Background-To date, there has been no systematic examination of the relationship between international normalized ratio (INR) control measurements and the prediction of adverse events in patients with atrial fibrillation on oral anticoagulation.

No abstract

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is the locus of Lesch-Nyhan syndrome, the activator of the prodrugs 6-mercaptopurine and allopurinol, and a target for antiparasitic chemotherapy. The three-dimensional structure of the recombinant human enzyme in complex with GMP has recently been solved [Eads, J., Scapin, G., Xu, Y., Grubmeyer, C., & Sacchettini, J. C. (1994) Cell 78, 325-334]. Here, ligand binding, pre-steady state kinetics, isotope trapping, and isotope exchange experiments are presented which detail the sequential kinetic mechanism of the enzyme. In the forward reaction, in which a base (hypoxanthine or guanine) reacts with PRPP to form nucleoside monophosphate and PPi, binding of PRPP precedes that of the base, and in the reverse direction, IMP binds first. Compared to k(cat), phosphoribosyl group transfer is rapid in both the forward (131 vs 6.0 s(-1)) and reverse (9 vs 0.17 s(-1)) directions. In the forward direction, product pyrophosphate dissociates rapidly (> 12 s(-1)) followed by release of IMP (6.0 s(-1)). In the reverse direction, Hx dissociates rapidly (9.5 s(-1)) and PRPP dissociates slowly (0.24 s(-1)). The more rapid rate of utilization of guanine than hypoxanthine in the forward reaction is the result of the faster release of product GMP rather than the result of differences in the rate of the chemical step. The kinetic mechanism, with rapid chemistry and slow product dissociation, accounts for the previously observed ability of the alternative product guanine to stimulate, rather than inhibit, the pyrophosphorolysis of IMP. The overall equilibrium for the hypoxanthine phosphoribosyl transfer reaction lies far toward nucleotide product (Keq approximately 1.6 x 10(5)), at the high end for PRPP-linked nucleotide formation. The three-dimensional structure of the HGPRTase x IMP complex has been solved to 2.4 A resolution and is isomorphous with the GMP complex. The results of the ligand binding and kinetic studies are discussed in light of the structural data.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyzes the reversible formation of IMP and GMP from their respective bases hypoxanthine (Hx) and guanine (Gua) and the phosphoribosyl donor 5-phosphoribosyl-1-pyrophosphate (PRPP). The net formation and cleavage of the nucleosidic bond requires removal/addition of a proton at the purine moiety, allowing enzymic catalysis to reduce the energy barrier associated with the reaction. The pH profile of kcat for IMP pyrophosphorolysis revealed an essential acidic group with pKa of 7.9 whereas those for IMP or GMP formation indicated involvement of essential basic groups. Based on the crystal structure of human HGPRTase, protonation/deprotonation is likely to occur at N7 of the purine ring, and Lys 165 or Asp 137 are each candidates for the general base/acid. We have constructed, purified, and kinetically characterized two mutant HGPRTases to test this hypothesis. D137N displayed an 18-fold decrease in kcat for nucleotide formation with Hx as substrate, a 275-fold decrease in kcat with Gua, and a 500-fold decrease in kcat for IMP pyrophosphorolysis. D137N also showed lower KD values for nucleotides and PRPP. The pH profiles of kcat for D137N were severely altered. In contrast to D137N, the kcat for K165Q was decreased only 2-fold in the forward reaction and was slightly increased in the reverse reaction. The Km and KD values showed that K165Q interacts with substrates more weakly than does the wild-type enzyme. Pre-steady-state experiments with K165Q indicated that the phosphoribosyl transfer step was fast in the forward reaction, as observed with the wild type. In contrast, D137N showed slower phosphoribosyl transfer chemistry, although guanine (3000-fold reduction) was affected much more than hypoxanthine (32-fold reduction). In conclusion, Asp137 acts as a general catalytic acid/base for HGPRTase and Lys165 makes ground-state interactions with substrates.

Escherichia coli transcription termination protein Rho aids in the release of newly synthesized RNA from paused transcription complexes (reviewed in Ref. 1). The homohexameric protein binds nascent RNA and, with the RNA-dependent hydrolysis of ATP, disrupts the ternary transcription complex, releasing product RNA and allowing RNA polymerase to recycle. The discovery of a 5Ј 3 3Ј RNA-DNA helicase activity of Rho (2) suggested that Rho might disrupt the RNA-DNA duplex of the transcription bubble. Recent studies of ternary transcription complexes (Refs. 3-5 and reviewed in Ref. 6) suggest that such disruption could be important in transcription termination, as could be the release of the nascent RNA just 5Ј of the RNA-DNA duplex from its interactions with RNA polymerase. As described by Nudler et al. (7), the interaction of RNA with RNA polymerase immediately 5Ј from the RNA-DNA hybrid may control the opening and closing of an RNA polymerase clamp around the DNA template near the leading edge of the enzyme, and contribute to the stability of the ternary transcription complex. An appealing model for Rho is one in which the enzyme binds to exposed mRNA behind RNA polymerase and travels 5Ј 3 3Ј along the RNA as it hydrolyzes ATP, binding and releasing RNA from different parts of the hexamer to accomplish movement (8). Such activity could release nascent RNA from RNA polymerase-binding sites and could constitute the basis for its RNA-DNA helicase activity, both of which might be involved in transcript release from paused ternary transcription complexes. The finding that the same number of ATP molecules per RNA length is hydrolyzed by Rho traveling along RNA and Rho unwinding RNA-DNA hybrids (8) supports this hypothesis.Rho binds single-stranded RNA, showing preferred entry regions on RNA upstream of eventual transcription termination sites. However, the characteristics of these regions, beyond low secondary structure and some preference for a C-rich, G-poor base composition (9), are too poorly understood to permit their identification by sequence inspection. When bound to RNA, Rho protects 80 bases from ribonuclease degradation (10, 11). The binding of Rho to 10-base RNA oligomers was reported as best fit by three tight and three weaker sites per hexamer (12, 13).The RNA-dependent hydrolysis of ATP is essential for the transcription termination function of Rho. Two components of ternary transcription complexes, the DNA template and RNA polymerase, are not required to elicit this ATPase activity, thus considerably simplifying study of the reaction (14). The reaction is particularly well stimulated by the RNA homopolymer poly(C), and Rho is frequently assayed in vitro by measuring its poly(C)-dependent ATPase activity. Previous work has shown that the Rho hexamer binds three molecules of MgATP in a single class of catalytically competent sites (15,16). An additional class of three ATP-binding sites of lower affinity has also been suggested (16), although the catalytic activity of these sites was not assessed. The stoic...

Site-directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase~HGPRTase! with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal~one-to threefold! increase in the K m values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies~k cat 0K m ! of the forward and reverse reactions were more severely reduced~6-to 30-fold!, and the mutant enzyme showed positive cooperativity in binding of a-d-5-phosphoribosyl-1-pyrophosphate~PRPP! and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7-hydroxy @4,3-d# pyrazolo pyrimidine~HPP! and Mg PRPP, and the refined structure reported. The PRPP molecule built into the @~F o Ϫ F c !f calc # electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop~residues Leu101 to Gly111! that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis~S103R!. In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.

Since it had been revealed that H. pylori infection pre-exists in gastric carcinoma and precancerous lesions, the results of Meta analysis present a strong evidence to support the conclusion that H. pylori infection is a risk factor for gastric carcinoma.

Background Exposure of the plasma protein factor XII to an anionic surface generates activated factor XII that not only triggers the intrinsic pathway of blood coagulation through the activatio of factor XI, but also mediates various vascular responses through activation of the plasma contact system. While deficiencies of factor XII are not associated with excessive bleeding, thrombosis models in factor deficient animals have suggested that this protein contributes to stable thrombus formation. Therefore, factor XII has emerged as an attractive therapeutic target to treat or prevent pathological thrombosis formation without increasing the risk for hemorrhage. Objectives Utilizing an in vitro directed evolution and chemical biology approach, we sought to isolate a nuclease resistant RNA aptamer that binds specifically to factor XII and directly inhibits factor XII coagulant function. Methods and Results Herein, we describe the isolation and characterization of a high affinity RNA aptamer targeting factor XII/XIIa that dose dependently prolongs fibrin clot formation and thrombin generation in clinical coagulation assays. This aptamer functions as a potent anticoagulant by inhibiting the autoactivation of factor XII, as well as inhibiting intrinsic pathway activation (factor XI activation). However, the aptamer does not affect the factor XIIa-mediated activation of the proinflammatory kallikrein-kinin system (plasma kallikrein activation). Conclusions We have generated a specific and potent factor XII/XIIa aptamer anticoagulant that offers targeted inhibition of discrete macromolecular interactions involved in the activation of the intrinsic pathway of blood coagulation.