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Biomedical subjects

T M Chiang

Publications and source records attributed to T M Chiang.

At least 19 recordsLinked to original sources
PubMed1992

Okadaic acid and vanadate inhibit collagen-induced platelet aggregation; the functional relation of phosphatases on platelet aggregation.

The different specific inhibitors for phosphoserine/threonine and phosphotyrosine protein phosphatases were used to study the role of these protein phosphatases in collagen-platelet interaction. The collagen-induced platelet aggregation and the release reaction as measured ATP release were inhibited in a dose-dependent fashion by the addition of okadaic acid, a specific inhibitor of phosphoserine/threonine protein phosphatase 1 and 2A. The inhibition was also observed by the addition of a phosphotyrosine protein phosphatase inhibitor, vanadate. Suboptimal concentrations of both inhibitors together also inhibited collagen-induced platelet aggregation and release reaction in a concentration-dependent fashion. These results suggest that collagen-platelet interaction is modulated by both protein phosphatases.

Adenosine Triphosphate↗
PubMed1991

Activation mechanisms of platelet-activating factor in U937 cells: possible involvement of protein kinase C.

We have previously demonstrated that platelet-activating factor (PAF) binds specifically on cell membranes isolated from U937 cells. We now describe biological evidence showing that the effect of PAF on U937 cells is a receptor-mediated event. myo-[3H]Inositol-labeled U937 cells were used to investigate the possible role of phosphoinositide metabolism in these cells after binding of PAF. Formation of inositol phosphates (IP1, IP2, and IP3) in response to PAF was increased two- to threefold more than in vehicle control in U937 cells. The effect of PAF on endogenous protein phosphorylation was also studied by using 32PO4-labeled cells. PAF stimulates the phosphorylation of a 45-kDa protein in a time-dependent and dose-related fashion. Since the phospholipase C-generated diglyceride is an important activator of protein kinase C, the phosphorylated 45-kDa protein could be the substrate of protein kinase C. In this regard, we were able to demonstrate that phorbol ester enhances the phosphorylation of the same 45-kDa protein band. In addition, sphingosine, a protein kinase C inhibitor, inhibits the phosphorylation of the same 45-kDa protein band. Down-regulation of the protein kinase C also inhibits the 45-kDa protein phosphorylation. These results suggest that protein kinase C is involved in the PAF-U937 cell interaction.

Diglycerides↗
PubMed1991

[Comparison of Bactec radiometric system and Lowenstein-Jensen medium for detection of mycobacterium tuberculosis and the susceptibility of isolates to antituberculosis drugs].

Tuberculosis is a chronic, durable but curable disease. Active cases are usually contagious and spread easily to the close intimates, especially in children causing catastrophic complications. Thereafter, early diagnosis and adequate treatment is critical for the control of the disease. Bactec 460 12B medium (Bactec system) provides a rapid, accurate and reliable method for the detection and differentiation of mycobacterium tuberculosis. It is better than the traditional method, Lowenstein-Jensen (L-J) medium. To prove this point, from Sept. 1990 to Jan. 1991, 341 specimens were sent to our laboratory for detection of mycobacterium tuberculosis. Each sample was cultured in both Bactec system and L-J medium. Meanwhile, each isolate was also cultured for the susceptibility to the antituberculosis drugs. In those 341 specimens, 45 samples were cultured as positive in either media. 43 samples were positive from Bactec system, the positive rate was 12.6% and the sensitivity rate was 95.6%. The positive cultures from L-J medium were 27 samples, the positive rate was 7.9% and the sensitivity rate was 60% (p less than 0.05). The positive culture duration in Bactec system was 5-33 days, the average was 13.05 days. But in L-J medium, it was 19-45 days and the average was 27.48 days (p less than 0.01). The susceptibility of the isolates to the common-use antituberculosis drugs was as follows: isoniazid, 47.8%; rifampin, 84.1%; ethambutol, 71%; streptomycin, 82.6%; (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Antitubercular Agents↗
PubMed1991

[Escherichia coli strains and their susceptibility to antibiotics in nosocomial infection].

Nosocomial infection may cause severe diseases and is sometimes life-threatening. Such infection may be caused by different bacteria strains, but in our pediatric ward, a large number of cases are infected by E. coli. We studied the cases hospitalized during the period from January 1986 to December 1988 in pediatric ward, sick baby room, baby room or intensive care unit. The most common microorganism of nosocomial infection was found to be Staphylococcus aureus (37 strains), followed by E. coli (32 strains). However, bacteremia, was more often caused by E. coli (13 strains) than by Staphylococcus aureus (4 strains). In patients with E. coli nosocomial infection the ratio of male to female was 2:1. Of them, 87.5% were below 1 month old, and 6.25% were from 1 month to 1 year or from 5 to 10 years of age. The antibiotics sensitivity in bacteremia strains of E. coli seemed to be the same as that in non-bacteremia strains. Both of them were less sensitive to aminoglycoside than the community-acquired strains. Their sensitivity to 3rd-generation cephalosporins was 100%.

Anti-Bacterial Agents↗
PubMed1991

[Pediatric bacteremia strains grow in blood culture media].

Bacteremia strains may influence the clinical therapy, the isolation of bacteremia strains is important for patients. In order to understand the distribution and the growth in the blood culture media of bacteremia strains, from January 1982 through December 1988 we studied the blood culture sent to us from the pediatric ward. These were three hundred and forty one bacterial strains (3.1%) isolated from patients. In the meantime we also studied the bacteremia strains growth in the blood culture media. In these positive cultures, there were 83 gram-positive, and 258 gram-negative strains. The 83 gram-positive included Staphylococcus aureus (38 strains), hemolytic streptococcus (22 strains), Enterococcus (13 strains), Streptococcus pneumoniae (8 strains) and others (2 strains). The 258 gram-negative strains included Escherichia coli (97 strains), Salmonella spp. (43 strains), Enterobacter spp. (31 strains). Klebsiella spp. (23 strains), Pseudomonas spp. (25 strains), Proteus spp. (17 strains), Citrobacter spp. (11 strains) and others (11 strains). The Growth of bacteremia strains in the blood culture medium, showed that first day isolation strains were 33.7%, second day strains 24.9%, third day were 19.9%, fourth day were 13.5%, fifth day were 3.2%, sixth day were 4.1% and seventh day were 0.6%. In conclusion, the bacteremia strains showed that the gram-negative, which were 75.7%, were more prevalent than the gram-positive which were 24.3%, the isolation of the strains was more in the first to fourth day and grew by 92%, on the fifth day it grew by 3.2% and on the seventh day by only 0.6%. On the fifth day the baby had recovered and we were able to discontinue antibiotic therapy.

Blood↗
PubMed1990

Activation of cyclic AMP phosphodiesterase by phorbol and protein kinase C pathway: differences in normal and diabetic tissue.

Diabetes mellitus is associated with high levels of adenosine 3',5'-cyclic monophosphate in tissue and plasma. Diabetes inhibits and insulin stimulates and restores low Km adenosine 3',5'-cyclic monophosphate phosphodiesterase activity. We recently reported that phorbol ester, a tumor promoting agent known to act through protein kinase C also stimulates phosphodiesterase. Here, we address the issue of whether or not the activation of phosphodiesterase by insulin and phorbol ester is different in streptozotocin diabetic adipose tissue. Rat adipose tissue was incubated with insulin, phorbol ester or other known components or effectors of the protein kinase C pathway, i.e. 1,2 dioleoyl-glycerol, 1- oleoyl, 2- acetylglycerol, Ca(++)-Ionophore A 23187, and nifedipine. After incubation, preparation and assay of adenosine 3',5'-cyclic monophosphate phosphodiesterase was made. As in previous data streptozotocin-diabetes inhibits basal phosphodiesterase by about 50% (P less than .02); insulin and phorbol ester each stimulate phosphodiesterase, in streptozotocin-diabetes less than normal (P less than .025); nifedipine inhibits phorbol stimulated phosphodiesterase in streptozotocin-diabetes but not normal (P less than .001); and nifedipine inhibits insulin stimulated phosphodiesterase in normal (84%) and diabetic (97%) (P less than .005). In normal and diabetic tissue, diacyl glycerol and oleoyl-acyl glycerol stimulate phosphodiesterase, are augmented by ionophore and inhibited by nifedipine. In addition 32P incorporation studies and measurements of tyrosine kinase activity are presented which support these differences between normal and diabetic. In summary then, these data suggest common pathways of activation for low Km adenosine 3',5'-cyclic monophosphate phosphodiesterase by insulin and phorbol ester; imply a relationship between two second messenger systems, phosphoinositides and adenosine 3',5'-cyclic monophosphate; and demonstrate a difference in activation of phosphodiesterase between normal and diabetic adipose tissue.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

Binding of lipoteichoic acid of group A streptococci to isolated human erythrocyte membranes.

The spontaneous binding of group A streptococcal lipoteichoic acid (LTA) to mammalian cell membranes was studied in isolated membranes of human erythrocytes. The binding of radiolabeled LTA to erythrocyte membranes was dependent on membrane concentration and time. Binding approached a maximum within 30 min of incubation. The bound LTA could be displaced by adding a 50-fold excess of unlabeled LTA. The displaced LTA was eluted from a column of Sepharose 6B in a position identical to that of authentic LTA, suggesting that binding did not alter the size of the molecule. A dissociation constant of 42 micrometers was calculated, and only one population of approximately 5.5 X 10(6) binding sites per erhtyrocyte membrane was detected. Since these results suggested that erythrocyte membranes possess specific binding sites for LTA, an attempt was made to localize the putative receptors to the outside or the inside surface of the erhtyrocyte membrane. Assays of the binding of LTA to resealed right-side-out and inside-out membrane ghosts demonstrated that the outside surface was able to bind over 10 times more LTA than the inside surface. These results support the concept that the membranes possess specific binding sites for LTA and inciate that these binding sites are located almost entirely on the outside surface of erythrocyte membranes.

Binding Sites↗
PubMed1978

Binding of chemotactic collagen-derived peptides to fibroblasts. The relationship to fibroblast chemotaxis.

We previously showed that collagen, alpha-chains, and collagen-derived peptide fragments induce chemotactic migration of human fibroblasts in vitro. We now describe biochemical and immunological evidence showing there are binding sites for collagen peptides on fibroblast membranes.By the use of (14)C-labeled alpha1(I) chain, binding to intact fibroblasts was demonstrated. The process was reversible, and time- and fibroblast concentration-dependent. Scatchard plot analyses of the data obtained for the binding of alpha1(I) suggested that there are congruent with 16 x 10(6) binding sites per fibroblast with an association constant of 1.1 x 10(7)/M for alpha1(I). Dissociation of the bound radioactivity and subsequent chromatographic analysis on agarose A-1.5 m revealed that the alpha1 was unaltered. The binding of (14)C-labeled alpha1 was inhibited by each of the CNBr peptides derived from alpha1 chain of chick skin collagen and CNBr peptide mixtures of various genetic types of collagen chains. Immunofluorescence studies with anti-alpha1 antibody showed that alpha1-treated fibroblasts exhibited strong immunofluorescence. The intensity of fluorescence was markedly diminished by prior absorption of the antibody with alpha1. The alpha1-treated cells stained with preimmune sera did not show significant fluorescence.Dose-response curves of fibroblast chemotaxis induced by alpha1 and the binding of alpha1 by fibroblasts correlate closely. Furthermore, the potency of alpha1-CNBr peptides as chemotactic agents correlates with their ability to inhibit the binding of labeled alpha1(I). These data suggest the hypothesis that collagenderived peptides cause fibroblast chemotactic migration by acting on fibroblast membranes.

Binding Sites↗
PubMed1977

Purification and properties of M protein extracted from group A streptococci with pepsin: covalent structure of the amino terminal region of type 24 M antigen.

M protein was extracted from type 24, group A streptococci with pepsin at pH 5.8 and was further purified by ammonium sulfate precipitation, ribonuclease digestion, ion-exchange chromatography, and isoelectric focusing. The purified pepsin extract of M (pep M) protein was shown to be free of nontype-specific immunoreactivity in (a) complement fixation tests with heterologous M antiserum, (b) skin tests in normal adult guinea pigs, and (c) passive hemagglutination tests for the presence of lipoteichoic acid sensitizing or antigenic activity. The pep M24 was highly immunogenic; two of three rabbits developed opsonic antibody titers of 1:256 and the third a titer of 1:32 6 wk after a single injection of 100-pg doses of pep M24 emulsified in complete Freund's adjuvant. The antisera lacked nontype-specific antibodies and produced single precipitin lines in agar gel diffusion tests against crude HC1 extracts of the homologous M protein. Thus, the type-specific antigenic determinant(s) of type 24 M protein appears to be separable from immunotoxic, cross-reactive antigens without loss of immunogenicity in rabbits. The mobility of pep M24 upon electrophoresis in 10 percent sodium dodecyl sulfate pelyacrylamide gel was consistent with an average mol wt of 33,500 daltons. Amino acid analysis demonstrated a predominance of alanine, followed by glutamic acid, lysine, leucine, and aspartic acid. Pep M24 contained an estimated six to seven methionine residues and approximately ten phenylalanine residues per molecule. No other aromatic amino acids were detected. Automatic Edman degradation of pep M24 yielded the sequence of the first 29 amino acids (the amino terminal amino acid being valine) of the amino terminal region of the molecule. The detection of only one new amino acid at each step of Edman degradation confirmed the homogeneity of the purified pep M24.

Amino Acid Sequence↗
PubMed1977

Interaction of lipoteichoic acid of group A streptococci with human platelets.

The interaction of group A streptococcal lipoteichoic acid (LTA) with mammalian cell membranes was studied in human platelets. The binding of LTA to platelets was platelet concentration and time dependent. Binding approached a maximum within 10 min of incubation. The bound LTA could be displaced by adding a 50-fold excess of unlabeled LTA. An association constant of 1.9 X 10(-7) M was calculated, and only one population of binding sites was detected. Immuno-ferritin labeling of LTA-treated platelets demonstrated a patchy distribution of LTA binding sites on the platelet surface. LTA inhibited collagen- and alpha1 chain-induced platelet aggregation, but not the platelet release reaction, suggesting that the LTA and collagen binding sites on human platelets are distinct. Apparently, LTA binds to platelets and interferes with collagen-induced aggregation although collagen is still able to attach to binding sites to trigger the release reaction.

Binding Sites↗
PubMed1977

Binding of collagen alpha1 chains to human platelets.

We previously reported that purified alpha1 chains of type 1 chick skin collagen induce platelet aggregation. We now describe immunological and biochemical evidence that the peptide binds to intact platelets as an early event in the induction of platelet aggregation and the release reaction. Antibody against alpha1 (I) was obtained by immunizing rabbits with complete Freund's adjuvant mixed with purified alpha1. Immunofluorescence studies showed that alpha1(I)-treated platelets exhibited strong immunofluorescence. The intensity of fluorescence was markedly decreased by the pretreatment of platelets with alpha1-CB5 and glucosylgalactosylhydroxylysine. Dose-response curves of platelet aggregation induced by alpha1 and the binding of alpha1 by washed intact platelets are correlated. The biochemical studies showed that the binding of the alpha1 chain to washed intact platelets was platelet concentration and temperature dependent, and that it reached a maximum in 10 min. The process was reversible and specific, with an association constant of 1.7 muM. The inhibitor of alpha1-induced platelet aggregation, glucosylgalactosyl hydroxylysine, inhibited the alpha1 binding. These results suggest that alpha1(I) chains bind to specific receptor site(s) on platelet membranes to trigger aggregation and the release reaction.

Binding Sites↗
PubMed1976

Binding of chick skin collagen alpha 1 chain by isolated membranes from human platelets.

Binding of a denaturated polypeptide chain derived from chick skin collagen, the alpha 1(I) chain, by isolated membranes of human platelets has been demonstrated. The process is reversible, and time- and protein concentration-dependent. The binding is specific, with an association constant of 1.88 X 10(-6) M. Prior treatment of the isolated membranes with trypsin, chymotrypsin, and pronase, resulted in significant inhibition of the 14C-labeled alpha 1 chain binding, but neuraminidase or collagenase treatment had no effect. Dissociation of the bound radioactivity and subsequent chromatographic analyses on carboxymethylcellulose and agarose A-1.5m revealed that the alpha 1 chain was unaltered. Scatchard plot analysis suggested that there are approximately 20,000 binding sites per platelet. The binding of the alpha 1 chain was inhibited by a glycopeptide derived from alpha 1, alpha 1-CB5 and by purified glucosylgalactosyl hydroxylysine, but was not affected by other cyanogen bromide peptides of alpha 1, namely alpha 1-CB3, -CB4, -CB7, and -CB8. Kinetic studies demonstrated that inhibition by the hydroxylysine glycoside is competitive. Dose-response curves of platelet aggregation induced by alpha 1 and the binding of alpha 1 by platelet membranes correlate closely. These results indicate that there are specific binding sites for collagen alpha 1 chain on platelet membranes, and that the carbohydrate moiety of the alpha 1 chain plays a role in the binding. The findings also support the hypothesis that the chick skin alpha 1 chain mediates platelet aggregation and the release reaction by acting on platelet membranes.

Animals↗
PubMed1976

Effect of cyclic 3',5'-guanosine monophosphate on human platelet function.

The relation of cyclic 3',5'-guanosine monophosphate (cGMP) to platelet function was studied by investigating the influence of this compound and its N2,O2'-dibutyryl derivative (DBcGMP) on platelet aggregation and the release reaction. Both cGMP and dibutyryl cGMP enhanced to an equal extent platelet aggregation induced by epinephrine or by chick skin collagen alpha1 chain. The platelet release reaction, as measured by the release of [14C]-labeled 5-hydroxytryptamine (serotonin) was also enhanced by the cyclic nucleotides. Both compounds were also able to partially overcome the inhibitory effect of N6,O2'-dibutyryl 3',5'-adenosine monophosphate (DBcAMP), prostaglandin E1, and theophylline. The effect of DBcGMP did not result from contamination with 5'-GMP. Butyric acid and 5'-GMP, either alone or in combination, had no detectable effect on platelet function. The in vitro effect of DBcGMP and cGMP was not dependent on preincubation of platelets with the compounds. This suggests that their effect is mediated by direct action on the platelet membranes. These data are consistent with previous observations that the platelet aggregating agents (epinephrine, ADP, collagen, and chick collagen alpha1 chains) cause an increase in the content of GMP, and support the hypothesis that platelet aggregation is favored by an increase in cGMP.

Animals↗
PubMed1975

Interaction of a chick skin collagen fragment (alpha1-CB5) with human platelets. Biochemical studies during the aggregation and release reaction.

The denatured alpha1(I) chain and the cyanogen bromide peptide, alpha1(I)-CB5, of chick skin collagen cause the release of serotonin and leakage of lactic dehydrogenase from human platelets in a manner similar to the release reaction mediated by adenosine diphosphate and native collagen. These peptides also cause a decrease in the level of adenosine 3':5'-monophosphate (cAMP) in platelets. Adenylate cyclase activity of platelets is partially inhibited by these peptides as well as by native collagen, ADP, and epinephrine, but cAMP phosphodiesterase activity is unaltered by these substances. In contrast, the level of platelet guanosine 3':5'-monophosphate (cGMP) is increased by the collagen peptides as well as the other aggregating agents. The increase is associated with increased guanylate cyclase, but normal cGMP phosphodiesterase activities of platelets. Optical rotatory and viscometric measurements of the alpha1 chains and alpha1-CB5 of chick skin in 0.01 M phosphate/0.15 M sodium chloride, pH 7.4, at various temperatures as a function of time indicate that no detectable renaturation occurs at 37 degrees for at least 30 min of observation. Molecular sieve chromatography of alpha1-CB5 in the phosphate buffer at 37 degrees shows that its elution position is identical to that performed under denaturing conditions (at 45 degrees) with no evidence of higher molecular weight aggregates, and the alpha1-CB5 glycopeptide fraction eluting from the column at the position of its monomer retains the platelet aggregating activity. Additionally, electron microscopic examination of the platelet-rich plasma that had been reacted with these peptides fail to show any ordered collagen structures. These data indicate that the denatured alpha1 chain and alpha1-CB5 glycopeptide of chick skin collagen mediate platelet aggregation through the "physiologic" release reaction in a manner similar to that induced by other aggregating agents such as ADP, epinephrine, or native collagen, and support the conclusion that the aggregating activity of the alpha1 chain and alpha1-CB5 is not likely to be due to the formation of polymerized products.

Animals↗