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Biomedical subjects

K K Wu

Publications and source records attributed to K K Wu.

At least 55 records · Page 3Linked to original sources
PubMed2000

Thrombomodulin: a linker of coagulation and fibrinolysis and predictor of risk of arterial thrombosis.

Thrombomodulin (TM) binds thrombin, changes thrombin conformation and allows thrombin to activate protein C and thrombin-activatable fibrinolysis inhibitor (TAFI). Activated protein C and TAFI inhibit coagulation and fibrinolysis, respectively. TM is, thus, a linker of endogenous control of coagulation and fibrinolysis. Plasma soluble TM, cleaved products of cellular TM, also have anticoagulant and antifibrinolytic properties. TM plays an important role in thromboresistance. Prospective studies show that a high plasma TM level is associated with a low risk of developing coronary heart disease. The plasma TM level may reflect the level of endothelial TM expression. TM expression levels are influenced by multiple gene polymorphisms. Several of the polymorphisms are probably associated with coronary heart disease.

Blood Coagulation↗
PubMed1999

Transcriptional regulation of endothelial nitric-oxide synthase by an interaction between casein kinase 2 and protein phosphatase 2A.

We previously demonstrated that lysophosphatidylcholine up-regulated endothelial nitric-oxide synthase promoter activity by increasing Sp1 binding via the action of protein serine/threonine phosphatase 2A (Cieslik, K., Zembowicz, A., Tang, J.-L., and Wu, K.K. (1998) J. Biol. Chem. 273, 14885-14890). To characterize the regulation of basal endothelial nitric-oxide synthase promoter activity and the signaling pathway through which lysophosphatidylcholine augments endothelial nitric-oxide synthase transcription, we used a casein kinase 2 inhibitor coupled with immunoprecipitation to demonstrate that basal Sp1 binding and endothelial nitric-oxide synthase promoter activity were controlled by casein kinase 2 complexed with protein serine/threonine phosphatase 2A. Casein kinase 2 catalyzed protein serine/threonine phosphatase 2A phosphorylation thereby inhibiting its activity. Lysophosphatidylcholine selectively activated p42/p44 mitogen-activated protein kinase. Purified extracellular regulated kinase 2 blocked casein kinase 2 activity and increased protein serine/threonine phosphatase 2A activity, resulting in an increased Sp1 binding and endothelial nitric-oxide synthase promoter activity. These results indicate that Sp1 binding to its cognate site on the endothelial nitric-oxide synthase promoter and its transactivation of endothelial nitric-oxide synthase is regulated by post-translational Sp1 phosphorylation and dephosphorylation through a dynamic interaction between casein kinase 2 and protein serine/threonine phosphatase 2A.

Casein Kinase II↗
PubMed1999

Prospective study of markers of hemostatic function with risk of ischemic stroke. The Atherosclerosis Risk in Communities (ARIC) Study Investigators.

BACKGROUND: Several markers of hemostatic function and inflammation have been associated with increased risk of coronary heart disease, but prospective evidence for their role in ischemic stroke is scant. METHODS AND RESULTS: The Atherosclerosis Risk in Communities (ARIC) Study measured several of these markers in more than 14 700 participants 45 to 64 years old who were free of cardiovascular disease and were followed up for 6 to 9 years for occurrence of ischemic stroke (n=191). There was no apparent association between ischemic stroke incidence and factor VIIc, antithrombin III, platelet count, or activated partial thromboplastin time. After adjustment for multiple cardiovascular risk factors, von Willebrand factor, factor VIIIc, fibrinogen, and white blood cell count were positively associated and protein C was negatively but nonsignificantly associated with ischemic stroke incidence in regression analyses based on either continuous variables or fourths of the variable distributions. The adjusted relative risk (and 95% CI) for ischemic stroke in those in the highest versus lowest fourth were: von Willebrand factor, 1.71 (1.1 to 2.7); factor VIIIc, 1.93 (1.2 to 3.1); white blood cell count, 1.50 (0.9 to 2.4); fibrinogen, 1.26 (0.8 to 2.0); and protein C, 0.65 (0.4 to 1.0). CONCLUSIONS: This study offers modest support for the hypothesis that some markers of hemostatic function and inflammation can identify groups of middle-aged adults at increased risk of stroke. These factors may play a role in the pathogenesis of ischemic stroke.

Arteriosclerosis↗
PubMed1999

Poly(ethylenimine) and its role in gene delivery.

Since the first published examination of poly(ethylenimine) (PEI) as a gene delivery vehicle, there has been a flurry of research aimed at this polycation and its role in gene therapy. Here we will briefly review PEI chemistry and the characterization of PEI/DNA complexes used for gene delivery. Additionally, we will note various PEI transfection considerations and examine findings involving other polycationic gene delivery vehicles used with cellular targeting ligands. The current state of our knowledge regarding the mechanism of PEI/DNA transfection will also be discussed. Finally, we will survey toxicity issues related to PEI transfection.

DNA Adducts↗
PubMed1999

Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle.

Poly(ethylenimine) (PEI) samples of various molecular weights and pHs were used to transfect endothelial cells to achieve levels of gene expression for comparison. PEIs with nominal molecular weights of 600, 1200, 1800, 10,000, and 70,000 Da were examined at pHs of 5. 0, 6.0, 7.0, and 8.0, and the results were recorded in terms of transfection efficiencies at 24, 48, 68, 92, and 120 h post-transfection. Trials were performed on the human endothelial cell-derived cell line EA.hy 926. We found that, for the polymers tested, transfection efficiency increased as the molecular weight of PEI increased. Representative values of PEIs at pH 6 and molecular weight 70,000 produced average transfection efficiencies of 25.6 +/- 7.9% (n = 8) at the greatest average expression levels, while PEI of molecular weight 10,000 yielded efficiencies of only 11.4 +/- 1.7% (n = 6). Transfection efficiencies for molecular weight 1,800 PEI were essentially zero, and PEIs of lower molecular weights produced no transfection at all. In contrast, the pH of the PEI solutions had no discernible effect on transfection. Optimal expression of the green fluorescent protein reporter occurred between 2 and 3 days post-transfection. The amount of reporter expression also was noted, as determined by the brightness of fluorescing cells under UV. The data obtained demonstrate that the molecular weight of the PEI carrier has an effect on transfection efficiency while the pH of the PEI solutions prior to DNA complexation has no such effect.

Chromatography, Gel↗
PubMed1999

Soluble thrombomodulin as a predictor of incident coronary heart disease and symptomless carotid artery atherosclerosis in the Atherosclerosis Risk in Communities (ARIC) Study: a case-cohort study.

BACKGROUND: Small amounts of soluble thrombomodulin in plasma are thought to reflect endothelial damage. In a case-cohort study, we examined whether soluble thrombomodulin is a predictor of incident coronary heart disease and carotid artery atherosclerosis. METHODS: The study population consisted of 14,170 black and white participants from the Atherosclerosis Risk in Communities (ARIC) study, who did not have cardiovascular disease at the start of the study and who we followed up for 6 years. After appropriate exclusions, we analysed 258 cases of incident coronary heart disease and 449 cases of carotid atherosclerosis. A stratified random sample of 753 individuals from the ARIC cohort was used as the comparison group. We used EIA to measure soluble thrombomodulin in plasma samples from both groups. For the analysis, we used quintiles of soluble thrombomodulin concentrations (< 24.7, 24.8-30.6, 30.7-40.2, 40.3-55.2, and > or = 55.3 ng/mL). FINDINGS: Soluble thrombomodulin showed a strong, graded, inverse association with incident coronary heart disease (p=0.005). The adjusted rate ratio of the highest quintile of soluble thrombomodulin compared with the lowest quintile was 0.29 (95% CI 0.15-0.57). The association with carotid atherosclerosis, however, tended to be positive, especially among white participants (odds ratio 2.94 [1.15-7.51] for highest vs lowest quintile). The relation of soluble thrombomodulin to incident coronary heart disease and carotid atherosclerosis was dependent on factor VIII coagulant activity (p=0.06 and 0.003, respectively). INTERPRETATION: The prospective association of soluble thrombomodulin with incident coronary heart disease differs from its cross-sectional association with carotid atherosclerosis. In healthy people, plasma concentrations of soluble thrombomodulin may reflect endothelial expression of thrombomodulin. High concentration of soluble thrombomodulin may be associated with decreased risk of coronary heart disease.

Analysis of Variance↗
PubMed1999

Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery.

Poly(ethylenimine) (PEI) is one of a number of polycations that has been used successfully to transfer genes into living cells. Although PEI shows promise in the field of gene therapy, to date no rigorous proof of mechanism has been published regarding the fate of PEI/DNA administered for transfection. Here we show, by using fluorescent labeling and confocal microscopy, the paths of PEI/DNA complexes from endocytosis to gene expression. We found that complexes attach to cell surfaces and migrate into clumps that are endocytosed. The endocytotic vesicles grow in number and size and are occasionally seen to lyse. Most interesting is the fact that endocytosed PEI, whether administered with or without DNA, undergoes nuclear localization in the form of ordered structures.

Cell Line↗
PubMed1999

Suppression of inducible cyclooxygenase 2 gene transcription by aspirin and sodium salicylate.

The pharmacological action of salicylate cannot be explained by its inhibition of cyclooxygenase (COX) activity. In this report, the effects of aspirin and sodium salicylate on COX-2 expressions in human umbilical vein endothelial cells and foreskin fibroblasts were evaluated. Aspirin and sodium salicylate at therapeutic concentrations equipotently blocked COX-2 mRNA and protein levels induced by interleukin-1beta and phorbol 12-myristate 13-acetate. The suppressing effect was more pronounced in cultured cells deprived of fetal bovine serum for 24 h, suggesting that it may be cell cycle related. Salicylate inhibited nascent COX-2 transcript synthesis but had no effect on COX-2 mRNA stability. It inhibited COX-2 promoter activity in a concentration-dependent manner. In mice pretreated with aspirin (10 and 30 mg/kg), followed by challenge with lipopolysaccharide, COX-2 mRNA expression in peritoneal macrophages was markedly suppressed. These findings suggest that salicylate exerts its antiinflammatory action in part by suppressing COX-2 induction, thereby reducing the synthesis of proinflammatory prostaglandins.

Animals↗
PubMed1999

Improved packing of poly(ethylenimine)/DNA complexes increases transfection efficiency.

We have developed a modified poly(ethylenimine) (PEI) transfection procedure that significantly increases PEI's transfection efficiency. While the basic transfection procedure had a transfection efficiency of 37%, our modified procedure yielded a 53% transfection efficiency. The altered procedure gives improved results because of two simultaneous actions: free polycations are removed from the transfecting solutions, and the composition of the PEI complexes that are administered to cells has been modified. The reduction in the amount of free polycations in transfecting solutions reduced the toxicity sometimes associated with the administration of polycations to cellular environments. The structural modification of PEI/DNA transfecting complexes involves improved PEI packing around the delivered plasmid to yield a greater buffering capacity without a change in the complex's surface charge concentration. These structural properties were confirmed by titration and zeta potential analyses. Whether the modified PEI/DNA complexes are more effective because of increased cellular uptake or an enhanced ability to escape from endolysosomes has been addressed. The increase in transfection efficiency was obtained when the buffering capacity of the PEI/DNA was increased without a change in surface charge concentration, which implies that it is the property of enhanced lysosomal buffering that is responsible for successful PEI transfection.

Cells, Cultured↗
PubMed1999

Factor VIII and other hemostasis variables are related to incident diabetes in adults. The Atherosclerosis Risk in Communities (ARIC) Study.

OBJECTIVE: Our objective was to evaluate whether selected hemostasis variables, some of which may reflect inflammation or endothelial dysfunction, are independently associated with the development of diabetes. RESEARCH DESIGN AND METHODS: We studied a biethnic cohort of 12,330 men and women, 45-64 years of age, of the Atherosclerosis Risk in Communities Study. New cases of diabetes were diagnosed by a reported physician diagnosis, hypoglycemic medication use, or a casual or fasting serum glucose level of > or = 11.1 or > or = 7 mmol/l, respectively. RESULTS: Over an average follow-up of 7 years, 1,335 new cases of diabetes were detected. The odds ratios (4th versus 1st quartile) of developing diabetes, adjusted by logistic regression for age, sex, race, study center, family history of diabetes, fasting glucose, physical activity, and smoking, were 1.2 (95% CI 1.0-1.5) for fibrinogen and 1.4 (1.1-1.6) for factor VII. Associations for factor VIII, von Willebrand factor, and activated partial thromboplastin time were found to be 1.8 (1.3-2.3), 1.4 (1.1-1.8), and 0.63 (0.49-0.82), respectively, in women. Although further adjustment for BMI and waist-to-hip ratio diminished the relationships, a highly statistically significant association (P = 0.001) remained for factor VIII (1.6 [1.2-2.1]) in women. CONCLUSIONS: Factor VIII and other hemostasis variables are associated with the development of diabetes in middle-aged adults. These findings support a role for inflammation and, particularly in women, endothelial dysfunction in the pathogenesis of type 2 diabetes.

Arteriosclerosis↗
PubMed1999

Parental anxiety associated with participation in anaesthetic induction in children: questionnaire survey.

OBJECTIVES: To determine the reasons why Chinese parents accompany their child during anaesthetic induction; to explore the level of anxiety experienced by the parents; and to evaluate the factors that contribute to parental anxiety. DESIGN: Questionnaire study of Chinese parents who had chosen to be present during the anaesthetic induction of their child between January 1997 and July 1997. SETTING: Day Surgery Unit at a public hospital, Hong Kong. PARTICIPANTS: One hundred and fifty-one Chinese parents of 151 children undergoing general anaesthesia for surgery. MAIN OUTCOME MEASURE: Parental anxiety, as evaluated by a self-reported rating system and the State-Trait Anxiety Scale. RESULT: More than half (56%) of the participants graded their experience as either "very anxious but tolerable" or "a little anxious", and only 2% graded the situation as "very anxious, intolerable". All participants were either "satisfied" or "very satisfied" with their experience. The type of surgery and any history of previous surgical intervention were found to be factors that determined the level of anxiety (P<0.05). CONCLUSIONS: Most Chinese parents decide to be present at their child's anaesthetic induction because of a sense of duty and concern. Parental presence does not provoke further or intolerable parental anxiety. One limitation of this study is that the level of anxiety measured might be due to the combination of anaesthetic induction and surgery rather than to concerns about anaesthetic induction alone.

Journal Article↗
PubMed1998

Effects of Asp-369 and Arg-372 mutations on heme environment and function in human endothelial nitric-oxide synthase.

Eight polar amino acid residues in the putative substrate-binding region from Thr-360 to Val-379 in human endothelial nitric-oxide synthase (eNOS) (Thr-360, Arg-365, Cys-368, Asp-369, Arg-372, Tyr-373, Glu-377, and Asp-378) were individually mutated. Only two of these residues, Asp-369 and Arg-372, were found to be essential for enzyme activity. A further series of mutants was generated by replacing these two residues with various amino acids and the mutant proteins were expressed in a baculovirus system. Mutant eNOS had a very low L-citrulline formation activity with the exception of D369E and R372K, which retained 27% and 44% of the wild-type enzyme activity, respectively. Unlike the wild-type enzyme, all mutants except D369E, R372K, and R372M had a low spin heme (Soret peak at 416 nm). All the Asp-369 mutants had higher Kd values for L-arginine (1-10 mM) than wild-type eNOS (0.4 microM) and an unstable heme-CO complex, and except for D369E, had a very low (6R)-5,6,7, 8-tetrahydro-L-biopterin (BH4) content. In contrast, each of Arg-372 mutants retained a considerable amount of BH4, had a moderate reduction in L-arginine affinity, and had a more stable heme-CO complex. 1-Phenylimidazole did not bind to wild-type eNOS heme, but bound to all Asp-369 and Arg-372 mutants (Kd ranged from 10 to 65 microM) except R372K. Heme spin-state changes caused by binding of 3, 5-lutidine appeared to depend on both charge and size of the side chains of residues 369 and 372. Furthermore, all Asp-369 and Arg-372 mutants were defective in dimer formation. These results suggest that residues Asp-369 and Arg-372 in eNOS play a critical role in oxygenase domain active-site structure and activity.

Amino Acid Sequence↗
PubMed1998

Cloning and expression analysis of a novel salicylate suppressible gene, Hs-CUL-3, a member of cullin/Cdc53 family.

By using a mRNA differential display technique to search for salicylate suppressible genes, we identified a cDNA in human foreskin fibroblasts, which by GenBankTM DNA data base search shows sequence homology to the recently reported cullin/Cdc53 (CUL) family genes, especially CUL-3. We have cloned the full-length human CUL-3 (Hs-CUL-3) cDNA. It encodes a 768-amino acid polypeptide and has a predicted molecular weight of 88,939. The amino acid sequence of Hs-CUL-3 shows 46% homology to that of its Caenorhabditis elegans ortholog, Ce-CUL-3, and 27 and 23% to that of Hs-CUL-1 and Hs-CUL-2, respectively. Northern blot analysis showed that phorbol 12-myristate 13-acetate increased the expression of Hs-CUL-3 mRNA in a concentration- and time-dependent manner, and this increase was inhibited by sodium salicylate. Hs-CUL-3 widely expressed in human tissues and its expression in cultured COLO205 colon cancer cells was increased when compared with that in normal colon cells. It is likely that Hs-CUL-3 is involved in cell proliferation control.

Amino Acid Sequence↗
PubMed1998

Transcriptional regulation of endothelial nitric-oxide synthase by lysophosphatidylcholine.

We have shown that lysophosphatidylcholine (lyso-PC) increases endothelial nitric-oxide synthase (eNOS) expression at the transcriptional level (Zembowicz, A., Tang, J.-L., and Wu, K. K. (1995) J. Biol. Chem. 270, 17006-17010). To elucidate the mechanism by which lyso-PC increases the eNOS transcription, we identified Sp1 sites at -104 to -90 and PEA3 sites at -40 to -24 as being involved in lyso-PC-induced promoter activity. Site-directed mutagenesis of Sp1 sites resulted in a marked reduction of basal and lyso-PC-induced activity whereas PEA3 site mutation abrogated response to lyso-PC. Band shift assays revealed that lyso-PC augmented Sp1 binding activity. Pretreatment of cells or nuclear extracts with okadaic acid reduced the Sp1 binding activity. Furthermore, okadaic acid treatment abrogated the lyso-PC induced promoter augmentation. Lyso-PC increased the nuclear extract protein phosphatase 2A (PP2A) activity, which was suppressed by okadaic acid treatment. These results suggest that lyso-PC up-regulates eNOS transcription by a PP2A-dependent increase in Sp1 binding activity.

Base Sequence↗