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K K Wu

Publications and source records attributed to K K Wu.

At least 73 records · Page 4Linked to original sources
PubMed1998

Characterization of the secondary structure and membrane interaction of the putative membrane anchor domains of prostaglandin I2 synthase and cytochrome P450 2C1.

Prostaglandin I2 synthase (PGIS) produces prostaglandin I2 (PGI2) which has opposite actions on platelet aggregatory and vasoconstrictive properties compared to thromboxane A2 (TXA2) produced from the same substrate by another P450 enzyme, thromboxane A2 synthase (TXAS). PGIS and TXAS have only 16% amino acid sequence identity. Hydropathy analysis suggests that the putative NH2-terminal membrane anchor domain of PGIS is similar to many other membrane-bound microsomal P450s, which are believed to be anchored by a single transmembrane segment, and thus different from the TXAS anchor, which appears to have two transmembrane segments. To characterize the membrane anchor function of the PGIS NH2-terminal region, we have used the peptidoliposome reconstitution assay to identify the membrane anchor segment in the PGIS NH2-terminal domain and compared it with the anchor segment of P450 2C1. Four peptides, mimicking putative NH2-terminal membrane anchor segments of PGIS and P450 2C1, containing residues 1-28 (PGIS-LP1 and P450 2C1-LP1) or residues 25-54 (PGIS-LP2 and P450 2C1-LP2), were synthesized and their ability to insert in a lipid bilayer was evaluated. The results indicated that both LP1 peptides of PGIS and P450 2C1 became bound to the lipid bilayer, whereas both LP2 peptides did not bind the lipid. The two LP1 peptides were further characterized as to their conformation using CD spectroscopy. Helical structure induced in these peptides by addition of trifluoroethanol, dodecylphosphocholine, or incorporation into liposomes indicated that these segments tend to adopt a helical structure in a hydrophobic environment and thus could function as membrane anchor segments. These results support the hypothesis that PGIS and TXAS interact with the endoplasmic reticulum membrane in different ways, in which the NH2-terminal anchor domain of PGIS, as with P450 2C1, appears to have a single transmembrane segment.

Amino Acid Sequence↗
PubMed1998

Biochemical pharmacology of nonsteroidal anti-inflammatory drugs.

Aspirin and conventional nonsteroidal anti-inflammatory drugs are nonselective inhibitors of cyclooxygenase-1 (COX-1) and COX-2 enzymes. Two classes of selective COX-2 inhibitors: (1) sulfonamides, such as L-745,337, and (2) tricyclic methyl sulfone derivatives, such as SC58125, have been developed. X-ray crystal structures of COX-1 and COX-2 have provided valuable information regarding the structural basis for their COX-2 selectivity. These compounds have less gastrointestinal complications in animal experiments. Their clinical efficacy and side-effects are being evaluated. Salicylate has very weak activity against either COX isoform and yet possesses anti-inflammatory actions. Recent studies indicate that it suppresses the expression of genes involved in inflammation. These activities may provide a plausible explanation for the pharmacological dilemma and, furthermore, may represent novel mechanisms for controlling inflammation.

Animals↗
PubMed1998

Detailed alignment of saccharum and sorghum chromosomes: comparative organization of closely related diploid and polyploid genomes.

The complex polyploid genomes of three Saccharum species have been aligned with the compact diploid genome of Sorghum (2n = 2x = 20). A set of 428 DNA probes from different Poaceae (grasses) detected 2460 loci in F1 progeny of the crosses Saccharum officinarum Green German x S. spontaneum IND 81-146, and S. spontaneum PIN 84-1 x S. officinarum Muntok Java. Thirty-one DNA probes detected 226 loci in S. officinarum LA Purple x S. robustum Molokai 5829. Genetic maps of the six Saccharum genotypes, including up to 72 linkage groups, were assembled into "homologous groups" based on parallel arrangements of duplicated loci. About 84% of the loci mapped by 242 common probes were homologous between Saccharum and Sorghum. Only one interchromosomal and two intrachromosomal rearrangements differentiated both S. officinarum and S. spontaneum from Sorghum, but 11 additional cases of chromosome structural polymorphism were found within Saccharum. Diploidization was advanced in S. robustum, incipient in S. officinarum, and absent in S. spontaneum, consistent with biogeographic data suggesting that S. robustum is the ancestor of S. officinarum, but raising new questions about the antiquity of S. spontaneum. The densely mapped Sorghum genome will be a valuable tool in ongoing molecular analysis of the complex Saccharum genome.

DNA, Plant↗
PubMed1998

Plasma fibrinogen and incident hypertension in the Atherosclerosis Risk in Communities (ARIC) Study.

BACKGROUND: Several cross-sectional studies have reported a positive association between plasma fibrinogen levels and prevalent hypertension. Other studies have reported a positive association between hypertension and whole-blood or plasma viscosity, to which fibrinogen contributes. To our knowledge, there has been no prospective study of fibrinogen and incident hypertension. SUBJECTS AND METHODS: We measured plasma fibrinogen levels in a population-based cohort study of middle-aged adults and related it to the occurrence of incident hypertension (systolic blood pressure > or = 140 mmHg or diastolic blood pressure > or = 90 mmHg or use of antihypertensive medication) over 6 years. RESULTS: There was a moderately strong positive association between fibrinogen levels and prevalent hypertension in both men and women, with the odds of hypertension elevated by 50% for the highest fibrinogen quartile versus the lowest. Among 7884 participants at risk, 1609 developed hypertension over 6 years. Adjusted for age, race, field center and baseline systolic blood pressure, the odds ratio of incident hypertension in relation to fibrinogen quartiles was 1.0, 1.07, 1.21 and 1.43 in men (P= 0.003 for trend) and 1.0, 0.92, 0.99 and 0.99 in women (P= 0.89 for trend). After adjustment for other risk factors, the odds ratios were 1.0, 1.03, 1.15 and 1.29 (P= 0.045 for trend) in men and remained nonsignificant in women. CONCLUSIONS: Despite a moderately strong positive association between fibrinogen levels and prevalent hypertension in both sexes, there was only a weak positive association between fibrinogen levels and incident hypertension in men and no association in women. Whether an elevated fibrinogen level is a risk factor for, or a consequence of, hypertension remains unclear.

Arteriosclerosis↗
PubMed1998

Interleukin-1 beta induces cyclooxygenase-2 gene expression in cultured endometrial stromal cells.

Increasing evidence indicates that PGs may play an obligatory role in blastocyst implantation. Cyclooxygenase (also known as PGH synthase) isozymes 1 and 2 catalyze the rate limiting steps in the biosynthesis of PGs. The ubiquitous cyclooxygenase-1 (COX-1) subserves housekeeping functions, whereas the inducible cyclooxygenase-2 (COX-2) is expressed by limited cell types and tightly controlled. Here we report the induction of COX-2 gene expression by interleukin-1 beta (IL-1 beta) in cultured human endometrial stromal cells. COX-2 activity was induced by IL-1 beta (1 ng/mL); conversion of exogenous arachidonic acid to PGF2 alpha increased from 2.6 +/- 0.6 ng/well (mean +/- SEM; n = 6) to 22.2 +/- 5.6 ng, but was completely blocked (2.8 +/- 0.7 ng/well) by NS-398, a specific COX-2 inhibitor. Undetectable in quiescent stromal cells, messenger ribonucleic acid for COX-2 was induced 30 min after IL-1 beta treatment, reached a maximum at 4 h, and decreased after 15 h. Protein synthesis was not required for induction of the COX-2 gene, as it was blocked by actinomycin D but not by cycloheximide. The 70-kDa COX-2 protein was not detected in quiescent cells, became detectable 6 h after IL-1 beta treatment, and remained detectable even after 15 h. IL-1 beta (0.1-100 ng/mL) increased the luciferase activity in promoterless luciferase reporter containing the 900-bp 5'-flanking sequence (-891 to +9) of the COX-2 gene in a dose-dependent manner, with an ED50 of 0.1-1 ng/mL.

Arachidonic Acid↗
PubMed1997

Prospective study of hemostatic factors and incidence of coronary heart disease: the Atherosclerosis Risk in Communities (ARIC) Study.

BACKGROUND: Although hemostatic factors contribute to acute coronary syndromes and atherogenesis, few studies have prospectively evaluated the association between multiple hemostatic factors and coronary heart disease incidence. METHODS AND RESULTS: The Atherosclerosis Risk in Communities Study recruited 14,477 adults from 45 to 64 years of age who were initially free of coronary heart disease. Coronary disease risk factors and several plasma hemostatic factors were measured, and incidence of coronary heart disease was ascertained during an average follow-up of 5.2 years. Age-, race-, and field center-adjusted relative risks of coronary heart disease were significantly elevated (P < or = .05) per higher value of fibrinogen (relative risk: men, 1.76; women, 1.54), white blood cell count (men, 1.68; women, 2.23), factor VIII coagulant activity (women, 1.25), and von Willebrand factor antigen (men, 1.20; women, 1.18). Adjustment for other risk factors attenuated these associations for fibrinogen (adjusted relative risk: men, 1.48; women, 1.21), and it eliminated the white blood cell count, factor VIII, and von Willebrand factor associations, consistent with the other risk factors either confounding or partly operating through their effects on the hemostatic variables. Adjusted standardized relative risks of total mortality, ranging from 1.13 to 1.37, were also elevated (P < .05) in relation to these four factors. There was no association of coronary disease incidence with factor VII, protein C, antithrombin III, or platelet count. CONCLUSIONS: Elevated levels of fibrinogen, white blood cell count, factor VIII, and von Willebrand factor are risk factors and may play causative roles in coronary heart disease. However, their measurement in healthy adults appears to add little to prediction of coronary events beyond that of more established risk factors.

Aged↗
PubMed1997

Involvement of two Sp1 elements in basal endothelial prostaglandin H synthase-1 promoter activity.

Prostaglandin H synthase-1 (PGHS-1) is a constitutively expressed key enzyme in the biosynthesis of physiologically important prostanoids. The promoter of the human PGHS-1 gene lacks a TATA box, has a very GC-rich region, and contains multiple transcription start sites. To identify the elements involved in the constitutive expression of the PGHS-1 gene, we constructed a 2075-base pair fragment (-2095 to -21 relative to the translation start codon) and a series of 5'-deletion mutants into a promoterless luciferase expression vector, which was transfected in HUVEC. Two important regions were identified. DNase I footprinting identified a protected segment, which contains an Sp1 binding site proximal to the transcription start sites. Band shift assays confirmed specific binding of Sp1 to this segment. Band shift assays further revealed specific binding of Sp1 to a distal region containing a canonical Sp1 site. Mutation of either Sp1 binding site significantly reduced the promoter activity. When both sites were mutated, the activity was reduced to 29% of that of the wild type. Mutation of Sp1 sites did not abrogate promoter activity stimulated by phorbol ester. These results indicate that binding of Sp1 or its related proteins to two widely separated Sp1 sites on the promoter region activates the basal PGHS-1 gene transcription.

Base Sequence↗
PubMed1997

Mutation of Glu-361 in human endothelial nitric-oxide synthase selectively abolishes L-arginine binding without perturbing the behavior of heme and other redox centers.

Nitric oxide (NO) and L-citrulline are formed from the oxidation of L-arginine by three different isoforms of NO synthase (NOS). Defining amino acid residues responsible for L-arginine binding and oxidation is a primary step toward a detailed understanding of the NOS reaction mechanisms and designing strategies for the selective inhibition of the individual isoform. We have altered Glu-361 in human endothelial NOS to Gln or Leu by site-directed mutagenesis and found that these mutations resulted in a complete loss of L-citrulline formation without disruption of the cytochrome c reductase and NADPH oxidase activities. Optical and EPR spectroscopic studies demonstrated that the Glu-361 mutants had similar spectra either in resting state or reduced CO-complex as the wild type. The heme ligand, imidazole, could induce a low spin state in both wild-type and Glu-361 mutants. However, unlike the wild-type enzyme, the low spin imidazole complex of Glu-361 mutants was not reversed to a high spin state by addition of either L-arginine, acetylguanidine, or 2-aminothiazole. Direct L-arginine binding could not be detected in the mutants either. These results strongly indicate that Glu-361 in human endothelial NOS is specifically involved in the interaction with L-arginine. Mutation of this residue abolished the L-arginine binding without disruption of other functional characteristics.

Amino Acid Sequence↗
PubMed1997

Prostacyclin synthase active sites. Identification by molecular modeling-guided site-directed mutagenesis.

Prostacyclin synthase (PGIS), a cytochrome P450 enzyme, catalyzes the biosynthesis of a physiologically important molecule, prostacyclin. In this study we have used a molecular modeling-guided site-directed mutagenesis to predict the active sites in substrate binding pocket and heme environment of PGIS. A three-dimensional model of PGIS was constructed using P450BM-3 crystal structure as the template. Our results indicate that residues Ile67, Val76, Leu384, Pro355, Glu360, and Asp364, which were suggested to be located at one side of lining of the substrate binding pocket, are essential for catalytic activity. This region containing beta1-1, beta1-2, beta1-3, and beta1-4 strands is predicted well by the model. At the heme region, Cys441 was confirmed to be the proximal axial ligand of heme iron. The conserved Phe and Arg in P450BM-3 were substituted by Leu112 and Asp439, respectively in PGIS. Alteration of Leu112 to Phe retained the activity, indicating that Leu112 is a functional substitution for Phe. In contrast, mutant Asp439 --> Ala exhibited a slight increase in activity. This result implies a difference in the heme region between P450BM-3 and PGIS. Our results also indicate that stability of PGIS expression is not affected by heme site or active site mutations.

Amino Acid Sequence↗
PubMed1997

Prostacyclin and nitric oxide-related gene transfer in preventing arterial thrombosis and restenosis.

Prostacyclin (PGI2) and nitric oxide (NO) are potent vascular mediators, playing key roles in protecting arterial wall from injury-induced lesions. The key enzyme that catalyzes PGI2 biosynthesis is cyclooxygenase (COX). COX-1 undergoes auto-inactivation, which severely limits PGI2 synthesis. Overexpression of COX-1 in cultured endothelial cells by COX-1 gene transfer was accompanied by a higher capacity for and sustained synthesis of PGI2. Adenovirus-mediated COX-1 gene transfer to angioplasty damaged carotid arteries in pigs augmented PGI2 synthesis and prevents thrombus formation. Transfer of endothelial NO synthase (eNOS) into angioplasty injured, carotid arteries was reported to suppress intimal hyperplasia in rats. Transfer of PGI2 and NO synthetic enzymes restores the vasoprotective properties and represents an exciting new strategy for treating arterial thrombotic disorders.

Animals↗
PubMed1997

Hemostatic tests in the prediction of atherothrombotic disease.

Hemostatic components play major roles in the pathogenesis of human atherothrombotic disease. There has been interest in determining whether tests for hemostatic components predict this disorder. Recent population-based and clinically-oriented prospective studies have begun to provide useful information. Northwick Park Heart (NPH) study made the initial observation that the plasma fibrinogen level is an independent risk factor for non-fatal and fatal coronary heart disease (CHD). This has been confirmed by other population-based studies. By contrast, factor VIIc levels which were reported by NPH to be an independent risk factor for CHD, especially fatal myocardial infarction (MI) has not been confirmed by other studies. Factor VIIIc, von Willebrand factor (vWF), tPA and PAI-1 are reported to be associated with CHD. Prospective angina pectoris studies have identified fibrinogen, C-reactive protein (CRP) and von Willebrand factor as independent risk factors for acute MI. These findings raise a possible mechanism of inflammation in CHD. A number of studies imply an association of platelet activation with CHD. A prospective study has shown that a persistent positive spontaneous platelet aggregation (SPA) test is an independent risk factor for recurrent MI. Hence, prospective studies indicate a potential value for certain hemostatic tests to predict atherothrombotic disorder. However, clinical utility of these tests remains to be established. Controlled clinical trials may be required to provide a more definite information regarding the use of these tests for selecting high risk patients for preventive strategies.

Angina Pectoris↗
PubMed1997

Modified Mitchell's bunionectomy (Wu's bunionectomy).

A standard Wu's bunionectomy consists of an oblique first metatarsal neck osteotomy with a lateral, proximal, and plantar displacement of the first metatarsal head and firm fixation of the osteotomy site with two Herbert screws. The outstanding features of this surgical technique include its surgical simplicity, secure fixation of the osteotomy site, no need for subsequent screw removal, ease of screw insertion, complete absence of screw breakage, little postoperative pain, and a relatively greater degree of correcting the various deformities associated with a hallus valgus foot than those normally achieved by either a Mitchell's bunionectomy or a Chevron bunionectomy.

Bandages↗
PubMed1997

Family history of coronary heart disease and hemostatic variables in middle-aged adults. Atherosclerosis Risk in Communities Investigators and Family Heart Study Research Group.

Individuals with a family history of coronary heart disease (CHD) may be predisposed to atherothrombosis. To investigate this hypothesis, a family CHD risk score was computed for approximately 13,000 men and women aged 45 to 64; hemostatic variables (fibrinogen, factor VIIc, factor VIIIc, von Willebrand factor, antithrombin III. protein C) were also measured in plasma. After adjustment for age and ethnicity, there was a statistically significant, positive association between the family risk score and four of the six hemostatic variables (fibrinogen, factor VIIc, factor VIIIc, von Willebrand factor) in women and all six hemostatic variables in men. In general, these associations were weak and substantially attenuated after adjustment for constitutional, lifestyle, and biochemical covariates. These results indicate that mean levels of selected hemostatic variables, like traditional CHD risk factors, are higher in individuals with a family history of heart disease.

Blood Coagulation↗
PubMed1997

Interferon-gamma in cerebrospinal fluid of children with aseptic meningitis.

BACKGROUND: Certain cytokines may contribute to the sequence of events that lead to meningeal inflammation in bacterial meningitis. However, their role in viral meningitis is not so less well defined. We determined the cytokines levels in the cerebrospinal fluid (CSF) of children with aseptic meningitis and discussed their relationship with clinical and laboratory findings. METHODS: We determined the concentrations of interleukin-1 beta (IL-1 beta), interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the CSF of 62 patients with aseptic meningitis including 17 patients with culture-proved enteroviral meningitis, and from 19 control acute febrile patients without meningitis. RESULTS: The GM-CSF in the cerebrospinal fluid was detected from one of the 62 patients with aseptic meningitis and none of the 19 controls. Fourteen (23%) of the 62 patients with aseptic meningitis and 2 (10.5%) of 19 controls had detectable IL-1 beta. There was no significant difference in IL-1 beta levels between patients with aseptic meningitis (4.4 +/- 11.4 pg/ml) and control group (2.4 +/- 7.7 pg/ml). The CSF IFN-gamma level was detectable in 40 (65%) of 62 patients and 6 (31.6%) of 19 controls. The mean CSF IFN-gamma concentration was significantly higher in patients with aseptic meningitis when compared with that in control group (37.9 +/- 48.8 pg/ml vs 17.5 +/- 29.7 pg/ml; p = 0.007). CONCLUSIONS: IFN-gamma was detectable in the CSF in 65% of patients with aseptic meningitis and the role of interferon-gamma remains to be determined.

Adolescent↗
PubMed1996

Characterization of the complete genomic structure of human thromboxane synthase gene and functional analysis of its promoter.

Thromboxane A2 (TXA) is a potent vasoconstrictor and mediator of platelet aggregation. Thromboxane synthase (TXAS), which catalyzes the biosynthesis of TXA, is a member of the cytochrome P450 superfamily. We report here the complete genomic structure of the human TXAS gene. The gene contains 13 exons and is 193 kb long, the largest P450 gene ever isolated. The physical localization of the TXAS gene on the genetic map of human chromosome 7 was established. A major transcription start site is located at a motif similar to the initiator element where the transcription factor TFII-I binds. We have sequenced up to 1.6 kb of the 5'-flanking region of the TXAS gene and characterized the promoter activity in a human megakaryocyte cell line and endothelial cells. Our results demonstrated that the nucleotides -248/+137 relative to the major transcription start site conferred a full promoter activity of the TXAS gene. This region was regulated negatively by cis-elements located between -1562 and -248. Moreover, the regions outside -1562/+137 might control tissue-specific TXAS expression.

Base Sequence↗
PubMed1996

Identification of thromboxane A2 synthase active site residues by molecular modeling-guided site-directed mutagenesis.

Human thromboxane A2 synthase (TXAS) exhibits spectral characteristics of cytochrome P450 but lacks monooxygenase activity. Its distinctive amino acid sequence makes TXAS the sole member of family 5 in the P450 superfamily. To better understand the structure-function relationship of this unusual P450, we have recently constructed a three-dimensional model for TXAS using P450BM-3 as the template (Ruan, K.-H., Milfeld, K., Kulmacz, R. J., and Wu, K. K. (1994) Protein Eng. 7, 1345-1551) and have identified a potential active site region. The catalytic roles of several putative active site residues were evaluated using selectively mutated recombinant TXAS expressed in COS-1 cells. Mutation of Ala-408 to Glu or Arg-413 to Gly led to a complete loss of enzyme activity despite expression of mutant protein levels equivalent to that of the wild-type TXAS. Mutation of Ala-408 to Gly or Leu retained the enzyme activity at levels of 30 or 40%, respectively. This suggests that Ala-408 provides a hydrophobic environment for substrate binding. Mutation of Arg-413 to Lys or Gln completely abolished the enzyme activity, indicating that this residue is essential to catalytic activity and supports its identification as an active site residue. Mutation of Arg-410 to Gly or Glu-433 to Ala resulted in >50% reduction in the enzyme activity without appreciably altering mutant protein expression, consistent with a more subtle effect of these residues on TXAS catalytic efficiency. Mutation of residues predicted to be involved in binding the heme prosthetic group, including the heme thiolate ligand Cys-480, Arg-478, Phe-127, and Asn-110, each markedly reduced the expressed protein level and abolished enzyme activity. This suggests that proper heme binding is important to synthesis or stability of recombinant TXAS. Mutation of Ile-346, which corresponds to P450cam-Thr-252, an essential amino acid involved in dioxygen bond scission, to Thr increased the enzymatic activity by 40%, suggesting that oxygen bond cleavage is not a rate-limiting step in thromboxane A2 biosynthesis. The present results from site-directed mutagenesis support the overall structure of the TXAS active site predicted by homology modeling and have allowed refinement of the position of bound substrate.

Amino Acid Sequence↗
PubMed1996

Endothelial nitric-oxide synthase. Evidence for bidomain structure and successful reconstitution of catalytic activity from two separate domains generated by a baculovirus expression system.

A baculovirus system was used to express the oxygenase and reductase domains of human endothelial nitric-oxide synthase (ecNOS) as distinct proteins. The oxygenase domain (residues 1-491) was expressed using a vector containing a His6 tag at the N terminus. The purified oxygenase domain had an apparent molecular mass of approximately 54 kDa, and retained the ability to bind L-arginine and form the ferrous CO complex. The purified reductase domain (residues 492-1244) had an apparent molecular mass of approximately 82 kDa and retained the ability to catalyze NADPH-dependent cytochrome c reduction, which was enhanced 10-fold by the presence of Ca2+/calmodulin. Both purified domains exhibited immunoreactivity to rabbit anti-ecNOS IgG. The NOS activity was successfully reconstituted by mixing the two domains. These results demonstrate for the first time that the two domains of ecNOS are catalytically intact and can be reconstituted in vitro.

Animals↗