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Biomedical subjects

K K Wu

Publications and source records attributed to K K Wu.

At least 19 recordsLinked to original sources
PubMed1992

Inhibition of endothelial cell prostaglandin H synthase gene expression by naproxen.

Naproxen is a potent anti-inflammatory drug whose action is attributed to inhibition of prostaglandin biosynthesis. In view of our recent discovery that aspirin and sodium salicylate are capable of reducing cellular levels of prostaglandin H (PGH) synthase mRNA, we have evaluated the effect of naproxen on PGH synthase protein and mRNA levels in cultured human umbilical vein endothelial cells (HUVEC). PGH synthase mRNA levels were quantified by a competitive polymerase chain reaction (PCR) assay; protein was assessed by Western blotting. Naproxen decreased the PGH synthase protein level in HUVEC in a concentration-dependent manner. It abolished entirely the 70 kDa PGH synthase subunit at 5 micrograms/ml. It appears more effective in blocking interleukin-1 inducible PGH synthase levels. Naproxen also inhibited the synthase mRNA level in a concentration-dependent manner; levels were reduced by 33% at 5 micrograms/ml and 60% at 30 micrograms/ml naproxen. These results indicate that naproxen, like the salicylates, inhibits PGH synthase levels in cultured endothelial cells either by inhibiting transcription of the PGH synthase gene or by destabilizing its messenger RNA.

Cells, Cultured↗
PubMed1992

Endothelial cells in hemostasis, thrombosis, and inflammation.

Once regarded as an inert lining, the vascular endothelium is now known to synthesize a variety of molecules that can protect against or promote disease. These important agents include prostacyclin, thrombomodulin, plasminogen activators, and ecto-enzymes. Our growing understanding of their protective and pathologic roles has already provided new therapeutic strategies.

Blood Viscosity↗
PubMed1992

Primary structure of human thromboxane synthase determined from the cDNA sequence.

Polymerase chain reaction techniques have been used to isolate a cDNA clone containing the entire protein coding region of thromboxane A2 synthase (EC 5.3.99.5) from a human lung cDNA library. The cDNA clone hybridizes with a single 2.1-kilobase mRNA species in phorbol ester-induced human erythroleukemia and monocytic leukemia cell lines. A second cDNA, differing only by an insert of 163 base pairs near the 3'-end of the translated region, was also found to be present in the same library. The proteins predicted from both nucleic acid sequences include the three polypeptide sequences determined from amino acid sequencing of the purified human platelet enzyme, five potential sites for N-glycosylation, and a hydrophobic region that may serve to anchor the synthase in the endoplasmic reticulum membrane. The longer predicted protein, designated thromboxane synthase-I, contains 534 amino acids, with a Mr of 60,684, whereas the shorter protein, designated thromboxane synthase-II, contains 460 amino acids and has a Mr of 52,408. Although thromboxane synthase-II lacks the conserved cysteine that serves as the proximal heme ligand in the other cytochromes, significant sequence similarities exist among thromboxane synthase-I and -II and several P450s, particularly those in family 3. The overall amino acid identity is considerably less than 40%, making it likely that thromboxane synthase represents a previously undefined family of cytochrome P450.

Amino Acid Sequence↗
PubMed1992

Association of coagulation factors and inhibitors with carotid artery atherosclerosis. Early results of the Atherosclerosis Risk in Communities (ARIC) Study.

Several population studies have shown that plasma levels of fibrinogen and factor VII are significantly associated with ischemic cardiovascular events. However, there is little information regarding the association of hemostatic factors with early atherosclerosis. To evaluate this, we compared the plasma concentrations of several coagulation proteins (fibrinogen, factor VII, factor VIII, von Willebrand factor, protein C, and antithrombin III) between 385 case patients, defined by high-resolution B-mode ultrasonography as having carotid arterial wall thickening, and 385 age-, race-, and sex-matched control subjects. These case patients and control subjects were selected from participants in a prospective population investigation, the Atherosclerosis Risk in Communities (ARIC) Study, who were examined between May 1987 and May 1989. Plasma fibrinogen, factor VII, protein C, and antithrombin III levels were significantly higher in case patients than in control subjects (P < 0.05). Factor VIII and von Willebrand factor were not different. These findings were supported by quartile distribution and univariate analysis. However, only fibrinogen remained significantly associated with carotid atherosclerosis on multivariate analysis taking other atherosclerosis risk factors into consideration. A one standard deviation increase in fibrinogen (67 mg/dL) was associated with a 1.6-fold increase in the odds of carotid atherosclerosis univariately (P < 0.001) and with a 1.3-fold increase in the odds multivariately (P = 0.010). Further analysis revealed that the association of fibrinogen with carotid atherosclerosis was somewhat stronger in cigarette smokers than in nonsmokers. This early case-control analysis of the ARIC Study demonstrates a significant association between plasma fibrinogen concentration and early atherosclerosis in the carotid arteries. In the context of published findings from population studies, our results indicate that plasma fibrinogen concentrations may be a useful marker for identifying individuals at high risk of developing arterial thrombotic disorders.

Antithrombin III↗
PubMed1992

Relations between hemostasis variables and cardiovascular risk factors in middle-aged adults. Atherosclerosis Risk in Communities (ARIC) Study Investigators.

The relations between hemostatic variables and cardiovascular risk factors were examined in a biracial population sample of middle-aged adults. Fibrinogen, factor VII, factor VIII, von Willebrand factor, protein C, and antithrombin III levels varied considerably by age, sex, and race. Hemostatic variables also were associated with several life-style and biochemical risk factors. For the most part, higher levels of the risk factors were associated with higher levels of the hemostatic variables. The findings point to potential confounders that warrant consideration in cardiovascular disease studies, and/or mechanisms by which cardiovascular risk is conferred. They also suggest that modification of the cardiovascular risk factors may have the potential to alter the risk of thrombosis.

Antithrombin III↗
PubMed1992

Distributions of hemostatic variables in blacks and whites: population reference values from the Atherosclerosis Risk in Communities (ARIC) Study.

The Atherosclerosis Risk in Communities Study measured hemostatic variables in nearly 16,000 men and women, aged 45 to 64 years, from four US communities. This report, based on the first 12,681 participants, presents distributions of fibrinogen concentration, factor VII activity, factor VIII activity, von Willebrand factor antigen, protein C antigen, antithrombin III activity, and activated partial thromboplastin time. Many of the hemostatic variables differed between blacks and whites, and by sex and age. For example, compared to whites, blacks had higher mean values of fibrinogen, factor VIII, von Willebrand factor, and antithrombin III, and lower mean values of protein C. Some seasonal fluctuations in hemostatic variables were noted; most notably, mean values of factor VII were lowest and protein C were highest in subjects examined in the summer compared to those examined during the other seasons. These results provide population-based reference values on blacks and whites for those interested in the relation of hemostasis to disease.

Arteriosclerosis↗
PubMed1992

Cell-specific metabolism in mouse bone marrow stroma: studies of activation and detoxification of benzene metabolites.

Two of the major cell types in bone marrow stroma, macrophages and fibroblasts, have been shown to be important regulators of both myelopoiesis and lymphopoiesis. The enzymology relating to cell-specific metabolism of phenolic metabolites of benzene in isolated mouse bone marrow stromal cells was examined. Fibroblastoid stromal cells had elevated glutathione-S-transferase (4.5-fold) and DT-diaphorase (4-fold) activity relative to macrophages, whereas macrophages demonstrated increased UDP-glucuronosyltransferase (UDP-GT, 7.5-fold) and peroxidase activity relative to stromal fibroblasts. UDP-GT and glutathione-S-transferase activities in macrophages and fibroblasts, respectively, were significantly greater than those in unpurified white marrow. Aryl sulfotransferase activity could not be detected in either bone marrow-derived macrophages or fibroblasts, and there were no significant differences in GSH content between the two cell types. Because UDP-GT activity is high in macrophages, these data suggest that DT-diaphorase levels would be rate limiting in the detoxification of benzene-derived quinones in bone marrow macrophages. The peroxidase responsible for bioactivation of benzene-derived phenolic metabolites in bone marrow macrophages is unknown but has been suggested to be prostaglandin H synthase (PGS). Hydrogen peroxide, but not arachidonic acid, supported metabolism of hydroquinone to reactive species in bone marrow-derived macrophage lysates. These data do not support a major role for PGS in peroxidase-mediated bioactivation of hydroquinone in bone marrow-derived macrophages, although PGS mRNA could be detected in these cells. Similarly, hydrogen peroxide, but not arachidonic acid, supported metabolism of hydroquinone in a human bone marrow homogenate. Peroxidase-mediated interactions between phenolic metabolites of benzene occurred in bone marrow-derived macrophages. Bioactivation of hydroquinone to species that would bind to acid-insoluble cellular macromolecules was increased by phenol and was markedly stimulated by catechol. Bioactivation of catechol was also stimulated by phenol but was inhibited by hydroquinone. These data define the enzymology and the cell-specific metabolism of benzene metabolites in bone marrow stroma and demonstrate that interactions between phenolic metabolites may contribute to the toxicity of benzene in this critical bone marrow compartment.

Aged↗
PubMed1991

Differentiation-associated expression of prostaglandin H and thromboxane A synthases in monocytoid leukemia cell lines.

To elucidate the differentiation-associated expression of enzymes catalyzing arachidonic acid metabolism, we measured arachidonate metabolites by reverse-phase high pressure liquid chromatography in monocytoid leukemia (ML-1, THP-1, and U937) and myeloid leukemia (KG-1) cell lines. Undifferentiated ML-1 or THP-1 cells produced trace amounts of eicosanoids via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. Upon differentiation induced by phorbol ester (phorbol 12-myristate 13-acetate [PMA]), metabolites via the COX pathway were increased by 100-fold in ML-1 and THP-1 cells, while the LOX products remained barely detectable. All the COX metabolites were elevated, but thromboxane A2 (TXA2) formation was threefold higher in ML-1 cells than in THP-1 cells. Similar time-related increases in COX metabolites were observed in THP-1 cells induced to differentiate with retinoic acid. Undifferentiated U937 cells were capable of generating a much higher quantity of COX products than ML-1 or THP-1 cells, but, upon PMA-induced differentiation, COX products were increased by only two-fold to threefold over the undifferentiated cells and the total COX products in differentiated U937 cells were only one-seventh of those produced by differentiated ML-1 or THP-1 cells. KG-1 cells had an entirely different metabolic profile. They produced a large quantity of a metabolite coeluted with prostaglandin D2, and PMA had no effect on inducing changes in arachidonic acid (AA) metabolism. Increased COX metabolite formation in differentiated THP-1 and ML-1 cells was due to an enhanced level of prostaglandin H synthase enzyme mass, as measured by Western blot analysis. The TXA synthase activity was also increased by approximately 100-fold in PMA-induced ML-1 cells and 10-fold in THP-1 cells. These findings indicate that increased expression of prostaglandin H and TXA synthase enzymes is a feature of differentiated monocytoid leukemia cell lines.

Arachidonic Acid↗
PubMed1991

Regulation of PGI2 activity by serum proteins: serum albumin but not high density lipoprotein is the PGI2 binding and stabilizing protein in human blood.

Although previous studies have shown that serum albumin binds PGI2 and protects it from rapid degradation, it remains debatable whether it is physiologically important due to its low binding affinity for PGI2. We were intrigued by the observations of Yui et al. (J. Clin. Invest. 82 (1988) 803-807) which suggested that apo A-I of the high density lipoprotein (HDL) is the "serum PGI2 stabilizing factor". To clarify this, we carried out experiments to determine the binding kinetics and parameters of HDL and albumin purified from normal pooled human serum. Despite the use of multiple binding assays, we could not detect any binding activity in HDL2, HDL3 or nascent HDL preparations, nor could we demonstrate any PGI2 protecting activity by these molecules. By contrast, purified albumin exhibited essentially identical binding parameters as the native serum from which the albumin was purified. The binding activity of various albumin preparations was not due to the contamination of apo A-I. Computer simulation analysis also failed to provide evidence to support the notion that HDL bound and prolonged PGI2 activity. To determine whether physiological concentrations of albumin influence PGI2 binding to platelet receptors, we measured PGI2 binding to platelet membrane in the absence and presence of albumin. Albumin at 40 mg/ml increased the KD of PGI2 binding to the receptors by 2-3 fold. These findings indicate that albumin plays a major role in protecting PGI2 activity and regulating its availability for platelet PGI2 receptors.

Apolipoprotein A-I↗
PubMed1991

Isolation of partial complementary DNA encoding human thromboxane synthase.

Thromboxane synthase catalyzes the biosynthesis of thromboxane A2 which plays a key role in the proaggregatory and vasoconstrictive processes. In this communication, we reported the successful cloning of thromboxane synthase cDNA from a human lung cDNA library. Oligonucleotides were synthesized according to the direct amino acid sequence of 2 peptides derived from purified human thromboxane synthase. Polymerase chain reaction was carried out using these oligonucleotides as primers to isolate a complementary DNA from human lung cDNA library. The longest cDNA thus obtained was 687 base pairs in length. Amino acid sequences deduced from the cDNA contained all three peptide sequences reported, confirming the authenticity of the cDNA clone.

Amino Acid Sequence↗
PubMed1991

Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells.

Prostaglandin H (PGH) synthase (EC 1.14.99.1) is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations (0.1-1 micrograms/ml) inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[35S]methionine incorporation into PGH synthase that was induced by IL-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.

Acetaminophen↗
PubMed1991

Conformation of receptor-associated PGI2: an investigation by molecular modeling.

To elucidate the conformation of receptor-associated prostacyclin (PGI2), we first performed structure-activity correlation analysis of over 200 PGI2 analogues and derived from this analysis several crucial features pertaining to structural requirements for PGI2 activity [Ah-lim Tsai and Kenneth K. Wu, Eicosanoids, 2 (1989) 131-143]. These structural features proved to be useful guidelines for selecting 'model molecules' for further investigations by molecular mechanics. By properly selecting four analogues with either rigid or uniquely oriented alpha-side chain structure for geometric fitting, we succeeded in maximally minimizing the degree of freedom of the carboxylate terminus of PGI2. We were able to define the spatial relationship among the four critical functional groups, i.e., C1-COOH, C6a-O, C11-OH and C15-OH. More information is needed, however, to define the geometry of the omega-side chain, particularly for the moiety beyond C15. Nevertheless, results from structure-activity correlation analysis and molecular modeling provide useful information regarding the conformation of receptor-associated PGI2, which assumes an 'elongated' conformation instead of the traditional 'hairpin' structure.

Binding Sites↗
PubMed1991

Population correlates of plasma fibrinogen and factor VII, putative cardiovascular risk factors.

Recent prospective investigations have reported that higher plasma fibrinogen concentrations and higher factor VII coagulant activity are associated with greater risk of cardiovascular disease. To discover what characteristics may influence fibrinogen and factor VII, we analyzed data from the Atherosclerosis Risk in Communities Study obtained from over 12,000 men and women, aged 45-64 years, from four communities in December 1986 to June 1989. Fibrinogen was higher in blacks than whites and in women than men; in general, it increased with age, smoking, body size, diabetes, fasting serum insulin, LDL cholesterol, lipoprotein(a), leukocyte count, and menopause, and it decreased with ethanol intake, physical activity, HDL cholesterol, and female hormone use. Factor VII was higher in women than men and, in women, increased with age; in both sexes, it increased with body size, triglycerides, LDL cholesterol, and HDL cholesterol, and it decreased with ethanol intake. These findings indicate that elevations in fibrinogen and factor VII may be modifiable through appropriate lifestyle changes.

Body Constitution↗
PubMed1990

ARIC hemostasis study--II. Organizational plan and feasibility study.

In our previous paper, we reported the development of a blood collection and processing system (BCPS) suitable for the ARIC multicenter hemostasis study. As an additional step of preparation for the ARIC study, we incorporated this BCPS into an organizational plan to increase efficiency and minimize errors. We initially designed organizational trays for blood collection tubes and aliquot tubes and developed a coordinated step-by-step plan for the orderly processing of blood samples. Once the plan was considered workable, we carried out a pilot study to test the feasibility of this integrated organizational plan. Included in the pilot study were 95 healthy subjects randomly selected from 4 ARIC field centers, whose age and gender were comparable to those projected for the ARIC population. We determined the time lapse of filling the first tube as an index of blood flow. The overall mean time-lapse was 23 s (S.D. = 5). There was no significant difference among the field centers. We also determined the entire time lapse required for completing the sample processing. The total processing time was always less than 60 min. By performing the processing of samples in pairs, all the samples from two subjects could be completely processed in 70 min. This greatly increased the efficiency of field center operation. We evaluated the potential in vitro hemostasis activation by measuring plasma beta-thromboglobulin and platelet factor 4 levels. The geometric means of both proteins were comparable to our previously reported results. Fibrinogen, factor VII, factor VIII, von Willebrand factor, antithrombin III, protein C and activated partial thromboplastin time were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

Arteriosclerosis↗