Importance of the Arg-Gly-Asp triplet in human thrombin for maintenance of structure and function

Site-directed mutagenesis was employed to assess the importance of the Arg-Gly-Asp triplet that comprises residues 197 to 199 in the B-chain of thrombin. Properties of the R197E and the D199E variants were compared with those of zeta-thrombin and the inactive S205A variant wherein the active site Ser is replaced by Ala. Relative to zeta-thrombin, the R197E thrombin variant under the assay conditions used exhibits 26% activity toward a small chromogenic substrate, 13% activity in the activation of protein C in the presence of thrombomodulin, < 3% activity in processing fibrinogen, and 1% activity in inducing platelet activation. Thus, the substrate specificity of thrombin was altered by the R197-->E replacement. The D199E variant was essentially inactive. It exhibited only 0.02% of the activity of thrombin toward the chromogenic substrate and its reactivity toward the active site-directed alkylating agent D-Phe-Pro-Arg-CH2Cl was 10,000-fold lower than that of thrombin. Like the inactive S205A thrombin variant, the D199E variant antagonized the interactions of thrombin with hirudin and thrombomodulin, but was a less effective antagonist. The dependence of the antagonism of the thrombin-thrombomodulin interaction on the concentration of D199E thrombin variant provided evidence suggesting the presence of two or more domains in thrombin that independently interact with their counterparts in thrombomodulin. Although the S205A thrombin variant antagonized the action of thrombin on platelets no such activity could be demonstrated for the D199E variant in the concentration range studied (< 800 nm). Comparison of the circular dichroism spectra of zeta-thrombin, the D199E, R197E, and S205A variants indicated that subtle differences in conformation exist between the D199E variant and the other thrombins. These differences in conformation might well account for the altered behavior of the D199E variant with respect to its interactions toward thrombomodulin, hirudin, and platelets.     

Further studies of the mixed acetals of nucleosides

We reported in 1988 on a new nucleoside modification reaction: the exocyclic amino groups of (d)adenosine and (d)cytidine react rapidly at ambient temperature with acetaldehyde and alcohols to give stable mixed acetals (N-ethylethoxy-acetal). NH2 + O = CH(CH3) + ROH-->NH-CH(CH3)-O-R + H2O. Here we report in detail on the occurrence of this reaction in very dilute aqueous solution (ie under biological conditions), on its mechanism and kinetics, on the mixed acetal formation with other aldehydes and other nucleic acid components, and on the question of whether these adducts are mutagenic.     

Superior vena cava syndrome after heart transplantation: percutaneous treatment of a complication of bicaval anastomoses

The aims of this study were to establish a working rabbit heart model of regional myocardial ischaemia in which electrophysiologic parameters and arrhythmogenesis could be correlated and to explore the mechanisms underlying the antiarrhythmic activity of lignocaine. Monophasic action-potential duration (MAPD90), effective refractory period (ERP), and conduction delay were measured at three ventricular sites in isolated hearts paced at 3.3 Hz. The hearts were treated before and throughout 30 min of ischaemia and 15 min of reperfusion with a vehicle or 20 microM lignocaine. In both groups, ischaemia produced a similar shortening in MAPD90. Lignocaine decreased ERP shortening during ischaemia from -56+/-4 to -32+/-6 ms. An ischaemia-induced increase in conduction delay was greater in the lignocaine than the control group (49+/-7 vs. 11+/-2 ms). Ischaemia-induced dispersion of repolarisation was reduced by lignocaine from 66+/-4 to 32+/-7 ms, and dispersion of refractoriness was decreased from 57+/-6 to 16+/-3 ms. Lignocaine decreased inducibility of ventricular fibrillation (VF) during ischaemia from 86 to 25%. We conclude that, in this model, the antiarrhythmic activity of lignocaine during regional ischaemia is associated with an increase in ischaemia-induced conduction delay and reduced dispersion of repolarisation and refractoriness.     

Masking-level differences in older adults: the effect of the level of the masking noise

In Experiment 1, masking-level differences (MLDs) for a 500-Hz tone at five masker levels were obtained from younger and older adults. For both age groups, there were no reliable increases in MLD once the spectrum level of the masker exceeded 27 dB SPL. MLDs were larger for younger than for older adults over the range of masker levels tested. In Experiment 2, the levels of both the signal and the masker in one ear were attenuated by either 15 or 30 dB relative to their level in the other ear, which was fixed at a spectrum level of 47 dB SPL. MLDs for both age groups declined with increasing IAA and age-related differences were observed in all conditions. The findings of these experiments indicate that (1) age-related differences in MLDs exist even when the level of the masker is sufficiently high that older adults achieve their plateau performance, and (2) older listeners are not disadvantaged more than younger listeners by interaural differences in the level of the input.     

Radioimmunoscintigraphy with In-111-labeled capromab pendetide predicts prostate cancer response to salvage radiotherapy after failed radical prostatectomy

PURPOSE: We investigated the ability of In-111-capromab pendetide to separate patients who have failed radical prostatectomy into categories of those who would versus those who would not respond to salvage radiotherapy. METHODS: Prostate-specific antigen (PSA) levels in 32 men with prostate cancer who had failed radical prostatectomy and had undergone a whole-body In-111-capromab pendetide scan were followed-up for 13 months (median) after salvage radiotherapy to the pelvis. A logistic regression model was used to determine whether the scan findings, as well as other clinical variables, were associated with a durable complete response (DCR), a nondurable response (NDR), or no response (NR). RESULTS: Sixteen of 23 (70%) men with a normal scan outside the prostatic fossa achieved a DCR after salvage radiotherapy versus two of nine (22%) who had a positive scan outside the prostate fossa and pelvis (P = .0225, Fisher's exact test). Predicted probability (95% confidence interval [CI]) that a DCR would be obtained with a normal scan was 0.88 (0.55 to 0.98); for men with a positive scan limited to the prostatic fossa it was 0.62 (0.42 to 0.79); and for men with a positive scan outside the pelvis it was 0.27 (0.09 to 0.58). No other variables before radiotherapy showed a significant association with the DCR rate. CONCLUSION: Salvage radiotherapy is statistically more likely to lead to a durable complete PSA response in men with prostate cancer who have failed radical prostatectomy and have a negative In-111-capromab pendetide scan outside the pelvis as compared with those who have a positive scan.     

Early child-care selection: variation by geographic location, maternal characteristics, and family structure

More than half of all U.S. infants and toddlers spend at least 20 hr per week in the care of a nonparent adult. This article uses survival analysis to identify which families are most likely to place their child in care and the ages when these choices are made, using data from a national probability sample of 2,614 households. Median age at first placement is 33 months, but age varies by geographic region, mother's employment status during pregnancy, mother's education level, and family structure (1 vs. 2 parents, mother's age at 1st birth, and number of siblings). Controlling for these effects, differences by race and ethnicity are small. Implications for studies of child-care selection and evaluations of early childhood programs are discussed.     

The use of controlled-release oxycodone for the treatment of chronic cancer pain: a randomized, double-blind study

1. We have studied the effects of muscarinic cholinoceptor agonists and subtype-preferring antagonists on the isometric contraction of smooth muscle strips from dog prostate. 2. Acetylcholine and carbachol induced contraction of prostate strips from the peripheral zone, ('the capsule'). Bethanechol contracted the tissue but not at lower doses. McN-A-343 and oxotremorine-M showed the same effects. 3. Blocking alpha- and beta-adrenoceptors with phentolamine and propranolol, respectively, did not modify carbachol-induced contractions. 4. The nicotinic receptor blocker, hexamethonium (10(-6)-10(-4) M) did not affect the contractile response evoked by a single dose of carbachol (10(-5) M), whilst the muscarinic receptor antagonist, atropine (10(-11)-10(-9) M), inhibited it in a competitive manner. 5. The muscarinic M1 (pirenzepine), M2 [AF-DX 116, himbacine (M2/M4) and methoctramine], M3 (HHSID and f-F-HHSID), and putative M4 (tropicamide) antagonists reduced significantly the carbachol-induced contractions. The pIC50 values were: atropine (10.01) > himbacine (8.3) > methoctramine (7.85) > AF-DX 116 (7.60) > HHSID (7.21) > p-F-HHSID (7.10) > pirenzepine (7.30) > tropicamide (7.00). 6. The antagonist profile indicates that an predominant M2 receptor subtype could mediate the muscarinic contraction in the canine prostate.