Biomedical subjects

Ta-Yuan Chang

Publications and source records attributed to Ta-Yuan Chang.

29 records · Page 2Linked to original sources

PubMed2003

Trafficking defects in endogenously synthesized cholesterol in fibroblasts, macrophages, hepatocytes, and glial cells from Niemann-Pick type C1 mice.

Niemann-Pick type C1 disease (NPC1) is an inherited neurovisceral lipid storage disorder, hallmarked by the intracellular accumulation of unesterified cholesterol and glycolipids in endocytic organelles. Cells acquire cholesterol through exogenous uptake and endogenous biosynthesis. NPC1 participation in the trafficking of LDL-derived cholesterol has been well studied; however, its role in the trafficking of endogenously synthesized cholesterol (endoCHOL) has received much less attention. Previously, using mutant Chinese hamster ovary cells, we showed that endoCHOL moves from the endoplasmic reticulum (ER) to the plasma membrane (PM) independent of NPC1. After arriving at the PM, it moves between the PM and internal compartments. The movement of endoCHOL from internal membranes back to the PM and the ER for esterification was shown to be defective in NPC1 cells. To test the generality of these findings, we have examined the trafficking of endoCHOL in four different physiologically relevant cell types isolated from wild-type, heterozygous, and homozygous BALB/c NPC1NIH mice. The results show that all NPC1 homozygous cell types (embryonic fibroblasts, peritoneal macrophages, hepatocytes, and cerebellar glial cells) exhibit partial trafficking defects, with macrophages and glial cells most prominently affected. Our findings suggest that endoCHOL may contribute significantly to the overall cholesterol accumulation observed in selective tissues affected by Niemann-Pick type C disease.

Animals↗

PubMed2003

Transport of plasma membrane-derived cholesterol and the function of Niemann-Pick C1 Protein.

To visualize the intracellular transport of plasma membrane-derived cholesterol under physiological and pathophysiological conditions, a novel fluorescent cholesterol analog, 6-dansyl cholestanol (DChol), has been synthesized. We present several lines of evidence that DChol mimics cholesterol. The cholesterol probe could be efficiently incorporated into the plasma membrane via cyclodextrin-donor complexes. The itinerary of DChol from the plasma membrane to the cell was studied to determine its dependence on the function of Niemann-Pick C1 (NPC) protein. In all cells, DChol moved from the plasma membrane to the endoplasmic reticulum. Its further transport to the Golgi complex was observed but with marked differences among various cell lines. DChol was finally transported to small (approximately 0.5 microm diameter) lipid droplets, a process that required functional acyl-CoA:cholesterol acyltransferase. In human NPC fibroblasts, NPC-like cells, or in cells mimicking the NPC phenotype, DChol was found in enlarged (>1 microm diameter) droplets. When the NPC-phenotype was corrected by transfection with NPC1, DChol was again found in small-sized droplets. Our data show that NPC1 has an essential role in the distribution of plasma membrane-derived cholesterol by maintaining the small size of cholesterol-containing lipid droplets in the cell.

Amides↗

PubMed2003

Biotinylated theta-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells.

BCtheta is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (theta-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCtheta can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCtheta signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCtheta. Under this condition, BCtheta mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCtheta staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to beta-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCtheta staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin. Thus, BCtheta is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.

Animals↗

PubMed2003

Cholesterol is superior to 7-ketocholesterol or 7 alpha-hydroxycholesterol as an allosteric activator for acyl-coenzyme A:cholesterol acyltransferase 1.

We compared the abilities of cholesterol versus various oxysterols as substrate and/or as activator for the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT), by monitoring the activity of purified human ACAT1 in response to sterols solubilized in mixed micelles or in reconstituted vesicles. The results showed that 5 alpha,6 alpha-epoxycholesterol and 7 alpha-hydroxycholesterol are comparable with cholesterol as the favored substrates, whereas 7-ketocholesterol, 7 beta-hydroxycholesterol, 5 beta,6 beta-epoxycholesterol, and 24(S),25-epoxycholesterol are very poor substrates for the enzyme. We then tested the ability of 7-ketocholesterol as an activator when cholesterol was measured as the substrate, and vice versa. When cholesterol was measured as the substrate, the addition of 7-ketocholesterol could not activate the enzyme. In contrast, when 7-ketocholesterol was measured as the substrate, the addition of cholesterol significantly activated the enzyme and changed the shape of the substrate saturation curve from sigmoidal to essentially hyperbolic. Additional results show that, as an activator, cholesterol is much better than all the oxysterols tested. These results suggest that ACAT1 contains two types of sterol binding sites; the structural requirement for the ACAT activator site is more stringent than it is for the ACAT substrate site. Upon activation by cholesterol, ACAT1 becomes promiscuous toward various sterols as its substrate.

Allosteric Regulation↗

PubMed2003

A population-based study on the immediate and prolonged effects of the 1999 Taiwan earthquake on mortality.

PURPOSE: To investigate the patterns of immediate seismic deaths and post-earthquake mortality changes in the disaster area after the September 21, 1999 Taiwan earthquake. METHODS: We used the data of 1826 seismic deaths to elucidate the immediate seismic effects on mortality patterns, and to determine the association between seismic death rates and house damage among 23 townships in the disaster area. We used standardized mortality ratios (SMRs) to estimate the changes in mortality of all natural causes (ICD-9: 1-799) in the 12 months after the earthquake. RESULTS: For the 1826 seismic deaths, two leading causes of death were asphyxiation and intracranial injury and the death rates were higher among the female and elderly population. Township-specific seismic death rates were proportional to the proportion of completely collapsed houses. SMRs decreased six months after the earthquake for all residents and female adults aged 45 years and over. CONCLUSIONS: The immediate effects of the Taiwan earthquake included a higher proportion of female and elderly seismic deaths and an association between seismic death rates and earthquake damages in the disaster area. The prolonged effect of the earthquake on mortality in the post-earthquake year was a decrease in mortality for all residents except male adults.

Aged↗

PubMed2003

Promotion of tau phosphorylation by MAP kinase Erk1/2 is accompanied by reduced cholesterol level in detergent-insoluble membrane fraction in Niemann-Pick C1-deficient cells.

Niemann-Pick type C (NPC) disease is a cholesterol-storage disease accompanied by neurodegeneration with the formation of neurofibrillary tangles, the major component of which is the hyperphosphorylated tau. Here, we examined the mechanism underlying hyperphosphorylation of tau using mutant Chinese hamster ovary (CHO) cell line defective in NPC1 (CT43) as a tool. Immunoblot analysis revealed that tau was hyperphosphorylated at multiple sites in CT43 cells, but not in their parental cells (25RA) or the wild-type CHO cells. In CT43 cells, mitogen-activated protein (MAP) kinase Erk1/2 was activated and the specific MAPK inhibitor, PD98059, attenuated the hyperphosphorylation of tau. The amount of protein phosphatase 2A not bound to microtubules was decreased in CT43 cells. CT43 cells but not 25RA cells were amphotericin B-resistant, indicating that cholesterol level in the plasma membrane of CT43 is decreased. In addition, the level of cholesterol in the detergent-insoluble, low-density membrane (LDM) fraction of CT43 cells was markedly reduced compared with the other two types of CHO cells. As LDM domain plays critical role in signaling pathways, these results suggest that the reduced cholesterol level in LDM domain due to the lack of NPC1 may activate MAPK, which subsequently promotes tau phosphorylation in NPC1-deficient cells.

Animals↗

PubMed2003

Effects of occupational noise exposure on blood pressure.

We measured 24-hour ambulatory blood pressure and 16-hour noise exposure continuously for 20 automobile workers, and used linear mixed-effects regression models to estimate transient and sustained effects of noise exposure on blood pressure. The occupational noise levels of the high-exposure workers with 85 +/- 8 dBA were significantly higher than those of the low-exposure workers with 59 +/- 4 dBA (P < 0.05). We found a significant difference of 16 +/- 6 mm Hg in sleep-time systolic blood pressure (SBP) existed between 2 exposure groups, and a marginal increase of 1 mm Hg SBP per 1-dBA increase in occupational noise exposure at a 60-minute lag time during work (P = 0.07). Occupational noise exposure had both transient and sustained effects on workers' SBP.

Adult↗

PubMed2003

Acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) is induced in monocyte-derived macrophages: in vivo and in vitro studies.

To test the possibility that acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) may be expressed in human macrophages under pathologic conditions, we employed specific anti-ACAT2 antibodies and found clear ACAT2 signals in lipid-laden as well as lipid-free macrophages under various disease conditions, including atherosclerosis. However, no ACAT2 signal was detectable in macrophages under normal physiologic conditions. Using cultured human macrophages derived from blood-borne monocytes, immunoblot and RT-PCR analyses demonstrated that immature macrophages expressed only ACAT1, but the fully differentiated macrophages expressed both ACAT1 and ACAT2. Furthermore, RT-PCR clearly revealed the presence of both ACAT1 and ACAT2 mRNAs in human atherosclerotic aorta. Double immunohistochemical staining indicated that in human atherosclerotic aorta, all macrophages expressed ACAT1, while approximately 70% to 80% of macrophages also expressed ACAT2. In congenital hyperlipidemic mice, immunohistochemistry and RT-PCR demonstrated that ACAT2 was also present in lipid-laden cells of the atheromatous plaques. Our results suggest that in atherosclerotic plaque, the ability of macrophage foam cell transformation may be augmented by the dual expressions of ACAT1 and ACAT2. Additional immunoblot and RT-PCR experiments showed that the ACAT2 signal was clearly detectable in thioglycollate-elicited exudate mouse macrophages but not in peritoneal resident macrophages. We conclude that under various pathologic conditions, fully differentiated macrophages express ACAT2 in addition to ACAT1.

Animals↗

PubMed2002

Role of the N-terminal hydrophilic domain of acyl-coenzyme A:cholesterol acyltransferase 1 on the enzyme's quaternary structure and catalytic efficiency.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an enzyme involved in cellular cholesterol homeostasis and atherosclerosis. ACAT1 is an allosteric enzyme responding to its substrate cholesterol in a sigmoidal manner. It is a homotetrameric protein that spans the membrane multiple times, with its N-terminal 131 hydrophilic amino acids residing at the cytoplasmic side of the endoplasmic reticulum. This region contains two closely linked putative alpha-helices. Our current studies show that this region contains a dimer-forming motif. Adding this motif to the bacterial glutathione S-transferase (GST) converted the homodimeric GST to a tetrameric fusion protein. Conversely, deleting this motif from the full-length ACAT1 converted the enzyme from a homotetramer to a homodimer. The dimeric ACAT1 remains enzymatically active. Its biochemical characteristics, including the sigmoidal response to cholesterol, the IC(50) value toward a specific ACAT inhibitor, and sensitivity toward heat inactivation, are essentially unaltered. On the other hand, the dimeric ACAT1 exhibits a 5-10-fold increase in the V(max) of the overall reaction and a 2.2-fold increase in the K(m) for oleoyl-coenzyme. Thus, deleting the dimer-forming motif near the N-terminus changes ACAT1 from its tetrameric form to a dimeric form and increases its catalytic efficiency.

Allosteric Regulation↗

PubMed2002

Synthesis and biochemical properties of a new photoactivatable cholesterol analog 7,7-azocholestanol and its linoleate ester in Chinese hamster ovary cell lines.

We report the chemical synthesis of a new photoactivatable cholesterol analog 7,7-azocholestanol (AC) and its linoleate ester (ACL). We also examined the biochemical properties of the sterol and its ester by employing several different mutant Chinese hamster ovary (CHO) cell lines with defined abnormalities in cholesterol metabolism as tools. AC mimics cholesterol in supporting the growth of a mutant cell line (M19) that requires cholesterol for growth. In normal cells, tritiated ACL present in low-density lipoprotein (LDL) was hydrolyzed and reesterified in a manner similar to tritiated cholesteryl linoleate (CL) in LDL. Also, in the mutant cell line (AC29) lacking the enzyme acyl-coenzyme A:cholesterol acyltransferase or in the mutant cell line (CT60) defective in the Niemann-Pick type C1 protein, the hydrolysis of ACL in LDL was normal, but the reesterification of the liberated AC was defective. Therefore, the metabolism of ACL in LDL is very similar to that of CL in LDL. Tritium-labeled AC delivered to intact CHO cells as a cyclodextrin complex was shown to photoaffinity label several discrete polypeptides, including caveolin-1. These results demonstrate AC as an effective reagent for studying cholesterol-protein interactions involved in intracellular cholesterol trafficking.

Animals↗

PubMed2001

Mutant acyl-coenzyme A:cholesterol acyltransferase 1 devoid of cysteine residues remains catalytically active.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis and in the early stages of atherosclerosis. ACAT1 is an integral membrane protein with multiple transmembrane domains. Human ACAT1 contains nine cysteine residues; its activity is severely inhibited by various thiol-specific modification reagents including p-chloromercuribenzene sulfonic acid, suggesting that certain cysteine residue(s) might be near or at the active site. We constructed various ACAT1 mutants that contained either single cysteine to alanine substitution at various positions, contained a reduced number of cysteines, or contained no cysteine at all. Each of these mutants retained 20% or more of the wild-type ACAT activity. Therefore, cysteine is not essential for ACAT catalysis. For the cysteine-free enzyme, its basic kinetic properties and intracellular localization in Chinese hamster ovary cells were shown to be very similar to those of the wild-type enzyme. The availability of the cysteine-free ACAT1 will facilitate future ACAT structure function studies. Additional studies show that Cys467 is one of the major target sites that leads to p-chloromercuribenzene sulfonic acid-mediated ACAT1 inactivation, suggesting that Cys467 may be near the ACAT active site(s).

Animals↗