[Unilateral compartment syndrome secondary to prolonged lithotomy positioning and peripheral vascular disease].
Explore the source record for details and available documents.
Adenocarcinoma↗
Biomedical subjects
A Planas
Publications and source records attributed to A Planas.
Hydrolase and glycosynthase activity of endo-1,3-beta-glucanase from the thermophile Pyrococcus furiosus.
Pyrococcus furiosus laminarinase (LamA, PF0076) is an endo-glycosidase that hydrolyzes beta-1,3-glucooligosaccharides, but not beta-1,4-gluco-oligosaccharides. We studied the specificity of LamA towards small saccharides by using 4-methylumbelliferyl beta-glucosides with different linkages. Besides endo-activity, wild-type LamA has some exo-activity, and catalyzes the hydrolysis of mixed-linked oligosaccharides (Glcbeta4Glcbeta3Glcbeta-MU (Glc = glucosyl, MU = 4-methylumbelliferyl)) with both beta-1,4 and beta-1,3 specificities. The LamA mutant E170A had severely reduced hydrolytic activity, which is consistent with Glu170 being the catalytic nucleophile. The E170A mutant was active as a glycosynthase, catalyzing the condensation of alpha-laminaribiosyl fluoride to different acceptors. The best condensation yields were found at pH 6.5 and 50 degrees C, but did not exceed 30%. Depending on the acceptor, the synthase generated either a beta-1,3 or a beta-1,4 linkage.
Amino Acid Substitution↗
[Prognostic factors of early morbidity and mortality after lung transplantation].
OBJECTIVES: Despite years of experience with lung transplantation, the rate of perioperative mortality remains high. The objective of this study was to look at our experience in the early postoperative period following lung transplantation in an effort to identify possible pre-, intra- and postoperative risk factors associated with mortality. PATIENTS AND METHODS: A retrospective study of 68 consecutive patients receiving lung transplants over a period of 56 months. The conditions that led to transplantation were obstructive disease (40%), interstitial disease (33%) and suppurative disease (27%). Pre-, intra- and postoperative characteristics of donors and recipients were analyzed for their relation to morbidity and mortality. Statistical studies were done using SPSS 10.0 software. A p-value less than.05 was considered significant. Univariate analysis identified variables associated with the incidence of mortality in the postoperative recovery unit, and the variables with statistically significant associations were entered into multivariate analysis, using a logistic regression model to calculate odds ratio (OR) and 95% confidence intervals (CI). RESULTS: No donor variables correlated with mortality. Patients with suppurative lung disease had a lower mortality rate (0% vs 30%; P = 0.04). Mortality was related to ischemic time longer than 300 minutes (OR = 2) and the use of extracorporeal circulation (OR = 4). A PaO2/FiO2 ratio less than 150 during the first 24 hours following transplantation (OR = 5) and reoperation due to bleeding (OR = 12) were the variables showing the highest correlations with mortality during the early postoperative period. CONCLUSIONS: The mortality rate during the early postoperative period in our series was 22%. The survival rate was better in patients with suppurative lung disease. Bleeding that required reoperation and early graft dysfunction (defined in part by a PaO2/FiO2 ratio less than 150 during the first 24 hours) were the variables that best predicted death in the early postoperative period following lung transplantation.
Adolescent↗
Oligosaccharide synthesis by coupled endo-glycosynthases of different specificity: a straightforward preparation of two mixed-linkage hexasaccharide substrates of 1,3/1,4-beta-glucanases.
Glycosynthases are engineered glycosidases which are hydrolytically inactive yet efficiently catalyse transglycosylation reactions of glycosyl fluoride donors, and are thus promising tools for the enzymatic synthesis of oligosaccharides. Two endo-glycosynthases, the E134A mutant of 1,3/1,4-beta-glucanase from Bacillus licheniformis and the E197A mutant of cellulase Cel7B from Humicola insolens, were used in coupled reactions for the stepwise synthesis of hexasaccharide substrates of 1,3/1,4-beta-glucanases. Because the two endo-glycosynthases show different specificity, towards laminaribiosyl and cellobiosyl donors, respectively, the target hexasaccharides were prepared by condensation of the corresponding disaccharide building blocks through sequential addition of the glycosynthases in a "one-pot" process. Different strategies were used to achieve the desired transglycosylation between donor and acceptor in each step, and to prevent unwanted elongation of the first condensation product and polymerization (self-condensation) of the donor: 1) selection of disaccharide donors differing in the configuration of the hydroxyl substituent normally acting as acceptor, 2) temporary protection of the polymerizable hydroxyl group of the donor, or 3) addition of an excess of acceptor to decrease the probability that the donor can act as an acceptor. The best procedure involved the condensation of alpha-lactosyl or 4II-O-tetrahydropyranyl-alpha-cellobiosyl fluorides with alpha-laminaribiosyl fluoride, catalyzed by E197A Cel7B, to give tetrasaccharide fluorides, which were then the donors for in situ condensation with methyl beta-cellobioside catalyzed by E134A 1,3/1,4-beta-glucanase. After isolation, the final hexasaccharides Gal/beta4Glcbeta4Glcbeta3Glcbeta4Glcbeta4Glcbeta-OMe and Glcbeta4Glcbeta4Glcbeta3Glcbeta4Glcbeta4-Glcbeta-OMe were obtained in 70-80% overall yields.
Carbohydrate Sequence↗
Isolation and characterization of a thermostable endo-beta-glucanase active on 1,3-1,4-beta-D-glucans from the aerobic fungus talaromyces emersonii CBS 814.70.
A novel endoglucanase active on 1,3-1,4-beta-D-glucans was purified to apparent homogeneity from submerged cultures of the moderately thermophilic aerobic fungus Talaromyces emersonii CBS 814.70. The enzyme is a single subunit glycoprotein with M(r) and pI values of 40.7 +/- 0.3 kDa and 4.4, respectively, and an estimated carbohydrate content of 77% (w/w). The purified beta-glucanase displayed activity over broad ranges of pH and temperature, yielding respective optima values of pH 4.8 and 80 degrees C. This enzyme was markedly thermostable with 15% of the original activity remaining after incubation for 15 min at 100 degrees C. Substrate specificity studies revealed the identity of the enzyme to be a 1,3-1,4-beta-D-glucanase. Identical K(m) values (13.38 mg.ml(-1)) were obtained with lichenan and BBG, while the V(max) value with lichenan (142.9 IU.mg(-1)) was approximately twice the value obtained with BBG (79.3 IU.mg(-1)). Time-course hydrolysis of barley-beta-glucan did not proceed linearly with respect to time indicating an 'endo' or more processive action for the enzyme. HPAEC fractionation of the products of hydrolysis yielded a range of oligosaccharides, with cellobiose, cellotriose and cellotetraose being the predominant oligosaccharide products.
Journal Article↗
Presteady-state kinetics of Bacillus 1,3-1,4-beta-glucanase: binding and hydrolysis of a 4-methylumbelliferyl trisaccharide substrate.
In the present study the first stopped-flow experiments performed on Bacillus 1,3-1,4-beta-glucanases are reported. The presteady-state kinetics of the binding of 4-methylumbelliferyl 3-O-beta-cellobiosyl-beta-D-glucoside to the inactive mutant E134A, and the wild-type-catalysed hydrolysis of the same substrate, were studied by measuring changes in the fluorescence of bound substrate or 4-methylumbelliferone produced. The presteady-state traces all showed an initial lag phase followed by a fast monoexponential phase leading to equilibration (for binding to E134A) or to steady state product formation (for the wild-type reaction). The lag phase, with a rate constant of the order of 100 s(-1), was independent of the substrate concentration; apparently an induced-fit mechanism governs the formation of enzyme-substrate complexes. The concentration dependencies of the observed rate constant of the second presteady-state phase were analysed according to a number of reaction models. For the reaction of the wild-type enzyme, it is shown that the fast product formation observed before steady state is not due to a rate-determining deglycosylation step. A model that can explain the observed results involves, in addition to the induced fit, a conformational change of the productive ES complex into a form that binds a second substrate molecule in a non-productive mode.
Amino Acid Substitution↗
Mechanism of the family 1 beta-glucosidase from Streptomyces sp: catalytic residues and kinetic studies.
The Streptomyces sp. beta-glucosidase (Bgl3) is a retaining glycosidase that belongs to family 1 glycosyl hydrolases. Steady-state kinetics with p-nitrophenyl beta-D-glycosides revealed that the highest k(cat)/K(M) values are obtained with glucoside (with strong substrate inhibition) and fucoside (with no substrate inhibition) substrates and that Bgl3 has 10-fold glucosidase over galactosidase activity. Reactivity studies by means of a Hammett analysis using a series of substituted aryl beta-glucosides gave a biphasic plot log k(cat) vs pK(a) of the phenol aglycon: a linear region with a slope of beta(lg) = -0.8 for the less reactive substrates (pK(a) > 8) and no significant dependence for activated substrates (pK(a) < 8). Thus, according to the two-step mechanism of retaining glycosidases, formation of the glycosyl-enzyme intermediate is rate limiting for the former substrates, while hydrolysis of the intermediate is for the latter. To identify key catalytic residues and on the basis of sequence similarity to other family 1 beta-glucosidases, glutamic acids 178 and 383 were changed to glutamine and alanine by site-directed mutagenesis. Mutation of Glu178 to Gln and Ala yielded enzymes with 250- and 3500-fold reduction in their catalytic efficiencies, whereas larger reduction (10(5)-10(6)-fold) were obtained for mutants at Glu383. The functional role of both residues was probed by a chemical rescue methodology based on activation of the inactive Ala mutants by azide as exogenous nucleophile. The E178A mutant yielded the beta-glucosyl azide adduct (by (1)H NMR) with a 200-fold increase on k(cat) for the 2,4-dinitrophenyl glucoside but constant k(cat)/K(M) on azide concentration. On the other hand, the E383A mutant with the same substrate gave the alpha-glucosyl azide product and a 100-fold increase in k(cat) at 1 M azide. In conclusion, Glu178 is the general acid/base catalyst and Glu383 the catalytic nucleophile. The results presented here indicate that Bgl3 beta-glucosidase displays kinetic and mechanistic properties similar to other family 1 enzymes analyzed so far. Subtle differences in behavior would lie in the fine and specific architecture of their respective active sites.
Amino Acid Substitution↗
Long-lived glycosyl-enzyme intermediate mimic produced by formate re-activation of a mutant endoglucanase lacking its catalytic nucleophile.
The mutant E134A 1,3-1,4-beta-glucanase from Bacillus licheniformis, in which the catalytic nucleophilic residue has been removed by mutation to alanine, has its hydrolytic activity rescued by exogenous formate in a concentration-dependent manner. A long-lived alpha-glycosyl formate is detected and identified by (1)H-NMR and matrix-assisted laser desorption ionization-time-of-flight-MS. The intermediate is kinetically competent, since it is, at least partially, enzymically hydrolysed, and able to act as a glycosyl donor in transglycosylation reactions. This transient compound represents a true covalent glycosyl-enzyme intermediate mimic of the proposed covalent intermediate in the reaction mechanism of retaining glycosidases.
Bacillus↗
Relationship of obesity distribution and peripheral arterial occlusive disease in elderly men.
OBJECTIVE: To examine the relationships between total body fatness and abdominal fat distribution with peripheral arterial disease. DESIGN: Cross-sectional. SUBJECTS: Population-based sample of 708 men aged 55-74. MEASUREMENTS: Body mass index (BMI) to estimate total body fatness and waist-to-hip ratio for abdominal fat distribution; peripheral arterial disease defined by ankle/brachial index <0.9; cardiovascular risk factors. RESULTS: Peripheral arterial disease was observed in 13.4% of subjects. BMI did not correlate with peripheral arterial disease, whereas an increased waist-to-hip ratio over 0.966 (median value) doubled the prevalence of arterial disease. After controlling for smoking, diabetes, hypertension, high-density lipoprotein cholesterol and triglycerides, increased waist-to-hip ratio was independently associated with peripheral arterial disease (odds ratio 1.68; 95% confidence interval 1.05-2.70). CONCLUSION: Abdominal fat distribution, but not total body fatness, is associated with peripheral arterial occlusive disease, independently of concurrent cardiovascular risk factors.
Adipose Tissue↗
Bacterial 1,3-1,4-beta-glucanases: structure, function and protein engineering.
1,3-1,4-beta-Glucanases (or lichenases, EC 3.2.1.73) hydrolyse linear beta-glucans containing beta-1,3 and beta-1,4 linkages such as cereal beta-glucans and lichenan, with a strict cleavage specificity for beta-1,4 glycosidic bonds on 3-O-substituted glucosyl residues. The bacterial enzymes are retaining glycosyl hydrolases of family 16 with a jellyroll beta-sandwich fold and a substrate binding cleft composed of six subsites. The present paper reviews the structure-function aspects of the enzymatic action including mechanistic enzymology, protein engineering and X-ray crystallographic studies.
Amino Acid Sequence↗
Pancreatitis induces HSP72 in the lung: role of neutrophils and xanthine oxidase.
The aim of this work was to evaluate the systemic Hsp72 expression in rat lung and liver in vivo in a model of acute pancreatitis and investigate the possible involvement of xanthine oxidase and neutrophils in this process. Pancreatitis was induced by intraductal administration of 5% sodium taurocholate and samples of lung and liver were obtained 1 and 3 h later. In some groups of rats circulating xanthine oxidase was inhibited with oxypurinol, and neutrophil recruitment was blocked with a monoclonal antibody against P-selectin. Hsp72 expression was assessed by means of Western blot and immunohistochemistry. Results showed Hsp72 induction in lung, but not in liver, shortly after pancreatitis. Hsp72-induced expression was located in bronchial epithelium, alveolar macrophages, infiltrating neutrophils, and blood vessels. Oxypurinol and the antibody against P-selectin prevented pancreatitis-induced lung Hsp72 overexpression suggesting that Hsp72 induction is mediated by neutrophil infiltration into the lungs.
Animals↗
Protein-carbohydrate interactions defining substrate specificity in Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases as dissected by mutational analysis.
The carbohydrate-binding site of Bacillus macerans 1,3-1, 4-beta-D-glucan 4-glucanohydrolase has been analyzed through a mutational analysis to probe the role of protein-carbohydrate interactions defining substrate specificity. Amino acid residues involved in substrate binding were proposed on the basis of a modeled enzyme-substrate complex [Hahn, M., Keitel, T., and Heinemann, U. (1995) Eur. J. Biochem. 232, 849-859]. The effects of the mutations at 15 selected residues on catalysis and binding were determined by steady-state kinetics using a series of chromogenic substrates of different degree of polymerization to assign the individual H-bond and hydrophobic contributions to individual subsites in the binding site cleft. The glucopyranose rings at subsites -III and -II are tightly bound by a number of H-bond interactions to Glu61, Asn24, Tyr92, and Asn180. From k(cat)/K(M) values, single H-bonds account for 1.8-2.2 kcal mol(-)(1) transition-state (TS) stabilization, and a charged H-bond contributes up to 3.5 kcal mol(-)(1). Glu61 forms a bidentated H-bond in subsites -III and -II, and provides up to 6.5 kcal mol(-)(1) TS stabilization. With a disaccharide substrate that fills subsites -I and -II, activation kinetics were observed for the wild-type and mutant enzymes except for mutations on Glu61, pointing to an important role of the bidentate interaction of Glu61 in two subsites. Whereas removal of the hydroxyl group of Tyr121, initially proposed to hydrogen-bond with the 2OH of Glcp-I, has essentially no effect (Y121F mutant), side-chain removal (Y121A mutant) gave a 100-fold reduction in k(cat)/K(M) and a 10-fold lower K(I) value with a competitive inhibitor. In subsite -IV, only a stacking interaction with Tyr22 (0.7 kcal mol(-)(1) TS stabilization) is observed.
Amino Acid Sequence↗
From beta-glucanase to beta-glucansynthase: glycosyl transfer to alpha-glycosyl fluorides catalyzed by a mutant endoglucanase lacking its catalytic nucleophile.
Removal of the catalytic nucleophile Glu134 of the retaining 1,3-1,4-beta-glucanase from Bacillus licheniformis by mutation to alanine yields an enzyme with no glycosidase activity. The mutant is able to catalyze the regio- and stereospecific glycosylation of alpha-laminaribiosyl fluoride with different glucoside acceptors through a single-step inverting mechanism. The main advantage of the mutant as glycosylation catalyst with respect to the kinetically controlled transglycosylation using the wild-type enzyme is that the reaction products cannot be hydrolyzed by the mutant enzyme, and glycosylation yields rise to 90%.
Amino Acid Substitution↗
Probing the mechanism of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases by chemical rescue of inactive mutants at catalytically essential residues.
The role of the key catalytic residues Glu134 and Glu138 in the retaining 1,3-1,4-beta-glucanase from Bacillus licheniformis is probed by a chemical rescue methodology based on enzyme activation of inactive mutants by the action of added nucleophiles. While Glu134 was proposed as the catalytic nucleophile on the basis of affinity labeling experiments, no functional proof supported the assignment of Glu138 as the general acid-base catalyst. Alanine replacements are prepared by site-directed mutagenesis to produce the inactive E138A and E134A mutants. Addition of azide reactivates the mutants in a concentration-dependent manner using an activated 2, 4-dinitrophenyl glycoside substrate. The chemical rescue operates by a different mechanism depending on the mutant as deduced from 1H NMR monitoring and kinetic analysis of enzyme reactivation. E138A yields the beta-glycosyl azide product arising from nucleophilic attack of azide on the glycosyl-enzyme intermediate, thus proving that Glu138 is the general acid-base residue. Azide activates the deglycosylation step (increasing kcat), but it also has a large effect on a previous step (as seen by the large decrease in KM, the increase in kcat/KM, and the pH dependence of activation), probably increasing the rate of glycosylation through Bronsted acid catalysis by enzyme-bound HN3. By contrast, azide reactivates the E134A mutant through a single inverting displacement to give the alpha-glycosyl azide product, consistent with Glu134 being the catalytic nucleophile. Formate as an exogenous nucleophile has no effect on the E138A mutant, whereas it is a better activator of E134A than azide. Although the reaction yields the normal hydrolysis product, a transient compound was detected by 1H NMR, tentatively assigned to the alpha-glycosyl formate adduct. This is the first case where a nonmodified sugar gives a long-lived covalent intermediate that mimics the proposed glycosyl-enzyme intermediate of retaining glycosidases.
Alanine↗
Synthesis of aryl 3-O-beta-cellobiosyl-beta-D-glucopyranosides for reactivity studies of 1,3-1,4-beta-glucanases.
A series of substituted aryl beta-glycosides derived from 3-O-beta-cellobiosyl-D-glucopyranose with different phenol-leaving group abilities as measured by the pKa of the free phenol group upon enzymatic hydrolysis has been synthesised. Aryl beta-glycosides with a pKa of the free phenol leaving group > 5 were prepared by phase-transfer glycosidation of the per-O-acetylated alpha-glycosyl bromide with the corresponding phenol, whereas the 2,4-dinitrophenyl beta-glycoside was obtained by condensation of 1-fluoro-2,4-dinitrobenzene with the partially acetylated trisaccharide followed by acid de-O-acetylation. The aryl beta-glycosides have been used for reactivity studies of the wild-type Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase. The Hammett plot log kcat versus pKa is biphasic with an upward curvature at low pKa values suggesting a change in transition-state structure depending on the aglycon.
Bacillus↗
Expeditious synthesis of a new hexasaccharide using transglycosylation reaction catalyzed by Bacillus (1-->3),(1-->4)-Beta-D-glucan 4-glucanohydrolase.
Enzymatic hydrolysis of barley (1-->3),(1-->4)-beta-D-glucan using a recombinant (1-->3),(1-->4)-beta-glucanase from Bacillus licheniformis gives Glc beta 4Glc beta 3Glc isolated after acetylation in 49% yield. Conventional treatment produced the corresponding beta-fluoride which was carefully de-O-acetylated. A transglycosylation reaction with this substrate, catalyzed by the title enzyme, gave Glc beta 4Glc beta 3Glc beta 4Glc beta 4Glc beta 3Glc in 20% yield.
Bacillus↗
Mechanism of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases: kinetics and pH studies with 4-methylumbelliferyl beta-D-glucan oligosaccharides.
The carbohydrate binding site of Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase was probed with a series of synthetic 4-methylumbelliferyl beta-D-glucan oligosaccharides (1a-e). The title enzyme is a retaining endo-glycosidase that has an extended carbohydrate binding site composed of four glucopyranosyl binding subunits on the non-reducing end from the scissile glycosidic bond, plus two or three subsites on the reducing end. Subsites -II to -IV have a stabilizing effect on the enzyme-substrate transition state complex in the rate-determining step leading to a glycosyl-enzyme intermediate, with subsite -III having a larger effect (-3.5 kcal mol-1). Since KM values decrease from the mono- to the tetrasaccharide, part of the effect is due to ground stabilization of the Michaelis complex. On the other hand, the chromophoric trisaccharide 1c and the homologous nonchromogenic tetrasaccharide 2b, which locates a glucopyranosyl unit in subsite +I, have almost identical KM values, the difference in reactivity being a consequence of an 18-fold increase of kcat for 2b. Therefore, interactions between subsite +I and the substrate appear to be mainly used to lower the energy of the transition state in the glycosylation step, rather than in the stabilization of the Michaelis complex. Finally, the pH dependence of the kinetic parameters for the hydrolysis of 1c, and the pH-dependent enzyme inactivation by a water-soluble carbodiimide (EAC) suggest two essential groups with pKa values of 5.5 and 7.0 in the free enzyme. The latter value is shifted up to 1.5 pH units upon binding of substrate in the non-covalent enzyme-substrate complex.
Bacillus↗
Mutational analysis of the major loop of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases. Effects on protein stability and substrate binding.
The carbohydrate-binding cleft of Bacillus licheniformis 1,3-1, 4-beta-D-glucan 4-glucanohydrolase is partially covered by the surface loop between residues 51 and 67, which is linked to beta-strand-(87-95) of the minor beta-sheet III of the protein core by a single disulfide bond at Cys61-Cys90. An alanine scanning mutagenesis approach has been applied to analyze the role of loop residues from Asp51 to Arg64 in substrate binding and stability by means of equilibrium urea denaturation, enzyme thermotolerance, and kinetics. The DeltaDeltaGU between oxidized and reduced forms is approximately constant for all mutants, with a contribution of 5.3 +/- 0.2 kcal.mol-1 for the disulfide bridge to protein stability. A good correlation is observed between DeltaGU values by reversible unfolding and enzyme thermotolerance. The N57A mutant, however, is more thermotolerant than the wild-type enzyme, whereas it is slightly less stable to reversible urea denaturation. Mutants with a <2-fold increase in Km correspond to mutations at residues not involved in substrate binding, for which the reduction in catalytic efficiency (kcat/Km) is proportional to the loss of stability relative to the wild-type enzyme. Y53A, N55A, F59A, and W63A, on the other hand, show a pronounced effect on catalytic efficiency, with Km > 2-fold and kcat < 5% of the wild-type values. These mutated residues are directly involved in substrate binding or in hydrophobic packing of the loop. Interestingly, the mutation M58A yields an enzyme that is more active than the wild-type enzyme (7-fold increase in kcat), but it is slightly less stable.
Bacillus↗