Detailed active site configuration of a new crystal form of methanol dehydrogenase from Methylophilus W3A1 at 1.9 A resolution

The three-dimensional structure of a new crystal form of methanol dehydrogenase from Methylophilus W3A1 has been obtained in the presence of substrate using data recorded at a synchrotron. The structure of this approximately 140 kDa heterotetramer, refined at 1. 9 A resolution, reveals the detailed configuration of its redox cofactor, pyrroloquinoline quinone (PQQ). C4, one of the oxygen-bearing atoms of this orthoquinone is in a planar configuration while C5, which bears the other quinone oxygen, is tetrahedral, suggesting that the PQQ is in the semiquinone redox state. The substrate binding site has been identified close to PQQ and to the side chain of Asp297, the putative active site base. The proximity of the hydroxyl of methanol to C5 of PQQ compared to the greater separation of the substrate methyl group from C5 supports the addition-elimination reaction mechanism involving a hemiketal intermediate.     

Crystal structure of TNF-alpha mutant R31D with greater affinity for receptor R1 compared with R2

Crystal structures have been determined of recombinant human tumor necrosis factor-alpha (TNF-alpha) and its R31D mutant that preferentially binds to TNF receptor R1 with more than seven times the relative affinity of binding to receptor R2. Crystals of the wild-type TNF were of space group P4(1)2(1)2 and had unit cell dimensions of a = b = 94.7 and c = 117.4 A. Refinement of the structure gave an R-factor of 22.3% at 2.5 A resolution. The crystals of TNF R31D mutant diffracted to 2.3 A resolution, and were of identical space group to the wild type with unit cell dimensions of a = b = 95.4 and c = 116.2 A, and the structure was refined to an R-factor of 21.8%. The trimer structures of the wild-type and mutant TNF were similar with a root mean square (r.m.s.) deviation of 0.56 A for Calpha atoms; however, the subunits within each trimer were more variable with an average r.m.s. deviation of 1.00 A on Calpha atoms for pairwise comparison of subunits. Model complexes of TNF with receptors R1 and R2 have been used to predict TNF-receptor interactions. Arg31 in all three subunits of wild-type TNF is predicted to form an ionic interaction with the equivalent glutamic acid in both receptors R1 and R2. Asp31 of the TNF R31D mutant is predicted to interact differently with the two receptors. The side chain of Asp31 in two subunits of the TNF mutant is predicted to form hydrogen bond interactions with Ser59 or Cys70 of R1, while it has no predicted interactions with R2. The loss of three strong ionic interactions of Arg31 and the electrostatic repulsion of Asp31 with Glu in the receptors is consistent with the reduced binding of the R31D mutant to both receptors relative to wild-type TNF. The replacement of these ionic interactions by two weaker hydrogen bond interactions between Asp31 of the R31D mutant and R1, compared with no interactions with R2, is in agreement with the observed preferential binding of the R31D mutant to R1 over R2. Analysis of the structure and function of receptor-discriminating mutants of TNF will help understand the biological role of TNF and facilitate its use as an antitumor agent.      

Comparison of the single-dose pharmacokinetics and tolerability of modafinil and dextroamphetamine administered alone or in combination in healthy male volunteers

An open-label, randomized, crossover study was performed in healthy male volunteers to evaluate the potential pharmacokinetic and pharmacodynamic interactions and tolerability of single oral doses of modafinil (200 mg) and dextroamphetamine (10 mg). Blood samples were collected for determination of plasma levels of modafinil, the acid and sulfone metabolites of modafinil, and dextroamphetamine at intervals through 48 hours after administration for each treatment. Vital signs (blood pressure and pulse rate) were measured through 48 hours, and electrocardiograms were measured through 24 hours after administration. Pharmacokinetic parameters were determined using noncompartmental methods. The data collected in this study of 24 healthy volunteers suggest that concomitant administration of single oral doses of modafinil and dextroamphetamine has no clinically significant effects on the pharmacokinetic profile of either agent. Although there was a slightly greater incidence of adverse events when modafinil and dextroamphetamine were administered together, the concomitant administration of the two drugs was well tolerated.     

Effect of medication on biomechanical properties of rabbit bones: heparin induced osteoporosis

BACKGROUND: We hypothesize that the integrity of the latissimus dorsi muscle graft used to wrap the heart may affect the clinical outcome of patients undergoing dynamic cardiomyoplasty. METHODS: By correlating the pathologic findings with their clinical course in five patients who died 1 month to 6 years after dynamic cardiomyoplasty operation, we sought to discern findings that might shed light on the pathophysiology of cardiomyoplasty. RESULTS: Of the two patients who had a limited clinical response, one had an atrophic, edematous latissimus dorsi muscle with fatty infiltration resulting from cardiac cachexia, and the other had insufficient length of latissimus dorsi muscle to cover a large heart. The remaining patients responded well clinically without signs of pump failure and died at various intervals, mostly of arrhythmias. Autopsy findings included the following: (1) one patient with ischemic cardiomyopathy as the underlying disease had development of rich vascularity in the interface between the muscle wrap and the epicardium; whereas in four others with idiopathic cardiomyopathy, such evidence of collateralization was far less evident. (2) There was a variation in the skeletal muscle transformation achieved, with the fraction type I fatigue-resistant fiber in the muscle wrap ranging from 60% to 100%, in spite of the identical transformation protocol used. Such variation is believed to be genetically based. (3) In one patient, the skeletal muscle was paced to contract at 30 to 50 times/minute (2:1 ratio) for more than 5 years. Nevertheless, the pathologic specimen of the muscle wrap showed only minimal interstitial fibrosis. (4) Relatively thin muscle wrap around the heart found at autopsy could be atrophy but most likely was related to muscle transformation, which is known to reduce muscle mass and increase capillary density. (5) All skeletal muscle grafts showed geometric conformation to the shape of the epicardium and grossly looked as if they were an additional layer of the ventricular wall. Such conformation may facilitate the modulation of the ventricular remodelling process in the failing heart, as has been described both in clinical and experimental studies. CONCLUSIONS: Our findings are consistent with and support a number of mechanisms proposed for cardiomyoplasty. Thus preservation of latissimus dorsi muscle graft integrity may be important in the success of dynamic cardiomyoplasty.