Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203580 and PD 98059. SB 203580 also inhibits thromboxane synthase

The kinase inhibitors SB 203580 and PD 98059 have been reported to be specific inhibitors of the 38- and 42/44-kDa mitogen-activated protein kinase (MAPK) pathways, respectively. In this study, the two inhibitors were found to decrease platelet aggregation induced by low concentrations of arachidonic acid, suggesting that they also interfere with the metabolism of arachidonic acid to thromboxane A2. In support of this, SB 203580 and PD 98059 inhibited the conversion of exogenous [3H]arachidonic acid to [3H]thromboxane in intact platelets. Measurement of platelet cyclooxygenase-1 activity following immunoprecipitation revealed that SB 203580 and PD 98059 are direct inhibitors of this enzyme. Both compounds were shown to inhibit purified cyclooxygenase-1 and -2 by a reversible mechanism. In addition, SB 203580 (but not PD 98059) inhibited platelet aggregation induced by prostaglandin H2 and the conversion of prostaglandin H2 to thromboxane A2 in intact platelets. SB 203580 also inhibited this pathway in platelet microsome preparations, suggesting a direct inhibitory effect on thromboxane synthase. These results demonstrate that direct effects of the two kinase inhibitors on active arachidonic acid metabolites have to be excluded before using these compounds for the investigation of MAPKs in signal transduction pathways. This is of particular relevance to studies on the regulation of cytosolic phospholipase A2 as these two MAPKs are capable of phosphorylating cytosolic phospholipase A2, thereby increasing its intrinsic activity.     

Efficacy of cobalt chelates in the rabbit eye model for epithelial herpetic keratitis

STUDY OBJECTIVE: Accurate diagnosis in emergency department patients with possible myocardial ischemia is problematic. Two-dimensional echocardiography has a high sensitivity for identifying patients with myocardial infarction (MI); however, few studies have investigated its diagnostic ability when used acutely in ED patients with possible myocardial ischemia. Therefore we investigated the ability of ED echocardiography for predicting cardiac events in patients with possible myocardial ischemia. METHODS: Echocardiography was performed within 4 hours of ED presentation in 260 patients with possible myocardial ischemia, and was considered positive if there were segmental wall motion abnormalities or the ejection fraction was less than 40%. ECGs were considered abnormal if there was an ST-segment elevation or depression of greater than or equal to 1 mm, or ischemic T-wave inversion. Cardiac events included MI and revascularization. RESULTS: Of the 260 patients studied, 45 had cardiac events (23 MI, 19 percutaneous transluminal angioplasty, 3 coronary bypass surgery). The sensitivity of echocardiography for predicting cardiac events was 91% (95% confidence interval 79% to 97%]), which was significantly higher than the ECG (40% [95% CI 27% to 55%]: P < .0001), although specificity was lower (75% [95% CI 69% to 81%] versus 94% [95% CI 90% to 97%]; P < .001). Addition of the echocardiography results to baseline clinical variables and the ECG added significant incremental diagnostic value (P < .001). With use of multivariate analysis, only male gender (P < .03, odds ratio [OR] 2.4 [1.1 to 5.3]), and a positive echocardiographic finding (P < .0001, OR 24 [9 to 65]) predicted cardiac events. Excluding patients with abnormal ECGs (N = 30) did not affect sensitivity (85%) or specificity (74%) of echocardiography. CONCLUSION: Echocardiography performed in ED patients with possible myocardial ischemia identifies those who will have cardiac events, is more sensitive than the ECG, and has significant incremental value when added to baseline clinical variables and the ECG.     

A natural kinase-deficient variant of fibroblast growth factor receptor 1

A fibroblast growth factor receptor 1 variant missing 37 amino acids from the carboxy-terminal tyrosine kinase catalytic domain was discovered in human lung fibroblasts and several other human cell lines. The receptor variant binds specifically to acidic fibroblast growth factor but has no tyrosine kinase activity. It was found that cellular transfectants expressing the fibroblast growth factor receptor 1 variant are mitogenically inactive and ligand binding to the receptor causes neither receptor autophosphorylation nor phospholipase C-gamma transphosphorylation. The fibroblast growth factor receptor 1 variant therefore represents an inactive receptor for acidic fibroblast growth factor. Since both kinase and kinase-deficient receptor forms are expressed in cells, it is conceivable that the kinase-deficient receptor plays an important role in regulating cellular responses elicited by acidic fibroblast growth factor stimulation.     

Identifying changes in gene expression in the nervous system: mRNA differential display

The majority of cellular and developmental processes are characterized by changes in gene expression. Recently, several polymerase chain reaction-based methods have been introduced for detecting genes whose expressions differ between cells or tissues. It is now possible to investigate whether novel gene expression occurs within many systems as a response to extracellular signals, and to identify systematically those genes. The characterization of cell-, tissue- or stage-specific gene expression will broaden our understanding of many complex biological processes.     

Initial processing of human proenkephalin in bovine chromaffin cells

The opioid peptide precursor preproenkephalin (PPE) contains seven enkephalin sequences and is synthesized by epinephrine-producing adrenal chromaffin cells and various peripheral and central neurons. After removal of its signal peptide, PPE undergoes processing at dibasic amino acid sites to yield its final opioid products-Met-enkephalin, Leu-enkephalin, and various larger, enkephalin-containing peptides. Processing of PPE was examined in bovine chromaffin cells using a plasmid containing the human PPE (hPPE) cDNA under the control of the cytomegalovirus immediate early enhancer/promoter. Following transfection of this hPPE-containing plasmid into bovine chromaffin cells, several proenkephalin-immunoreactive bands were observed on western blots with monoclonal antibodies that recognize human, but not bovine, proenkephalin sequences. The pattern of hPPE-derived peptides observed was similar to that of bovine PPE processing products. A series of recombinant plasmids containing mutations in the hPPE sequence at putative processing sites was then constructed. Conversion of Lys-Lys and Lys-Arg sequences to Lys-Gln and of Arg-Arg to Arg-Gln altered initial hPPE processing at only three of the putative processing sites. When hPPE cDNA containing mutations at all of these initially processed sites was expressed, one or more alternative processing sites were revealed. These data suggest the importance of structural features in addition to the dibasic sequences that limit the processing of proenkephalin.     

On the genetics of hypodontia and microdontia: synergism or allelism of major genes in a family with six affected members

Plasma beta-endorphin (beta-E) concentration was determined before, during, and after a standardized incremental exercise test to maximal capacity in eight type I diabetic patients and eight normal control subjects. Diabetic patients were studied under normoglycemic and hyperglycemic conditions in a single-blind random fashion to differentiate between the effects of acute hyperglycemia and of diabetes per se on the beta-E response to exercise. The perceived magnitude of leg effort elicited by exercise was evaluated using a category scale. Whereas plasma beta-E concentrations increased in control subjects with increasing workload, causing significantly higher beta-E levels at the end of exercise than at the beginning (P < .001), no such increase could be observed in the diabetic patients under normoglycemic and hyperglycemic conditions. In addition, baseline plasma beta-E concentrations were significantly lower in normoglycemic (P < .01) and hyperglycemic (P < .001) diabetic patients than in control subjects. Even during the recovery period, patients' beta-E levels remained significantly lower than those of control subjects. At submaximal levels of power output, the perceived intensity of leg effort was significantly higher in normoglycemic and hyperglycemic diabetic patients than in control subjects. We conclude that in type I diabetic patients, the ability of the endogenous opioid system to respond to exercise-induced stress is impaired under hyperglycemic and even under normoglycemic conditions. Considering the effect of endogenous opioids on stress tolerance, such changes may compromise exercise performance in diabetic patients.