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Daniel Tsun-Yee Chiu

Publications and source records attributed to Daniel Tsun-Yee Chiu.

12 recordsLinked to original sources
PubMed2006

Flow cytometry for assessment of the efficacy of siRNA.

BACKGROUND: Small interfering RNA (siRNA) has emerged as a powerful tool to study the loss-of-function phenotype by specifically silencing a target gene. The success of gene silencing depends on the choice of appropriate target sequence, and requires a rapid and sensitive assay for quantifying siRNA efficiency. Conventional assays include Western blotting and reverse transcription coupled with polymerase chain reaction. However, these methods are somewhat inaccurate owing to the variation in transfection efficiency. To avoid this, we have developed a flow cytometric method to provide a quantitative analysis of siRNA efficacy. METHODS: We constructed a novel vector pHyper1G, which can express enhanced green fluorescent protein (EGFP). It contains a H1 promoter, which can drive expression of short hairpin RNA (shRNA) directed against a target gene. The target gene was cloned into pDsRed1-N1. The resulting construct can express a fusion protein between target protein and DsRed. These vectors were co-transfected into 293T cells. The transfected cells were analyzed by flow cytometry. The percentage of EGFP+, DsRed+ cells and the change in mean fluorescence intensity (MFI) of DsRed channel indicate changes in expression of target gene in a cell population, and hence the efficacy of the corresponding shRNA. In addition, the cells transfected with pHyper1G derivative were sorted, and analyzed for the activity of target gene. RESULTS: We designed an oligonucleotide duplex encoding shRNA against glucose-6-phosphate dehydrogenase (G6PD) gene, and cloned this into pHyper1G. The resulting vector pHyper1G101 effectively knocked down the expression of G6PD-DsRed from pDsRed301, as shown by significant reduction in both the percentage of EGFP+, DsRed+ cells, and MFI. Changes in these parameters were consistent with decreases in protein level and activity of G6PD. Moreover, albeit at low suboptimal transfection efficiency, cells transfected with pHyper1G101 alone were successfully sorted for those expressing shRNA, and their G6PD activity was found to be suppressed. CONCLUSION: Flow cytometric analysis provides a reliable assessment of the efficiency of siRNA in a cell population. This method can be easily automated and used for screening of appropriate shRNAs against certain genes. Moreover, using the present system, we can deliberately sort for and analyze those cells expressing shRNA. This can be applied to the hard-to-transfect cell types, and greatly facilitates the analyses of gene silencing in these cells.

Cell Line↗
PubMed2006

Green tea polyphenol epigallocatechin-3-gallate protects cells against peroxynitrite-induced cytotoxicity: modulatory effect of cellular G6PD status.

Glucose-6-phosphate dehydrogenase (G6PD) plays important roles in the maintenance of cellular redox balance. It was not until recently that the importance of G6PD in regulation of cellular growth and apoptosis emerged. In the present study, we found that G6PD-deficient fibroblasts were more susceptible to peroxynitrite-induced cytotoxicity. Treatment with peroxynitrite generator 3-morpholinosydnonimine (SIN-1) hydrochloride caused apoptosis in human fibroblast in a dose-dependent manner. This was preceded by a decrease in the intracellular level of glutathione (GSH) as well as accumulation of p53. The extent of apoptosis and glutathione depletion were greater in G6PD-deficient fibroblasts than in the normal counterpart. Pretreatment with green tea polyphenol epigallocatechin-3-gallate (EGCG) effectively blocked peroxynitrite-induced glutathione depletion, p53 accumulation, and apoptosis in both normal and G6PD-deficient cells. EGCG, administered to cells alone or as pretreatment, caused activation of Akt. The protective effect was abolished by phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin, and LY294002. Our findings suggest that G6PD deficiency enhances the toxicity of peroxynitrite and that EGCG initiates cell survival signaling via the PI3K/akt pathway.

Apoptosis↗
PubMed2006

Decreased glucose-6-phosphate-dehydrogenase (G6PD) activity and risk of senile cataract in Taiwan.

G6PD deficiency may render afflicted individuals more susceptible to certain degenerative diseases. To clarify the relationship between G6PD deficiency and cataract, blood G6PD activity was determined in patients with cataract in Taiwan. The cases and hospital-based controls were recruited from the medical outpatient department and from the physical checkup department at Chang Gung Memorial Hospital, respectively. A questionnaire survey was used to determine associations between cataracts and their risk factors. G6PD activity in fresh RBC was quantitatively measured and genomic DNA was extracted from lymphocyte nuclei. The mean blood G6PD activity among cataract patients (278.1 U/10[12] RBC) was similar to that of normals (288.0 U/10[12] RBC). No statistically significant difference in the distribution of G6PD activities as grouped by an increment of 100 U/[10, 12] RBC was observed between cataract patients and normal subjects. The predominant forms of G6PD gene mutation (cDNA 1376 G to T and 95 A to G) were both found in the patients with cataract. The adjusted odds ratio for cataract was 1.21 for every decrement of 100 U/10[12] RBC of G6PD activity in these subjects. These data indicate that G6PD activity is not a potential risk factor for senile cataract in Taiwan.

Aged↗
PubMed2005

Rapid detection of glucose-6-phosphate dehydrogenase gene mutations by denaturing high-performance liquid chromatography.

OBJECTIVES: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide. Different kinds of G6PD mutations may result in variable severity of clinical onset in G6PD-deficient individuals. In this study, a reliable molecular diagnostic method was developed for rapid detection of G6PD gene mutation. DESIGN AND METHODS: Primers were designed to amplify G6PD gene fragments that were subjected to mutation screening using denaturing high-performance liquid chromatography (DHPLC) analysis. Mutations were identified by their distinct elution peak patterns and were confirmed by DNA sequencing. The assay was further validated against 29 samples from individuals with G6PD deficiency. RESULTS: A DHPLC-based assay for G6PD mutation detection was established. The 9 common G6PD mutations in the Taiwanese and Chinese population could be distinguished through the analysis of DNA elution patterns. During the validation test with the 29 G6PD deficiency specimens, two additional rare mutations, T517C and C519G, were unveiled. Overall, the DHPLC-based mutation detection was 100% concordant with the DNA sequencing results. CONCLUSION: Compared to other genotyping techniques, this method requires significantly less technical time to perform and has a greatly increased throughput capacity. Hence, the DHPLC method represents a major technical advance for G6PD genotyping and should benefit G6PD-deficient individuals for proper clinical care.

Chromatography, High Pressure Liquid↗
PubMed2005

Antioxidant deficit and enhanced susceptibility to oxidative damage in individuals with different forms of alpha-thalassaemia.

alpha-Thalassaemia is a common red cell disorder in Taiwan, affecting 6-8% of Taiwanese. Previous studies have shown that reactive oxygen species are generated in increased amounts in thalassaemic red cells. This implies the possible alteration of redox status in thalassaemic patients, which may adversely affect their health. In the present study, the redox status of patients with alpha-thalassaemia trait and haemoglobin H (Hb H) disease was investigated. Lipid peroxidation, as measured by the level of plasma thiobarbituric acid reactive substances (TBARS), was increased in alpha-thalassaemic patients, with the highest level of TBARS in Hb H disease patient. The plasma levels of vitamin A, C, and E were significantly lower in alpha-thalassaemic patients than in controls. The overall antioxidant capacity in plasma was inversely correlated with the severity of alpha-globin gene defect: the more severe the form of alpha-thalassaemia, the lower the overall antioxidant capacity in plasma. Erythrocytes isolated from alpha-thalassaemia patients had lower levels of vitamin E, glutathione, catalase and superoxide dismutase. In addition, these alpha-thalassaemic red cells were more susceptible to hydrogen peroxide-induced lipid peroxidation and decrease in deformability. All these data suggest that the alpha-thalassaemic patients suffer from increased oxidative stress and antioxidant deficit, which may complicate the pathophysiology of alpha-thalassaemia.

Adult↗
PubMed2005

G6PD--an old bottle with new wine.

The major role of glucose-6-phosphate dehydrogenase (G6PD) is to generate reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is indispensable to reductive metabolism and maintenance of cellular redox homeostasis. Most advances in this field have been made in the pathophysiology of G6PD-deficient erythrocytes and the molecular characterization of different G6PD variants. Recently, numerous studies have shown the importance of G6PD in cell growth, development and disease progression.

Genetic Variation↗
PubMed2004

Glucose-6-phosphate dehydrogenase-deficient cells show an increased propensity for oxidant-induced senescence.

Glucose-6-phosphate dehydrogenase (G6PD) is involved in the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and the maintenance of cellular redox balance. We previously showed that G6PD-deficient fibroblasts undergo growth retardation and premature cellular senescence. In the present study, we demonstrate abatement of both the intracellular G6PD activity and the ratio NADPH/NADP(+) during the serial passage of G6PD-deficient cells. This was accompanied by a significant increase in the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). This suggests that the lowered resistance to oxidative stress and accumulative oxidative damage may account for the premature senescence of these cells. Consistent with this, the G6PD-deficient cells had an increased propensity for hydrogen peroxide (H(2)O(2))-induced senescence; these cells exhibited such senescent phenotypes as large, flattened morphology and increased senescence-associated beta-galactosidase (SA-beta-Gal) staining. Decreases in both the intracellular G6PD activity and the NADPH/NADP(+) ratio were concomitant with an increase in 8-OHdG level in H(2)O(2)-induced senescent cells. Exogenous expression of G6PD protected the deficient cells from stress-induced senescence. No significant telomere shortening occurred upon repetitive treatment with H(2)O(2). Simultaneous induction of p16(INK4a) and p53 was detected in G6PD-deficient but not in normal fibroblasts during H(2)O(2)-induced senescence. Our findings support the notion that G6PD status, and thus proper redox balance, is a determinant of cellular senescence.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2003

Disabled-2 small interfering RNA modulates cellular adhesive function and MAPK activity during megakaryocytic differentiation of K562 cells.

Previous studies have shown that Disabled-2 (DAB2) is up-regulated during megakaryocytic differentiation of human K562 cells. To delineate the consequences of DAB2 induction, a DNA vector-based small interfering RNA (siRNA) was designed to intervene in DAB2 expression. We found that DAB2 siRNA specifically inhibited DAB2 induction, resulting in the modulation of cell-cell adhesion and mitogen-activated protein kinase (MAPK) phosphorylation. The morphological changes and beta3 integrin expression associated with megakaryocytic differentiation were not affected. Since the MAPK pathway has been shown to involve DAB2 induction [Tseng et al., Biochem. Biophys. Res. Commun. 285 (2001) 129-135], our results suggest a reciprocal regulation between DAB2 and MAPK in the differentiation of K562 cells. In addition, we have demonstrated for the first time that DAB2 siRNA is a valuable tool for unveiling the biological consequences of DAB2 expression.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Palladium and platinum ions interfere with the measurement of erythrocyte vesiculation by inhibiting the acetylcholinesterase activity of the released spectrin-depleted microvesicles.

Palladium (Pd(2+)) and platinum (Pt(2+)) ions were found to inhibit erythrocyte membrane-bound acetylcholinesterase (AChE) with Ki values of 6.0 and 6.5 microg/ml, respectively. Lineweaver-Burke plots revealed that the inhibition of erythrocyte AChE by both metal ions was competitive in nature. Binding studies using alkaline phosphatase as a reporting enzyme confirmed that both metal ions indeed did bind to the enzyme molecules. In the process of red cell vesiculation, membrane-bound AChE is shed along with vesicles. The measurement of AChE activities in the medium containing vesiculated RBC could potentially be served as an index of vesiculation. Inhibition of AChE activities by both metal ions can thus constitute a potential source of error in vesiculation measurement. To illustrate these effects, a simulated vesiculation system, using green tea polyphenol in the presence (25 microg/ml) or absence of Pd(2+) ion was simultaneously examined by the electronmicrography and the AChE method. We observed vesiculation under the experimental condition in Pd(2+)-free controls that was associated with a time-dependent increase in AChE activity were barely detected in the Pd(2+)-spiked specimen because of the masking effect exerted by the metal ions themselves.

Acetylcholinesterase↗
PubMed2003

Humic acid induces oxidative DNA damage, growth retardation, and apoptosis in human primary fibroblasts.

Humic acid (HA) has been implicated as an etiological factor of Blackfoot disease endemic in the southwest coast of Taiwan. Dysfunction of endothelial cells and vasculopathy have been proposed to explain the onset of ulcerous changes at extremities. However, little is known about the effect of HA on activities of cells in these nonhealing wounds. In the present study, we demonstrate that HA adversely affects the growth properties of fibroblasts, one of the key players in wound repair. HA treatment caused growth arrest and apoptosis in human foreskin fibroblasts (HFF). This was accompanied by a significant increase in the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in cellular DNA. The increased fluorescence in dichlorofluorescin (H2DCF)-stained and HA-treated cells suggests the involvement of reactive oxygen species (ROS) in HA-induced biological effects. Conversely, vitamin E pretreatment, which significantly reduced the 8-OHdG formation in HA-treated cells, alleviated the growth-inhibitory and apoptosis-inducing effects of HA. These results indicate that HA initiates oxidative damages to fibroblasts, and leads to their dwindling growth potential and survival. The present study suggests that HA-induced growth retardation and apoptosis of fibroblasts may play a role in the pathogenesis of Blackfoot disease.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2002

Decreased blood activity of glucose-6-phosphate dehydrogenase associates with increased risk for diabetes mellitus.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency predisposes affected individuals highly susceptible to oxidative stress, which is one of the risk factors for diabetes. To evaluate the relationship between blood level of G6PD activity and diabetes in Taiwan, blood G6PD activity was analyzed among 237 patients with diabetes and 656 healthy subjects. A significant difference in the distribution of G6PD activities as grouped by an increment of 100 U/10(12) red blood cells (RBCs) was observed between diabetic patients and healthy subjects. The odds ratio for diabetes was 1.46 (95% confidence interval = 1.11-1.92) for every decrement of 100 U/10(12) RBC G6PD activities in these subjects. The data indicate that low G6PD activity is another risk factor for diabetes.

Aged↗