Other divalent compounds such as MgCl2, Mg(Oac)2, MnCl2 and BaCl2 were less effective.

Blood Proteins↗
PubMed1986

Characterization of the major phosphoprotein and its kinase on the surface of the rat adipocyte.

Intact rat fat cells exposed to 12.5 microM [gamma-32P]ATP incorporate label into specific proteins within minutes. By solubilizing the reaction mixture with SDS which by passes the subcellular fractionation steps, the labeled proteins can be identified in autoradiographs of SDS-PAGE gels. The most prominently labeled protein has an Mr of 42,000. Localization of this component to the cell surface can be made on the basis of inhibition of phosphorylation by addition of a protein derived from the rat brain with protein kinase inhibitory property, susceptibility of the phosphorylated protein to tryptic digestion, whereas the unphosphorylated protein is unaffected by digestion with trypsin (15 min), inhibition of phosphorylation of this protein after brief exposure to melittin, and the consistent observation that more label is associated with the 42,000 Mr band in homogenates and permeabilized cells than in comparable numbers of intact cells exposed to the same amount of label. A 42,000 Mr phosphoprotein is also present in mitochondria which is most likely the alpha subunit of pyruvate dehydrogenase. To rule out the possibility that the cell surface protein might be a mitochondrial contaminant from broken cells, 32Pi-labeled and [gamma-32P]ATP-labeled cells were solubilized with Triton and chromatographed on a rabbit anti-pyruvate dehydrogenase antibody-Sepharose 4B column. A single labeled peak was detected upon elution of the bound fraction only in the 32Pi-labeled sample, and not in the [gamma-32P]ATP-labeled sample. Subcellular fractionation studies of intact cells labeled with [gamma-32P]ATP showed differences in the recovery of phosphoproteins of 42,000 Mr depending on whether a continuous sucrose gradient (27.6-54.1%, g/ml) or a discontinuous sucrose gradient (16, 35 and 48%, g/ml) was used. Phosphoproteins of 42,000 Mr were located in the mitochondrial and membrane fractions collected by discontinuous sucrose gradient separation, whereas a phosphoprotein of 42,000 Mr was found primarily in the mitochondrial fraction after continuous sucrose gradient separation. By 5'-nucleotidase activity measurements, the latter approach appears to result in the isolation of a heavy fragment of the plasma membrane with the mitochondrial light fraction which is 42,000 in Mr and labeled. Finally, comparison of the autoradiographs of two-dimensional (2D) gels (isoelectric focusing followed by 10% SDS-PAGE) show different isoelectric points for 42,000 Mr components in [gamma-32P]ATP- and 32Pi-labeled cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1985

Effect of the C1q on the soluble collagen-platelet interaction.

The mechanism by which C1q inhibited soluble collagen-induced platelet aggregation was examined. Platelet aggregation induced by soluble collagen in gel filtrated platelets was inhibited by the addition of C1q. There were no cross-reactions between C1q, purified soluble collagen receptor and both of their polyclonal antibodies in enzyme-linked immunosorbent assays. These results suggest that the effect of C1q on soluble collagen-induced platelet aggregation does not compete at the same binding site on the platelet surface.

Animals↗
PubMed1985

Identification, purification, and partial characterization of plasma protein kinases.

Two isomeric forms of protein kinases, FI and FII, were isolated from human plasma. These two isomeric enzymes were isolated to apparent homogeneity on NaDodSO4-PAGE by using (NH4)2SO4 fractionation, DEAE-cellulose, hydroxylapatite, Affi-Gel blue, and high-pressure liquid column chromatography. Polyclonal antibodies were obtained from immunized rabbits and both enzymes cross-reacted with each other. Furthermore, immunoaffinity-purified anti-FI and anti-FII antibodies inhibited the enzyme activity of both FI and FII. These enzymes are cyclic nucleotides, Ca2+, calmodulin and phosphatidylserine-independent enzymes which can phosphorylate exogenously added histone, casein, protamine, phosvitin, and platelet surface proteins. The phosphorylated proteins of intact platelets by these enzymes in the presence of exogenously added [gamma-32P]ATP ranged in apparent molecular weights from 13.5K to 200K, as estimated by their mobility during NaDodSO4-PAGE. Trypsin removed the label from the platelet surface phosphoproteins without affecting the intracellularly located phosphoproteins labeled endogenously by 32PO4-prelabeling of intact platelets. These observations raise the possibility that these enzymes could play a role in modulating the properties of platelets through phosphorylation of the platelet surface proteins.

Amino Acids↗
PubMed1984

Immunochemical studies of the purified human platelet receptor for the alpha 1(I)-chain of chick skin collagen.

The membrane receptor of human platelets that binds the alpha 1(I)-chain of chick skin collagen was purified to homogeneity by molecular sieve chromatography, affinity chromatography, and preparative gel-electrophoresis. Rabbits that were immunized with the purified receptor emulsified in complete Freund's adjuvant produced antibody, as measured by an enzyme-linked immunosorbent assay. The antiserum was shown to be highly receptor-specific by immunoprecipitation of solubilized platelet membrane protein that had been labeled with (gamma-32P)ATP and protein kinase. Platelet aggregation induced by alpha 1(I) as well as native chick skin collagen was inhibited by both the purified receptor and antibody against the receptor in a dose-dependent fashion. In contrast, neither the receptor nor the antiserum inhibited ADP-induced aggregation. These studies provide the most definitive evidence that alpha 1(I) and native collagen interact specifically with a cell surface receptor on human platelets to induce aggregation.

Animals↗
PubMed1982

Isolation and purification of collagen alpha 1(I) receptor from human platelet membrane.

We have previously demonstrated that chick-skin type I collagen and the alpha 1(I) chain mediate platelet aggregation. Aggregation was associated with specific binding of these substances by platelet membranes. We now describe the isolation and purification of the receptor from isolated human platelet membrane. The receptor can be solubilized from platelet membranes with 0.5% Triton or 0.5% sodium deoxycholate. Using the combination of gel filtration, affinity column chromatography on alpha 1-Sepharose 4B, and preparative slab gel electrophoreses, the receptor protein can be purified to a single band as judged on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its activity sulfate-polyacrylamide gel electrophoresis. Its activity was destroyed by incubation with trypsin or Pronase. The apparent molecular weight was estimated to be 65,000 by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bound [14C]glycine-labeled alpha 1(I) is displaced by unlabeled alpha 1 chain. The binding of [14C]glycine-labeled alpha 1(I) by the purified alpha 1(I) receptor can also be inhibited by the receptor isolated from type I fibrillar collage-Sepharose affinity chromatography. The data suggest that the alpha 1(I) binding site is identical with the type I fibrillar collagen binding site.

Animals↗
PubMed1982

The effect of myelin basic protein on the endogenous phosphorylation of platelets.

Bovine myelin basic protein (BP) induced a shape change and endogenous phosphorylation of a 45,000 (45K) molecular weight protein of intact human platelets. This effect occurred rapidly over an effective concentration range of 5-100 microM BP. BP peptides encompassing residues 1-42, 43-88, and 89-169 from the BP molecule of 169 residues, neither induced phosphorylation of platelets nor blocked the effect of intact BP. Subfractionation of disrupted platelets demonstrated the phosphorylated 45K protein in the 100,000 xg supernate. When isolated platelet membranes were used, no BP induced phosphorylation of a 45K protein could be detected. The amino acid composition of the purified, phosphorylated 45K protein differed from those of other known platelet proteins. BP itself was also phosphorylated by an endogenous platelet protein kinase(s) present both in the 100,000 xg supernatant and in the isolated membrane fraction of platelets. These results indicate that the normal or pathological release of BP from myelin may lead to phosphorylation of an internal protein of platelets and possibly other tissue elements with resultant metabolic and functional changes.

Amino Acids↗
PubMed1980

Effect of peptides of bovine myelin basic protein on dermal fibroblasts.

Bovine basic protein (BP) was digested with purified bovine brain cathepsin D to produce well defined BP derived peptides 1-42, 43-88, 43-169, and 89-169. BP and these BP peptides were tested for their effects on cultured human dermal fibroblasts using a concentration range of 0.01-1,000 ng/ml. No effect was found on cellular proliferation, and neither total protein nor collagen synthesis was altered in the presence of these substances. Although preparations of brain which contain a fibroblast-growth factor also contain BP peptides, these results indicate that the purified BP peptides studied have no detectable biological effect on the growth of human dermal fibroblasts.

Cells, Cultured↗
PubMed1980

Collagen-platelet interaction. Type V(A-B) collagen induced platelet aggregation.

One of the more recently discovered collagens, type V (or A-B) collagen, in its native fibillar form mediates human platelet aggregation and the release of serotonin. In agreement with a recent report, it has no detectable effect on human platelets in the soluble or amorphous form. The possibility that the observed results might be due to contaminating interstitial collagens was eliminated by taking advantage of unusual solubility properties of type V collagen. Type V collagen dissolved in 0.1M acetic acid formed native-type fibrils when dialyzed against PBS and amorphous fibrils when dislyzed against 0.05M Tris/0.13M NaCl, pH 7.4, at 4 degrees C. Interstitial collagens remained in solution under both of these conditions. In addition, type V collagen treated with sufficient, purified synovial collagenase to digest all contaminating interstitial collagen retained its platelet-aggregating properties. The purity of type V collagen was confirmed by SDS-PAGE of CNBr digests. These data indicate that the quaternary structure of type V collagen is important in its recognition by platelet membranes.

Amino Acids↗