No abstract

A comparative theoretical study of a bimolecular reaction in aqueous solution and catalyzed by the enzyme catechol O-methyltransferase (COMT) has been carried out by a combination of two hybrid QM/MM techniques: statistical simulation methods and internal energy minimizations. In contrast to previous studies by other workers, we have located and characterized transition structures for the reaction in the enzyme active site, in water and in a vacuum, and our potential of mean force calculations are based upon reaction coordinates obtained from features of the potential energy surfaces in the condensed media, not from the gas phase. The AM1/CHARMM calculated free energy of activation for the reaction of S-adenosyl methionine (SAM) with catecholate catalyzed by COMT is 15 kcal mol(-1) lower the AM1/TIP3P free-energy barrier for the reaction of the trimethylsulfonium cation with the catecholate anion in water at 300 K, in agreement with previous estimates. The thermodynamically preferred form of the reactants in the uncatalyzed model reaction in water is a solvent-separated ion pair (SSIP). Conversion of the SSIP into a contact ion pair, with a structure resembling that of the Michaelis complex (MC) for the reaction in the COMT active site, is unfavorable by 7 kcal mol(-1), largely due to reorganization of the solvent. We have considered alternative ways to estimate the so-called "cratic" free energy for bringing the reactant species together in the correct orientation for reaction but conclude that direct evaluation of the free energy of association by means of molecular dynamics simulation with a simple standard-state correction is probably the best approach. The latter correction allows for the fact that the size of the unit cell employed with the periodic boundary simulations does not correspond to the standard state concentration of 1 M. Consideration of MC-like species allows a helpful decomposition of the catalytic effect into preorganization and reorganization phases. In the preorganization phase, the substrates are brought together into the MC-like species, either in water or in the enzyme active site. In the reorganization phase, the roles of the enzymic and aqueous environments may be compared directly because reorganization of the substrate is about the same in both cases. Analysis of the electric field along the reaction coordinate demonstrates that in water the TS is destabilized with respect to the MC-like species because the polarity of the solute diminishes and consequently the reaction field is also decreased. In the enzyme, the electric field is mainly a permanent field and consequently there is only a small reorganization of the environment. Therefore, destabilization of the TS is lower than in solution, and the activation barrier is smaller.

The aims of the present study were (a) to maintain the structure and function of the small intestine of the piglet after weaning, and (b) to compare the capacity in vivo of sucking and weaned piglets to digest oral boluses of lactose and sucrose and absorb their monosaccharide products. Piglets were fed on cows' whole milk ad libitum every 2 h for 5 d after weaning. Physiological doses of lactose plus fructose (treatment LAC + FRU) and sucrose plus galactose (treatment SUC + GAL) were administered on day 27 of lactation and on the fifth day after weaning, after which time piglets were killed. Villus height and crypt depth were maintained (P > 0.05) by feeding cows' milk after weaning. The areas under the curves (AUC) for galactose and glucose, adjusted for live weight and plasma volume, increased (P < 0-05) after weaning. Despite the enhancement of gut function after weaning, the galactose index (Gall: AUC for galactose ingested as lactose divided by the AUC for the same dose of galactose ingested as the monosaccharide) and fructose index (FruI: AUC for fructose ingested as sucrose divided by the AUC for the same dose of fructose ingested as the monosaccharide), which are indices of digestive and absorptive efficiency, both decreased after weaning. This apparent anomaly may be reconciled by increased growth, and hence surface area, of the small intestine between weaning and slaughter such that 'total' digestion and absorption most probably increased despite apparent decreases in GalI and FrnI.Positive correlations (P < 0.05) between villus height and GalI are consistent with the maximum activity of lactase occurring more apically along the villus. Significant linear relationships (P < 0.05) were recorded between villus height at the proximal jejunum and adjusted AUC for galactose and glucose following treatment LAC + FRU, and between villus height at the proximal jejunum and adjusted glucose AUC following treatment SUC + GAL. These relationships suggest that maximum digestion and absorption occurs at increasing distances along the crypt:villus axis in the weaned pig.

(3) and (4) (P < 0-05 and P = 0-073, respectively). In turn, villous height was significantly correlated (r = 0-78 to 0-87, P < 0-05) with the rate of bodyweight gain after weaning in these two groups. For piglets offered ewes' milk plus glutamine, an increase in DM intake was associated only with increases in crypt depth (P < 0-01). These data show that the structure and function of the small intestine can be preserved when a milk diet is given after weaning, and suggest an association between food intake and villous height in determining post-weaning weight gain. were offered ewes' fresh milk every 2 h in a feeding schedule that increased from 1-2 I per piglet on the 1st day after weaning to 2-4 I on days 4 and 5. On the 5th day all piglets were killed and samples of small intestine were taken for histological and biochemical examination. Feeding ewes' milk or ewes' milk plus 20 g L-glutamine per I maintained (P > 0-05) villous height and crypt depth compared with piglets killed at weaning. In contrast, piglets given a dry starter diet had shorter villi (P < 0-001), deeper crypts (P < 0-001), and proportionately 0-21 to 0-28 less protein (P>0-05) in their intestinal mucosa. Piglets given the starter diet proportionately grew from 0-49 to 0-62 more slowly (P < 0-01), ate the same amount of dry matter (DM; P > 0-05), but consumed proportionately 0-30 less energy (P < 0-001) than their counterparts given the milk diets. No treatment differences in the specific activity of lactase and sucrase were observed (P>0-05). Significant correlations existed between voluntary food intake and villous height at the proximal jejunum for piglets given the starter diet and ewes' milk

The hypothesis tested in this experiment was that the structure and function of the small intestine of piglets given a milk liquid diet after weaning depends on their level of energy intake. At weaning (28 days)

Intracellular accumulation of the protease inhibitors (PIs) saquinavir (SQV), ritonavir (RTV), and indinavir (IDV) was determined in 50 human immunodeficiency virus-positive patients. Following extraction, PIs were quantified by mass spectrometry. Paired plasma and intracellular samples were collected over a full dosing interval from patients (13 on SQV, 6 on RTV, 8 on IDV, 16 on SQV plus RTV, 7 on IDV plus RTV) with a plasma viral load of <400 copies/ml. Data were expressed as intracellular/plasma drug concentration ratios. A hierarchy of intracellular accumulation was demonstrated by the following medians: 9.45 for SQV > 1.00 for RTV > 0.51 for IDV. Coadministration of RTV did not boost ratios of SQV or IDV within the cell or in plasma, although absolute plasma and intracellular SQV concentrations were increased by RTV. Seven individuals receiving SQV in hard-gel capsule form (median, 32 months) had higher intracellular/plasma drug ratios than all other patients receiving SQV (median, 17.62 versus 4.83; P ‫؍‬ 0.04), despite consistently low plasma SQV concentrations. How this occurs may provide insight into the mechanisms that limit adequate drug penetration into sanctuary sites.The failure to achieve and maintain suppression of human immunodeficiency virus (HIV) replication is emerging as a major problem in antiretroviral therapy (ART). One large cohort study has recently reported a mean time before virological failure on first-ART-regimen drugs of 12 months but with markedly decreasing durability for each successive regimen (F. Palella, J. Chmiel, M. Deloria-Knoll, A. Moorman, S. Holmberg, and the HIV Outpatient Investigators, 8th Conf. Retrovir. Opportunistic Infect., abstr. 268B, 2001). Treatment failure is multifactorial and includes viral resistance, poor adherence, and pharmacological and host factors. Much interest has been generated by potential pharmacological mechanisms of failure. HIV replicates within cells; therefore, drugs must penetrate intracellularly at concentrations sufficient to inhibit viral replication. Failure to do so results in the establishment of a sanctuary site where virus may evolve in the absence of selection pressure from the drug (22) or where subtherapeutic levels generate drug-resistant virus with subsequent "seeding" into plasma. Virus from sanctuary sites, such as the central nervous system and seminal fluid, can exhibit genotypic resistance profiles, which differ from peripheral blood isolates (6, 21). Pharmacological studies that examine the cellular and tissue penetration of HIV drugs are crucial to the understanding of sanctuary sites and the subsequent evolution of drug resistance and the failure of ART. This understanding may inform the design of strategies to maximize drug potency. In particular, the roles of cellular efflux transporters such as the P-glycoprotein (P-gp) in limiting the intracellular penetration of drugs and the potential for ritonavir (RTV) to boost intracellular drug accumulation within sanctuary sites deserve further investigation.We have previou...

Increased expression of P-glycoprotein and MRP1 on lymphocytes is associated with lower intracellular accumulation of saquinavir and ritonavir. These two transporters may play a role in the efflux of ritonavir and saquinavir from lymphocytes in vivo.

Surface-active asphaltene molecules are naturally found in crude oil, causing serious problems in the petroleum industry by stabilizing emulsion drops, thus hindering the separation of water and oil. Asphaltenes can adsorb at water-oil interfaces to form viscoelastic interfacial films that retard or prevent coalescence. Here, we measure the evolving interfacial shear rheology of water-oil interfaces as asphaltenes adsorb. Generally, interfaces stiffen with time, and the response crosses over from viscous-dominated to elastic-dominated. However, significant variations in the stiffness evolution are observed in putatively identical experiments. Direct visualization of the interfacial strain field reveals significant heterogeneities within each evolving film, which appear to be an inherent feature of the asphaltene interfaces. Our results reveal the adsorption process and aged interfacial structure to be more complex than that previously described. The complexities likely impact the coalescence of asphaltene-stabilized droplets, and suggest new challenges in destabilizing crude oil emulsions.