Biomedical subjects

T Y Chang

Publications and source records attributed to T Y Chang.

At least 73 records · Page 4Linked to original sources

PubMed1995

Translocation of both lysosomal LDL-derived cholesterol and plasma membrane cholesterol to the endoplasmic reticulum for esterification may require common cellular factors involved in cholesterol egress from the acidic compartments (lysosomes/endosomes).

Using a stable cell line 25-RA derived from wild-type Chinese hamster ovary (CHO) cells as the parental cell, this laboratory previously reported the isolation and characterization of CHO cell mutants (cholesterol-trafficking or CT) defective in transporting LDL-derived cholesterol out of the acidic compartment(s) (lysosomes/endosomes) to the endoplasmic reticulum (ER) for esterification. In this report, we show that the CT mutation can be complemented by fusion with human cells; however, attempts to complement the CT defect through DNA transfection have resulted in a collection of stable cell lines designated as ST cells. Under cholesterol starvation condition, the ST cells exhibit an elevated rate of cholesterol ester biosynthesis (by 3- to 5-fold) compared to both the parental CHO cells and the CT cells. The phenotypes of the ST cells are stable. ST cells are thus new cell lines arisen from the CT cells. When the plasma membranes of the parental, CT, and ST cells are labelled with [3H]cholesterol, ST cells show rates of [3H]cholesterol esterification much higher than that observed in CT cells but lower than that observed in the parental CHO cells. This result shows that translocation of plasma membrane cholesterol to the ER for esterification is defective in the CT cells. This result also suggests that ST cells acquire increased cholesterol trafficking activity between the lysosome and the ER without mixing the plasma membrane cholesterol pool. The characteristics of CT cells and ST cells reported here suggest that translocation of both lysosomal LDL-derived cholesterol and plasma membrane cholesterol to the ER for esterification may require common cellular factors involved in cholesterol egress from the acidic compartment(s) (lysosomes/endosomes).

Animals↗

PubMed1995

Activation of acyl-coenzyme A:cholesterol acyltransferase by cholesterol or by oxysterol in a cell-free system.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the conjugation of long chain fatty acid and cholesterol to form cholesteryl esters. It is an integral membrane protein located in the endoplasmic reticulum. Experiments performed in intact mammalian cells have shown that the rate of cholesteryl ester synthesis in intact cells, as well as the ACAT activity from cell extracts, are greatly activated by the addition of low density lipoprotein (LDL) or oxygenated sterols such as 25-hydroxycholesterol to the growth medium. However, the molecular mechanism(s) by which sterol(s) stimulate the ACAT activity remains to be elucidated. Recently, our laboratory reported the expression cloning of human ACAT cDNA (Chang, C. C. Y., Huh, H. Y., Cadigan, K. M., and Chang, T. Y. 1993) J. Biol. Chem. 268, 20747-20755). In the current study, we report the expression of human ACAT cDNA in insect Sf9 cells. Uninfected Sf9 cells do not express detectable ACAT-like activity. Infecting these cells with recombinant virus containing ACAT cDNA caused these cells to express high levels of ACAT protein and high levels of ACAT activity when assayed in vitro. The catalytic properties of ACAT expressed in these cells were found to be similar to those found in human tissue culture cells. The combination of high level of ACAT protein expression and the low level of cellular cholesterol content in the infected cells have provided us a novel opportunity to establish a simple cell-free system, whereby stimulation of ACAT by sterols can be readily demonstrated. Using this system, we have shown that cholesterol itself can serve as an ACAT activator in vitro, in addition to its role as an ACAT substrate. The current work provides the experimental basis to hypothesize that, inside mammalian cells, cholesterol itself may serve as a physiological regulator of ACAT.

Amino Acid Sequence↗

PubMed1995

Assessing ozone-precursor relationships based on a smog production model and ambient data.

A semi-empirical model, Johnson's smog production model (SPM), which relates precursor emissions to ozone levels and estimates the relative effectiveness of volatile organic compounds (VOC) and NOx emission controls, has been evaluated and a modified version of SPM has been introduced. Both versions have been applied to routine data from 1989-1991 in five areas in the United States. In particular, extent parameters, which reveal the relative merit of VOC and NOx controls in reducing high ozone levels, have been calculated. Preliminary applications of SPM reveal interesting features with respect to VOC vs. NOx controls in reducing high ozone levels. For hourly data with ozone > or =0.08 ppm, distributions of extent parameters resulting from the modified SPM show the effectiveness of VOC controls at more monitoring sites than those from Johnson's SPM; however, relative features between the two versions are similar. On the other hand, for hourly data with ozone > or =0.12 ppm, the two SPM versions show very similar relative effectiveness of VOC and NOx controls with chosen values of model parameters. To improve the credibility of SPM, the range of validity of relationships between maximum smog produced or maximum ozone and NOx concentrations must be determined, and the parameters in these relationships must be better determined for typical VOC mixtures. Another essential parameter, which determines the fractional loss of NOy (NO and its oxidation products) from the gas phase must be better determined.

Air Pollutants↗

PubMed1995

Female genital tuberculosis: report of seven cases.

Seven cases of histologically proven genital tuberculosis (TB) encountered at the National Taiwan University Hospital in the past 16 years were reviewed. From a total of 54,576 gynecologic specimens submitted for pathologic examination, the occurrence of genital TB was 0.01%. Of these seven patients, one suffered from primary infertility and one from secondary infertility. The patient with primary infertility had been married for 29 years. The patient with secondary infertility underwent a tuboplasty due to bilateral hydrosalpinx. Genital TB was not diagnosed preoperatively in any of the seven cases. The preoperative diagnoses included: postmenopausal spotting, ovarian malignancy, cervicitis, ectopic pregnancy and hydrosalpinx with secondary infertility. This review suggests that genital TB is becoming rare in Taiwan. It is difficult to diagnose from clinical symptoms and is usually discovered on pathologic examination.

Adult↗

PubMed1994

Somatic cell genetic and biochemical characterization of cell lines resulting from human genomic DNA transfections of Chinese hamster ovary cell mutants defective in sterol-dependent activation of sterol synthesis and LDL receptor expression.

We have isolated several non-leaky mutant Chinese hamster ovary (CHO) cell clones (M4, M19, and M21) requiring cholesterol and unsaturated fatty acid for growth. These mutants belong to the same complementation group as the mutant M1 cells previously reported from this laboratory. M19 cells reverted to lipid prototrophy at very low frequency and were chosen as recipients to perform DNA-mediated gene-transfer experiments using total human genomic DNAs. Biochemical characterization of these transfectant clones indicated that, unlike their parental M19 cells, they were able to exhibit activation of cholesterol biosynthesis and LDL receptor expression in response to sterol removal from the growth medium. RNA blotting analysis indicated that these transfectants were able to increase HMG-CoA synthase gene transcripts in response to sterol removal. From the genomic DNAs of a representative secondary transfectant cells, we cloned a unique human DNA fragment (designated as h lambda 2) and showed that h lambda 2 closely linked with the presumptive human M1 gene.

Amphotericin B↗

PubMed1994

Somatic cell genetic analysis of two classes of CHO cell mutants expressing opposite phenotypes in sterol-dependent regulation of cholesterol metabolism.

Two different classes of hamster cell mutants (25RA cells and M1 cells) express opposite phenotypes in sterol dependent regulation. In 25RA cells, sterols added in growth medium fail to cause down-regulation of sterol synthesis rate and low density lipoprotein (LDL) receptor activity, while in M1 cells, removal of lipids from growth medium fail to cause up-regulation of sterol synthesis rate and LDL receptor activity. Cell hybridization analysis showed that the 25RA phenotype is semidominant, while the M1 phenotype is recessive. Using 25RA as the parental cells, we isolated eight independent mutant cells (DM cells) and showed that all of them belong to the same genetic complementation group as the M1 mutant, indicating that the normal (unmutated) M1 gene product(s) is required to express the 25RA phenotype. We next performed gene transfer experiments using hamster cell genomic DNAs containing the functional human M1 gene as the donor, and the double mutant cell DM7 as the recipient. The resultant transfectant cells express the 25RA cell phenotype (instead of the wild-type cell phenotype). This result, along with the results obtained from cell hybridization analysis, shows that the 25RA and M1 cell phenotypes are caused by mutations at two different genes.

Animals↗

PubMed1994

Localization of acyl coenzyme A:cholesterol acyltransferase gene to human chromosome 1q25.

Acyl coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the formation of cholesterol esters from cholesterol and long-chain fatty acyl-coenzyme A. It is believed that ACAT plays a key role in lipoprotein metabolism and atherogenesis. Recently our laboratory succeeded in molecular cloning and functional expression of human macrophage ACAT cDNA. We have now mapped the ACAT gene to chromosome 1, band q25 by using fluorescence in situ hybridization to metaphase chromosomes, and by Southern blotting analysis of human--hamster somatic cell hybrid panels.

Animals↗

PubMed1994

Using a radioactive and non-radioactive exchangeable labeling system for laboratory diagnosis.

In this communication a safe and simple molecular diagnosis strategy was described, which can be applied for the identification of pathogens or oncogenes in clinical studies. In Southern or Northern analysis, we demonstrated the feasibility to exchange labeling probes from non-radioactive to radioactive or vice versa in a single membrane simply by stripping. We chose non-radioactive labeling because it is safe and easy to operate, but when the sensitivity is insufficient, it can be replaced by radioactive labeling. Our data show that radioactive or non-radioactive labeling is exchangeable in a single membrane and the sensitivity was comparable by either method under optimal conditions. The clinical implication is that for general hybridization starts with non-radioactive labeling, radioactive labeling is not needed unless the signals cannot be detected by non-radioactive labeling. Moreover, one membrane can be labeled repeatedly by different probes.

Blotting, Northern↗

PubMed1993

Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells.

Accumulation of cholesterol esters as cytoplasmic lipid droplets within macrophages and smooth muscle cells is a characteristic feature of early lesions of atherosclerotic plaque. Intracellularly, an essential element in forming cholesterol ester from cholesterol is the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT). ACAT is a membrane protein located in the endoplasmic reticulum. The ACAT protein has never been purified to homogeneity, and no antibodies directed against ACAT have been reported. The gene(s) encoding this enzyme had not been isolated. This laboratory had previously reported the isolation of Chinese hamster ovary cells expressing human ACAT activity. From DNAs of these cells, we have cloned a 1.2-kb exonic human genomic DNA. This led to the eventual cloning of a 4-kb cDNA clone (K1) from a human macrophage cDNA library. Transfection of K1 in ACAT-deficient mutant Chinese hamster ovary cells complemented the mutant defect and resulted in the expression of human ACAT activity. K1 contained an open reading frame of 1650 bp encoding an integral membrane protein of 550 amino acids. Protein homology analysis showed that the predicted K1 protein shared homologous peptide sequences with other enzymes involved in the catalysis of acyl adenylate formation followed by acyl thioester formation and acyl transfer. These results indicate that K1 encodes a structural gene for ACAT. The cDNA reported here should facilitate future molecular studies on ACAT.

Amino Acid Sequence↗

PubMed1993

Comparison between the effects of crude saikosaponin and 16, 16-dimethyl prostaglandin E2 on tannic acid-induced gastric mucosal damage in rats.

Hung, C.R., T.S. Wu and T.Y. Chang. The comparison between the effects of ethanol-extracted saikosaponin (SS) and 16, 16-dimethyl prostaglandin E2 (dmPGE2) on gastric acid back diffusion and mucosal damage induced by 100 mg/kg of acidified tannic acid (tannic acid dissolved in 100 mm HCl + 54 mM NaCl solution) was studied in the vagotomized rat. Crude saikosaponin (500 mg/kg) given by intragastric irrigation (I.G.) produced a significant inhibition (p < 0.05), while dmPGE2 (100 micrograms/kg) provoked a significant increase (p < 0.05) in acid back diffusion induced by acid solution. When graded doses of SS (100-1000 mg/kg) were added to acidified tannic acid solution and instilled to the stomach, a dose-dependent inhibition in acidified tannic acid-induced mucosal ulceration and acid back diffusion was achieved. The decrease in the H+ concentration and the increase in the Na+ concentration in the final samples induced by acidified tannic acid were also significantly (p < 0.05) inhibited by the same doses of SS. However, neither intraduodenal (I.D.) nor intravenous (i.v.) administration of SS was effective in inhibiting these ulcerogenic parameters. When dmPGE2 (3-100 micrograms/kg) was given concomitantly with acidified tannic acid solution, the acid back diffusion as well as mucosal ulceration provoked by acidified tannic acid were not significantly improved. The volume of luminal contents but not electrolyte concentrations in the final sample was considerably increased by adding dmPGE2 to the acidified tannic acid solution. The failure of I.D. or i.v. of dmPGE2 in inhibiting tannic acid-induced acid back diffusion and mucosal ulceration was also observed in other series of experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

16,16-Dimethylprostaglandin E2↗

PubMed1993

[Clinical observation and analysis of febrile children].

Fever is an indication of acute disease, and needs special attention. To better understand the causes of fever in children, we analysed the records of 100 febrile children who were admitted to this Hospital from June 1987 to December, 1989. In these 100 cases, 7 days' fever prior to admission included 32 cases (32%); 7 to 14 days' fever prior to admission, 43 cases (43%); over 14 days' pre-admission, 25 cases (25%). The age distribution was younger than 1 month, 3 cases; 1 month to 1 year, 52 cases; 1 year to 2 1/2 years, 20 cases; 2 1/2 years to 5 years, 16 cases; 5 to 10 years 6 cases; older than 10 years included 3 cases. Post-admission diagnoses of these children showed most had an infectious disease: 86 cases; "summer fever": 5 cases; perineal abscess: 1 case; cellulitis: 1 case; leukemia, 2 cases; 5 were from other causes. Fevers were more prevalent in summer. In conclusion, the most common cause of prolonged fever in children is infectious disease 86%, with the highest incidence of age distribution under 2 1/2 years old, with 73%. The case distribution showed a prevalence in summer 43%. Almost all prognoses were good.

Age Factors↗

PubMed1991

High-efficiency stable gene transfection using chloroquine-treated Chinese hamster ovary cells.

We describe a highly efficient stable gene transfection procedure for Chinese hamster ovary (CHO) cells using a modification of the calcium phosphate-DNA coprecipitation method. We have found that treatment of CHO cells with chloroquine increases the efficiency of gene transfer by up to 20-fold (from approx. 0.01% to approx. 0.2%) when transfection is done using the pSV2-neo plasmid. The optimized transfection procedure requires that CHO cells to be incubated with calcium phosphate-DNA coprecipitate and chloroquine (100 microM) for a total of 16 h. By using high-molecular-weight human genomic DNA as a DNA source for transfection, we show that this procedure is equally efficient for stably transferring a much larger gene, such as the 49-kb human hypoxanthine phosphoribosyltransferase gene.

Animals↗

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

Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking.

This paper reports the isolation and characterization of Chinese hamster ovary cell mutants defective in low density lipoprotein (LDL)-cholesterol trafficking. The parental cell line was 25-RA, which possesses LDL receptors and various cholesterogenic enzyme activities that are partially resistant to down regulation by exogenous sterols (Chang, T. Y., and J. S. Limanek. 1980. J. Biol. Chem. 255:7787-7795). Because these cells accumulate a large amount of intracellular cholesteryl ester when grown in medium containing 10% fetal calf serum, mutagenized populations of 25-RA cells were grown in the presence of a specific inhibitor of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which depleted their cholesteryl ester stores. Without this cholesterol ester storage, 99% of 25-RA cells die after 5-d growth in cholesterol starvation medium, while the mutant cells, which accumulate free cholesterol intracellularly, survived. In two mutant clones chosen for characterization, activation of cholesteryl ester synthesis by LDL was markedly reduced in the mutant cells compared with 25-RA cells. This lack of activation of cholesterol ester synthesis in the mutant cells could not be explained by defective uptake and/or processing of LDL or by a decreased amount of ACAT, as determined by in vitro enzyme activity. Mutant cells grown in the presence of LDL contain numerous cytosolic particles that stain intensely with the fluorescent compound acridine orange, suggesting that they are acidic. The particles are also stained with filipin, a cholesterol-specific fluorescent dye. Indirect immunofluorescence with a monoclonal antibody specific for a lysosomal/endosomal fraction revealed a staining pattern that colocalized with the filipin signal. The mutant phenotype was recessive. The available evidence indicates that the mutant cells can take up and process LDL normally, but the hydrolyzed cholesterol accumulates in an acidic compartment, probably the lysosomes, where it can not be transported to its normal intracellular destinations.

Animals↗

PubMed1990

Carbamazepine drug interactions: the influence of concurrent drug therapy on serum concentrations of carbamazepine and its epoxide metabolite.

The influence of concurrent drug therapy (phenytoin, valproate, phenobarbital, primidone) on serum concentrations of total and free carbamazepine (CBZ) and its active metabolite carbamazepine-10, 11-epoxide (CBZ-E) in 135 epileptic patients was studied. Serum CBZ and CBZ-E levels were analyzed by high performance liquid chromatography, whereas serum levels of the anticonvulsants were determined by fluorescence polarization immunoassay. Ultrafiltration was used to separate the free drugs from the protein-bound drugs in serum. Patients were divided into five groups according to the medication they received. Linear regression analyses revealed that concurrent drug therapy affected the metabolic rate of CBZ and CBZ-E in various ways. In patients on CBZ monotherapy, 78.3% of CBZ and 52.4% of CBZ-E were bound to plasma proteins. The total serum CBZ and CBZ-E concentrations significantly correlated with their respective free levels in serum. Compared with the CBZ monotherapy group, patients receiving concurrent drug therapy showed higher CBZ clearance and had elevated CBZ-E/CBZ ratios. Although the decrease in the total CBZ concentration depended on the simultaneous phenytoin and valproate concentrations (p less than 0.05), the serum levels of phenobarbital and primidone appeared to have no significant influence on the CBZ concentration. Since great interindividual variations were found in the serum concentration after a given dose, routine monitoring of the CBZ and CBZ-E serum concentrations is essential in designing a safe and effective therapeutic regimen for epileptic patients, especially for those on polytherapy.

Adolescent↗

PubMed1989

Comparison of phosphatidylethanolamine and phosphatidylcholine vesicles produced by treating cholate-phospholipid micelles with cholestyramine.

We have previously reported the preparation and characterization of unilamellar phosphatidylcholine vesicles from cholate-phospholipid micelles treated with the bile-salt sequestrant cholestyramine (Ventimiglia, J.B., Levesque, M.C., and Chang, T.Y. (1986) Anal. Biochem. 157, 323-330). We now describe a slightly modified procedure for forming unilamellar vesicles consisting of phosphatidylethanolamine, and the characterization of the resultant vesicles by gel exclusion chromatography. In contrast to phosphatidylcholine vesicles, the formation of phosphatidylethanolamine vesicles is highly pH dependent; pH 9.2 is superior to pH 8.1 or pH 7.1. Via the dialysis step, the final pH of the vesicles could be altered to be at 8.1 or at 7.1, although decreasing the pH from 9.2 resulted in the loss of approx. 20% of the total lipid as large aggregates. Residual cholate was still present in the resultant vesicles after cholestyramine treatment; the low levels of cholate, removable by dialysis, was found to stabilize the phosphatidylethanolamine vesicles formed at pH 8.1. These results suggest that the majority of the amino groups of the phosphatidylethanolamine molecules should either be in the deprotonated form, or be neutralized and/or restricted by the anionic cholate monomers in order to facilitate the vesicle formation. Phosphatidylethanolamine vesicles were found to be much more permeable to small ions than phosphatidylcholine vesicles. The incorporation of phosphatidylserine, but not phosphatidylinositol, into the phosphatidylethanolamine vesicles at 10% resulted in decreased permeability of the bilayer against the cobalt ion influx, suggesting cooperative and complementary packing of phosphatidylethanolamine and phosphatidylserine molecules within the bilayer.

Cholestyramine Resin↗

PubMed1989

Isolation of Chinese hamster ovary cell lines expressing human acyl-coenzyme A/cholesterol acyltransferase activity.

We have previously reported the isolation of Chinese hamster ovary cell mutants deficient in acylcoenzyme A/cholesterol acyltransferase (ACAT) activity (Cadigan, K. M., J. G. Heider, and T. Y. Chang. 1988, J. Biol. Chem. 263:274-282). We now describe a procedure for isolating cells from these mutants that have regained the ability to synthesize cholesterol esters. The protocol uses the fluorescent stain Nile red, which is specific for neutral lipids such as cholesterol ester. After ACAT mutant populations were subjected to chemical mutagenesis or transfected with human fibroblast whole genomic DNA, two revertants and one primary transformant were isolated by virtue of their higher fluorescent intensities using flow cytofluorimetry. Both the revertants and transformant have regained large amounts of intracellular cholesterol ester and ACAT activity. However, heat inactivation experiments revealed that the enzyme activity of the transformant had heat stability properties identical to that of human fibroblasts, while the ACAT activities of the revertants were similar to that of other Chinese hamster ovary cell lines. These results suggest that the molecular lesion in the ACAT mutants resides in the structural gene for the enzyme, and the transformant has corrected this defect by acquiring and stably expressing a human gene encoding the ACAT polypeptide. Secondary transformants were isolated by transfection of ACAT mutant cells with primary transformant genomic DNA. Genomic Southern analysis of the secondary transformants using a probe specific for human DNA revealed several distinct restriction fragments common to all the transformants which most likely comprise part or all of the human ACAT gene. The cell lines described here should facilitate the cloning of the gene encoding the human ACAT enzyme.

Animals↗