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T Y Chang

Publications and source records attributed to T Y Chang.

At least 91 records · Page 5Linked to original sources
PubMed1989

[Detection of heat-labile enterotoxin produced by E. coli with reversed passive latex agglutination].

From June 1987 to March 1988, 103 E. coli strains were collected from non-foodborne samples, foodborne samples, FDA (USA) etc. and analyzed for heat-labile E. coli enterotoxin (LT) by reversed passive latex agglutination (RPLA). The results showed that 84 strains were LT-positive, 19 strains were LT-negative. Forty six strains of enteropathogenic E. coli isolated from foodborne samples showed that 34 strains were LT-positive, 12 strains were negative. Four enterotoxigenic E. coli strains from FDA (USA) used as positive controls, can produce heat-labile E. coli enterotoxin and give positive results.

Bacterial Toxins↗
PubMed1988

Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A:cholesterol acyltransferase activity.

A protocol has been developed for isolating cholesterol ester-deficient cells from the Chinese hamster ovary cell clone 25-RA. This cell line previously was shown to be partially resistant to suppression of cholesterogenic enzyme activities by 25-hydroxycholesterol and to accumulate a large amount of intracellular cholesterol ester when grown in medium containing 10% fetal calf serum (Chang, T. Y., and Limanek, J. S. (1980) J. Biol. Chem. 255, 7787-7795). The higher cholesterol ester content of 25-RA is due to an increase in the rate of cholesterol biosynthesis and low density lipoprotein receptor activity compared to wild-type Chinese hamster ovary cells, and not due to an abnormal acyl-CoA:cholesterol acyltransferase enzyme. The procedure to isolate cholesterol ester-deficient mutants utilizes amphotericin B, a polyene antibiotic known to bind to cholesterol and to form pore complexes in membranes. After incubation in cholesterol-free medium plus an inhibitor of endogenous cholesterol biosynthesis, 25-RA cells were found to be 50-500 times more sensitive to amphotericin B killing than were mutant cells containing reduced amounts of cholesterol ester. Twelve amphotericin B-resistant mutants were isolated which retained the 25-hydroxycholesterol-resistant phenotype. These mutants did not exhibit the perinuclear lipid droplets characteristic of 25-RA cells, and lipid analysis revealed a large (up to 40-fold) reduction in cellular cholesterol ester. The acyl-CoA:cholesterol acyltransferase activities of these cholesterol ester-deficient mutants were markedly lower than 25-RA when assayed in intact cells or in an in vitro reconstitution assay. The tightest mutant characterized, AC29, was found to have less than 1% of the parental acyl-CoA:cholesterol acyltransferase activity. These mutants all have reduced rates of sterol synthesis and lower low density lipoprotein receptor activity compared to 25-RA, probably as a consequence of their reduced enzyme activities. Cell fusion experiments revealed that the phenotypes of all the mutants examined are not dominant and that the mutants all belong to the same complementation group. We conclude that these mutants contain a lesion in the gene encoding acyl-CoA:cholesterol acyltransferase or in a gene encoding a factor needed for enzyme production.

Amphotericin B↗
PubMed1988

A simple method for reconstitution of CHO cell and human fibroblast acyl coenzyme A: cholesterol acyltransferase activity into liposomes.

A new method for reconstituting acyl coenzyme A: cholesterol acyltransferase (ACAT) activity from either Chinese hamster ovary (CHO) or human fibroblast cell extracts into cholesterol-phosphatidylcholine liposomes is described. The method is rapid (less than 60 min) and easy to perform. The procedure involves solubilizing the cell extracts with deoxycholate followed by dilution into preformed liposomes. Ficoll gradient analysis demonstrated that, after reconstitution, almost all of the detectable ACAT activity co-migrated with the liposomes. Exogenous cholesterol in the liposomes was absolutely necessary for providing ACAT activity, but not for incorporation of the ACAT enzyme into the vesicle bilayer. Human fibroblast cell extracts prepared from cells grown in medium containing 10% fetal calf serum were found to contain a 10-fold higher microsomal ACAT activity compared to extracts from cells grown in 10% delipidated fetal calf serum. In contrast, when the ACAT activity from these extracts was measured using the reconstitution assay, there was no difference in the specific activities. These results support our previous work (Doolittle, G. M., and T. Y. Chang. 1982. Biochim. Biophys. Acta. 713: 529-537; and Chang, C. C. Y., et al. 1986. Biochemistry. 25: 1693-1699), and suggest that cholesterol regulates ACAT activity in CHO cells and human fibroblasts by mechanism(s) other than modulation of the amount of enzyme.

Acyl Coenzyme A↗
PubMed1986

Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in Chinese hamster ovary cells. 1. Effect of exogenous sterols.

Chinese hamster ovary cells grown in medium containing low-density lipoprotein (LDL) express high acyl coenzyme A:cholesterol acyltransferase (ACAT) activity as measured by an [3H]oleate pulse. Removal of LDL from the medium causes rapid inactivation of ACAT activity; the t1/2 for the initial inactivation rate is 0.8 h. Preincubation with protein synthesis inhibitors (cycloheximide or emetine) for 2 h or longer lengthens the t1/2 for the initial inactivation rate to approximately 2.1 h. When LDL is removed for more than 10 h, the cells contain only 3% of the original ACAT activity. Cycloheximide under this condition causes an 8-fold increase in ACAT activity; the increase approaches a maximum in 6-8 h. The extent of ACAT activation by cycloheximide inversely depends on exogenous sterol present in the medium; LDL diminishes the activation, while cationized LDL or 25-hydroxycholesterol completely abolishes the activation. Adding LDL back to the sterol-free medium causes a 40-70-fold increase in ACAT activity; however, the activation of LDL is not further augmented if the cells are pretreated with cycloheximide. The above observations are qualitatively confirmed by ACAT assays in vitro with cell homogenates. LDL or cycloheximide has no effect on the rates of 3H-labeled triglyceride and 3H-labeled polar lipid synthesis. Efflux of prelabeled cholesterol from cells is cycloheximide-insensitive. Rates of degradation of [3H]-leucine-pulse-labeled total protein in cells grown with or without LDL are identical. The above results imply the existence of at least one specific short-lived factor that directly or indirectly inhibits ACAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in Chinese hamster ovary cells. 2. Effect of sterol endogenously synthesized.

We reported in another paper [Chang, C. C. Y., Doolittle, G. M., & Chang, T. Y. (1986) Biochemistry (preceding paper in this issue)] that in Chinese hamster ovary (CHO) cells activation of acyl coenzyme A:cholesterol acyltransferase (ACAT) activity by treating cells with cycloheximide was abolished by providing exogenous sterol in the medium. We now report that providing 20 mM DL-mevalonate to cells grown in sterol-free medium increases the ACAT activity by approximately 6-fold and diminishes the cycloheximide activation effect. The mevalonate supplement has no significant effect on the rate of triglyceride or polar lipid synthesis, [3H]cholesterol efflux, or bulk protein degradation in cells. The activation of ACAT by mevalonate is prevented by adding a specific squalene oxide cyclase inhibitor to cells, indicating the requirement for endogenous sterol synthesis to mediate the mevalonate effect. In sterol-free medium, if sterol synthesis is blocked by specific enzyme inhibitors or through mutation, the ACAT activation by cycloheximide is again abolished. These results support the hypothesis that there may exist a short-lived factor(s) serving directly or indirectly as an endogenous ACAT inhibitor(s), the inhibitory action of this (these) factor(s) is (are) abolished, and its (their) turnover rate(s) is (are) increased by either exogenous sterol or by sterol endogenously synthesized. Conversely, removing exogenous sterol coupled with blocking endogenous sterol synthesis decreases the turnover rate(s) of the inhibitor(s), rendering its (their) action insensitive to intracellular degradation over the time period studied.

Animals↗
PubMed1986

Preparation and characterization of unilamellar vesicles from cholate-phospholipid micelle treated with cholestyramine.

Cholestyramine, a well-known bile-salt sequestrant, can be used effectively to remove cholate or deoxycholate from a solution of phosphatidylcholine-bile salt mixed micelle. Upon removal of the bile salt, unilamellar phospholipid vesicles form essentially instantaneously. Cholestyramine resin could be pelleted and removed from the vesicle solution after a low speed centrifugation. Based on phosphate analyses, the recovery of vesicles was approximately 60% of the starting material. The average diameter of these vesicles, as estimated by gel exclusion chromatography on sephacryl S-1000 beads and by trapped volume measurement using [3H]sucrose, ranged between 85 to 121 nm. Phosphatidylethanolamine, cholesterol, or n-alkane such as tetradecane can be incorporated into the vesicles without any selective loss; however, selective loss was experienced when negatively charged phospholipid species such as phosphatidylglycerol or phosphatidylserine was included in vesicle formation.

Cholestyramine Resin↗
PubMed1982

Acyl-CoA:cholesterol acyltransferase in Chinese hamster ovary cells. Enzyme activity determined after reconstitution in phospholipid/cholesterol liposomes.

Acyl-CoA:cholesterol acyltransferase from Chinese hamster ovary (CHO) cells was solubilized by deoxycholate, and then reconstituted in phosphatidylcholine/cholesterol liposomes. This reconstituted activity was totally dependent upon the cholesterol content of the mixture and showed saturation for cholesterol. Analysis of the reconstituted enzyme on linear Ficoll gradients shows that the enzyme has been incorporated into phospholipid/cholesterol liposomes. The CHO cell enzyme activity as measured by conventional assay (using cellular cholesterol as the substrate) was activated approximately 20-fold by low density lipoprotein. This activation process was independent of protein synthesis. When the above cell homogenates were assayed after optimal reconstitution, the activation produced by low density lipoprotein was essentially completely abolished. There was also no change in enzyme activity measured after reconstitution when cells were switched from sterol-containing medium to sterol-free medium, in contrast to a more than 7-fold drop in enzyme activity when assayed without reconstitution. These results suggest that the enzyme activity in intact cells is controlled by the content and composition of cellular lipids associated with the enzyme molecule. Since the intracellular messenger of low density lipoprotein is known to be cholesterol, it is likely that this enzyme activity in intact cells is primarily controlled by the cholesterol content in the vicinity of the enzyme molecule.

Acyltransferases↗
PubMed1982

Revertants of a Chinese hamster ovary cell mutant resistant to suppression by an analogue of cholesterol: isolation and partial biochemical characterization.

A highly efficient selection procedure was developed for isolating revertants of Chinese hamster ovary (CHO) cell mutants resistant to suppression by 25-hydroxy-cholesterol. The procedure is based on the fact that the specific polyene antibiotic amphotericin B caused a lethal porous complex formation with membrane cholesterol only in cholesterol-rich cells. The wild-type cells and the revertant cells switched to grow from fetal calf serum medium to delipidated fetal calf serum medium for approximately 1 day became deficient in cellular cholesterol content. These cells, unlike the cholesterol-rich mutant cells, became much less sensitive to amphotericin B cytotoxicity. The spontaneous reversion frequency of a previously reported 25-hydroxycholesterol-resistant cell clone, 25-RA [Chang, T.-Y., & Limanek, J.S. (1980) J. Biol. Chem. 255, 7787-7795], was found to be approximately 3 X 10(-6), a frequency comparable to other single gene mutations of CHO cells. Biochemical analyses of three of these revertants showed that all defects manifested in 25-RA cells reverted back in parallel, a result suggesting that these observed defects in 25-RA cells are due to a single mutation event, thus supporting the hypothesis (Chang & Limanek, 1980) that a common controlling factor may be involved in mediating the suppressive action(s) of the cholesterol analogue on various cholesterogenic enzyme activities. The function of this common controlling factor is rendered abnormal in 25-RA cells by mutation.

Amphotericin B↗
PubMed1982

Solubilization, partial purification, and reconstitution in phosphatidylcholine-cholesterol liposomes of acyl-CoA:cholesterol acyltransferase.

Acyl-CoA:cholesterol acyltransferase (ACAT) was solubilized from pig liver microsomes with a combination of 1:1% deoxycholate and 1 M potassium chloride. This solubilized activity was then reconstituted in lipid vesicles by diluting the extract into a solution of phosphatidylcholine, cholesterol, and sodium cholate, followed by dialysis. The reconstituted activity was shown to be dependent upon cholesterol in the reconstitution mixture and also shown to vary with changes in the phospholipid headgroup: phosphatidylethanolamine was most active, phosphatidylcholine was next, and phosphatidylserine or phosphatidylinositol was inhibitory. The reconstituted activity showed a migration pattern of ficoll gradients that was distinct from that of the unreconstituted enzyme and similar to that of phospholipid-cholesterol liposomes. These method provide a technique to assay the ACAT activity in defined lipid environment. The solubilized ACAT fraction was further purified by ammonium acetate fractionation and Sepharose 4B column chromatography. The entire purification procedure yielded a 150-fold increase in ACAT specific activity with 40% of the original activity recovered.

Acyltransferases↗
PubMed1982

Further characterization of a Chinese hamster ovary cell mutant defective in lanosterol demethylation.

Sensitive in vitro lanosterol 14 alpha- and 4 alpha-methylsterol oxidase assays, particularly suitable for cell extracts of tissue culture cells, were developed and validated. Using these assays, we showed that the biochemical lesion of mutant 215, a cholesterol-requiring Chinese hamster ovary cell auxotroph isolated and partially characterized previously [Chang, T. Y., Telakowski, C., Vanden Heuvel, W., Alberts, A. W., & Vagelos, P. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 832-836], was localized at the 4 alpha-methylsterol oxidase enzyme system. The defect in 4 alpha-methylsterol oxidase activity in mutant 215 cells could be demonstrated by using either 4,4-dimethylcholestanol or 4 alpha-methylcholestanol as the substrate, suggesting that the enzyme systems responsible for 4 alpha-methyl- and 4,4-dimethylsterols may share a common component. However, demethylation of the C-14 alpha methyl group was found to occur at identical rates in wild-type and mutant 215, suggesting that C-14 alpha demethylation and C-4 alpha demethylation may occur by separate enzyme systems. A [3H]dihydrolanosterol incorporation experiment in intact cells of wild-type and mutant 215 supported these conclusions. Despite these results, a [14C]acetate pulse experiment indicated that [14C]lanosterol, instead of its 14C-labeled 14-demethylated sterol derivative(s), accumulated in intact cells of mutant 215. Possible implications of these findings for the mechanisms of lanosterol demethylation reactions are discussed.

Animals↗
PubMed1981

Evidence indicating that inactivation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by low density lipoprotein or by 25-hydroxycholesterol requires mediator protein(s) with rapid turnover rate.

The half-life (t 1/2) of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of Chinese hamster ovary cells grown in fetal calf serum medium is approximately 2 h. When cells are switched to grow in delipidated serum medium (DeL-M) for more than 24 h, the t 1/2 of the enzyme is found to be drastically altered to approximately 13 h. Exposure of low density lipoprotein (LDL) (100 micrograms of protein/ml) or 25-hydroxycholesterol (1 microgram/ml) to cells grown in DeL-M suppresses reductase activity more rapidly than would be expected solely if reductase synthesis were suppressed, showing that inactivation of reductase activity by sterols, previously demonstrated using only analogs of cholesterol, is a normal mechanism for regulation of HMG-CoA reductase activity by the physiologically important sterol source (LDL). This inactivation effect by LDL or by 25-hydroxycholesterol is shown to be at least in part due to acceleration of reductase degradation rate. Furthermore, the inactivation effect by sterols is shown to be largely abolished if cycloheximide (250 micrograms/ml) is added simultaneously to the growth medium, indicating that continuous synthesis of a class of mediator protein(s) is necessary in mediating the effect of LDL or 25-hydroxycholesterol. Two different protein synthesis inhibitors (emetine and puromycin) were used and gave essentially identical results. Preincubation of cell culture with cycloheximide for 2 h essentially completely abolishes the effect of 25-hydroxycholesterol, indicating that the mediator protein(s) turns over rapidly, with t 1/2 less than 3 or 4 h.

Animals↗
PubMed1981

Evidence for coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase ad low density lipoprotein binding activity.

A Chinese hamster ovary cell mutant that requires both cholesterol and unsaturated fatty acids for growth (Limanek, J. S., Chin, J., and Chang, T. Y. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 5452-5456) has been further characterized with respect to its dependence on cholesterol. Upon removal of serum lipids from the growth medium, the activity of the important cholesterogenic enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and the low density lipoprotein (LDL) binding activity both increase significantly in the normal cell. Both these increases were much less in the mutant cell. Studies in vitro with NaF indicate that the differences in reductase activities between normal and mutant cells are not due to differences in activation by a dephosphorylation mechanism. Heat inactivation profiles and Km for HMG-CoA of both cell reductases were found to be identical, thus reducing the possibility that the mutant cell contains a mutation in the polypeptide chain of reductase. The fact that in lipid-deficient medium both reductase and LDL binding activities are low in the mutant strongly suggests that the expression of these activities is controlled in a coordinate manner. This conclusion is supported by parallel studies on a spontaneous revertant of the mutant in which the expression of reductase and LDL binding activities have both reverted to normal. These results indicate that the phenotypic abnormalities seen in the mutant are probably caused by a single mutation. A common factor is postulated to mediate this coordinate expression, and the function of such a factor is altered in the mutant cell.

Animals↗