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S K Wright

Publications and source records attributed to S K Wright.

17 recordsLinked to original sources
PubMed2001

Alteration of the specificity of malate dehydrogenase by chemical modulation of an active site arginine.

Malate dehydrogenase from Escherichia coli is highly specific for the oxidation of malate to oxaloacetate. The technique of site-specific modulation has been used to alter the substrate binding site of this enzyme. Introduction of a cysteine in place of the active site binding residue arginine 153 results in a mutant enzyme with diminished catalytic activity, but with K(m) values for malate and oxaloacetate that are surprisingly unaffected. Reaction of this introduced cysteine with a series of amino acid analog reagents leads to the incorporation of a range of functional groups at the active site of malate dehydrogenase. The introduction of a positively charged group such as an amine or an amidine at this position results in improved affinity for several inhibitors over that observed with the native enzyme. However, the recovery of catalytic activity is less dramatic, with less than one third of the native activity achieved with the optimal reagents. These modified enzymes do have altered substrate specificity, with alpha-ketoglutarate and hydroxypyruvate no longer functioning as alternative substrates.

Arginine↗
PubMed2001

Structural analyses of a malate dehydrogenase with a variable active site.

Malate dehydrogenase specifically oxidizes malate to oxaloacetate. The specificity arises from three arginines in the active site pocket that coordinate the carboxyl groups of the substrate and stabilize the newly forming hydroxyl/keto group during catalysis. Here, the role of Arg-153 in distinguishing substrate specificity is examined by the mutant R153C. The x-ray structure of the NAD binary complex at 2.1 A reveals two sulfate ions bound in the closed form of the active site. The sulfate that occupies the substrate binding site has been translated approximately 2 A toward the opening of the active site cavity. Its new location suggests that the low catalytic turnover observed in the R153C mutant may be due to misalignment of the hydroxyl or ketone group of the substrate with the appropriate catalytic residues. In the NAD.pyruvate ternary complex, the monocarboxylic inhibitor is bound in the open conformation of the active site. The pyruvate is coordinated not by the active site arginines, but through weak hydrogen bonds to the amide backbone. Energy minimized molecular models of unnatural analogues of R153C (Wright, S. K., and Viola, R. E. (2001) J. Biol. Chem. 276, 31151-31155) reveal that the regenerated amino and amido side chains can form favorable hydrogen-bonding interactions with the substrate, although a return to native enzymatic activity is not observed. The low activity of the modified R153C enzymes suggests that precise positioning of the guanidino side chain is essential for optimal orientation of the substrate.

Binding Sites↗
PubMed2001

Factors influencing the antibiotic-prescribing decisions of nurse practitioners.

Decision-making about treatment options is a complex process that is influenced by many variables. An instrument for studying this decision-making process and the factors influencing it was designed. A survey of the antibiotic-prescribing practices of nurse practitioners (NPs) was then conducted via mail and the World Wide Web. NPs who treat adult patients were presented with scenarios in which antibiotic prescription would be an appropriate choice. Participants were asked to evaluate the clinical scenarios and describe their decision-making about treatment and the rationale for treatment. Responses were analyzed to determine the factors that played a role in antibiotic decision-making and their relative importance in that process. The ranks of categories of factors by the respondents were similar across the 4 scenarios. Antibiotic-prescribing patterns and the rationale of the antibiotic-prescribing decision of the respondents for the presented scenarios are also described.

Adult↗
PubMed2000

From malate dehydrogenase to phenyllactate dehydrogenase. Incorporation of unnatural amino acids to generate an improved enzyme-catalyzed activity.

Malate dehydrogenase (MDH) from Escherichia coli is highly specific for its keto acid substrate. The placement of the active site-binding groups in MDH effectively discriminates against both the shorter and the longer keto dicarboxylic acids that could potentially serve as alternative substrates. A notable exception to this specificity is the alternative substrate phenylpyruvate. This aromatic keto acid can be reduced by MDH, albeit at a somewhat slower rate and with greatly diminished affinity, despite the presence of several substrate-binding arginyl residues and the absence of a hydrophobic pocket in the active site. The specificity of MDH for phenylpyruvate has now been enhanced, and that for the physiological substrate oxaloacetate has been diminished, through the replacement of one of the binding arginyl residues with several unnatural alkyl and aryl amino acid analogs. This approach, called site-specific modulation, incorporates systematic structural variations at a site of interest. Molecular modeling studies have suggested a structural basis for the affinity of native MDH for phenylpyruvate and a rationale for the improved catalytic activity that is observed with these new, modified phenyllactate dehydrogenases.

Alkylation↗
PubMed1999

Introduction of histidine analogs leads to enhanced proton transfer in carbonic anhydrase V.

The rate-limiting step in the catalysis of the hydration of CO2 by carbonic anhydrase involves transfer of protons between zinc-bound water and solution. This proton transfer can be enhanced by proton shuttle residues within the active-site cavity of the enzyme. We have used chemical modulation to provide novel internal proton transfer groups that enhance catalysis by murine carbonic anhydrase V (mCA V). This approach involves the site-directed mutation of a targeted residue to a cysteine which is then subsequently reacted with an imidazole analog containing an appropriately positioned leaving group. Compounds examined include 4-bromoethylimidazole (4-BEI), 2-chloromethylimidazole (2-CMI), 4-chloromethylimidazole (4-CMI), and a triazole analog. Two sites in mCA V, Lys 91 and Tyr 131, located on the rim of the active-site cavity have been targeted for the introduction of these imidazole analogs. Modification of the introduced Cys 131 with 4-BEI and 4-CMI resulted in enhancements of up to threefold in catalytic activity. The pH profiles indicate the presence of a new proton shuttle residue of pKa near 5.8, consistent with the introduction of a functional proton transfer group into the active site. This is the first example of incorporation by chemical modification of an unnatural amino acid analog of histidine that can act as a proton shuttle in an enzyme.

Animals↗
PubMed1998

Evaluation of methods for the quantitation of cysteines in proteins.

Several methods for the quantitation of cysteines in proteins have been evaluated and compared. Titration of protein sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) under carefully controlled conditions has extended the detection limits of this method with high accuracy and reproducibility. Results are reported for a variety of enzymes containing a range of total cysteines with different degrees of solvent accessibility and reactivity. A papain amplification assay has also been examined, in which reactivation of the disulfide-blocked active site cysteine of papain can be achieved by a coupled reaction with protein sulfhydryl groups. Detection of sulfhydryls by this amplification assay can be extended, by increasing the enzyme assay times, to achieve over a 40-fold increase in sensitivity over the improved DTNB titration method. Alternatively, titration of enzyme cysteinyl residues with either bromobimane or a maleimide derivative of naphthopyranones has the advantage that a fluorescent product results upon modification of the sulfhydryl group. Reaction of bromobimane with several different enzymes results in nonspecific background fluorescence that limits the detection range of this method unless the products are separated. In contrast, low background fluorescence and high quantum yields with maleimide naphthopyranoses has allowed detection of protein cysteinyl residues with very high sensitivities.

Cysteine↗
PubMed1998

Chemotherapy does not nullify the ability of donor lymphocyte infusions to mediate graft-versus-host reactions.

Two patients with multiple myeloma in relapse after allogeneic BMT received donor lymphocyte infusions (DLI) but later required chemotherapy for treatment of myeloma-related complications. In both patients, recovery from chemotherapy-induced aplasia was accompanied by manifestations of graft-versus-host reactions. The first patient developed grade II acute GVHD and a complete remission which has lasted >22 months. The second patient developed grade III acute GVHD but died with co-existing GVHD and extensive extramedullary myeloma. These results demonstrate that chemotherapy does not nullify the ability of donor lymphocytes to mediate graft-versus-host reactions.

Adult↗
PubMed1996

Empirical treatment of severe acute community-acquired gastroenteritis with ciprofloxacin.

We conducted a randomized controlled trial to determine whether empirical treatment of severe acute community-acquired gastroenteritis (four fluid stools per day for > 3 days) with ciprofloxacin reduces the duration of diarrhea and other symptoms and to determine what effect ciprofloxacin has on the duration of long-term fecal carriage of gastrointestinal pathogens. A total of 173 patients were recruited for the study and received either ciprofloxacin (500 mg b.i.d.) or placebo for 5 days, during which time they recorded the duration of diarrhea and other symptoms (fever, abdominal pain, vomiting, and myalgia). Fecal samples were collected before treatment and regularly after treatment to determine the duration of carriage of gastrointestinal pathogens. Antibiotic susceptibility tests were performed, and the minimum inhibitory concentrations (MICs) of ciprofloxacin were determined. A significant reduction in the duration of diarrhea and other symptoms was observed after treatment, regardless of whether a pathogen was detected (P = .0001). Treatment failure occurred in 3 of 81 patients in the ciprofloxacin group and 17 of 81 patients in the placebo group. Significant pathogens were detected in 87% of patients, 85.5% of whom had cleared the pathogen at the end of treatment with ciprofloxacin, as compared with 34% who received placebo. Six weeks after treatment, there was no difference between the two groups in terms of the pathogen carriage rate (12%). Treatment with ciprofloxacin did not prolong carriage. High-level resistance to ciprofloxacin (MIC, > 32 mg/L) was detected in three strains (4%) of Campylobacter species.

Adult↗
PubMed1995

Mechanistic studies on malate dehydrogenase from Escherichia coli.

Kinetic studies and chemical modification studies using diethylpyrocarbonate and iodoacetate were performed on malate dehydrogenase isolated from Escherichia coli. Product inhibition experiments indicate that this enzyme follows an ordered Bi Bi kinetic mechanism, similar to other dehydrogenases, while log V/K profiles reveal that one ionizing group with a pKa between 7.8 and 8.7 acts as a general acid/general base in the catalytic mechanism. Log V profiles indicate that malate binds to the correctly protonated form of the enzyme while binding of OAA is pH-independent. Chemical modification experiments implicate an activate site histidine residue is essential for catalytical activity. A primary kinetic isotope effect of 1.44 +/- 0.14 on V/K using malate-2-d at pH 9.0 was measured while no isotope effect was observed on Vmax which is, again, similar to other dehydrogenases. This implies that proton abstraction is partially rate determining under nonsaturating conditions. Within experimental error, small isotope effects were observed on V and V/K (1.219 +/- 0.188 and 1.078 +/- 0.064, respectively) when NADD was utilized indicating that release of cofactor may be rate limiting.

Binding Sites↗
PubMed1995

Large-volume leukapheresis using regional citrate anticoagulation to collect peripheral blood progenitor cells.

The use of peripheral blood progenitor cells (PBPC) for hematopoietic rescue after high-dose chemotherapy is limited by the number of leukaphereses required to collect an adequate number of hematopoietic progenitors. To optimize the collection of PBPC, we evaluated a single large-volume leukapheresis protocol with citrate anticoagulation. A group of 23 patients received cyclophosphamide (4 g/m2) and GM-CSF (5 micrograms/kg/day for 15 days) as PBPC mobilization, with a single outpatient 6 h leukapheresis performed on the COBE Spectra 15 days later. Citrate (0.190 mmol/ml) was infused at 1.2 ml/L of blood/minute with a whole blood to citrate ratio between 17:1 and 25:1. Calcium chloride (50 mM) was administered at a citrate to calcium molar ratio between 10:1 and 5:1 to prevent hypocalcemia. A median 36.6 L (range 24.4-46.4) blood was processed using 338 mM citrate (269-473) and 50 mM calcium (25-75). A median 5 x 10(6) CD34+ cells/kg (< 0.3-24) and 6.2 x 10(5) CFU-GM/kg (< 0.001-29) were collected, representing 5.6 and 5.9 more PBPC, respectively, than were in circulation at the initiation of leukapheresis. We conclude that a 6 h large-volume leukapheresis following cyclophosphamide and GM-CSF mobilization is safe, can recruit hematopoietic progenitors into the circulatory compartment, and allows the collection of high numbers of PBPC in a single procedure.

Adolescent↗
PubMed1995

Predictive factors for peripheral-blood progenitor-cell collections using a single large-volume leukapheresis after cyclophosphamide and granulocyte-macrophage colony-stimulating factor mobilization.

PURPOSE: (1) To study the ability of mobilized peripheral-blood progenitor cells (PBPC) collected in a single large-volume leukapheresis performed on a predetermined date to accelerate engraftment after high-dose cyclophosphamide and thiotepa; (2) to establish the minimum dose of PBPC associated with early engraftment; and (3) to identify parameters predictive of collection of large numbers of PBPC. PATIENTS AND METHODS: Twenty-three patients with breast cancer received cyclophosphamide (4 g/m2) and granulocyte-macrophage colony-stimulating factor ([GM-CSF] 5 micrograms/kg/d x 15 days) for PBPC mobilization. A single leukapheresis was performed 15 days after cyclophosphamide administration. Then, patients received high-dose cyclophosphamide and thiotepa followed by reinfusion of PBPC and 4-hydroperoxycyclophosphamide (4HC)-purged bone marrow. PBPC concentration was measured in serial peripheral-blood samples and in the leukapheresis product. Correlation analysis between PBPC dose and engraftment and between leukapheresis yield and patient characteristics was attempted. RESULTS: A single leukapheresis processed a median 36 L (range, 24 to 46) blood and collected 5 x 10(6) CD34+ cells/kg (< 0.3 to 24) and 6.2 x 10(5) colony-forming units granulocyte-macrophage (CFU-GM)/kg (< 0.001 to 29). All sixteen patients (70%) reinfused with > or = 2.9 x 10(6) CD34+ cells/kg reached a level of greater than 1,000 leukocytes/microL by day 13 and greater than 50,000 platelets/microL by day 15. All of these patients had a percentage of peripheral-blood CD34+ cells > or = 0.5%, and all but one, a level of greater than 100,000 platelets/microL, on the day of leukapheresis. The bone marrow CD34+ cell percentage at study entry predicted the number of CD34+ cells collected after PBPC mobilization (R2 = .42, P = .002). All patients with > or = 2.5% bone marrow CD34+ cells experienced early engraftment. CONCLUSION: Reinfusion of PBPC collected in a single leukapheresis accelerates engraftment in the majority of patients. Pretreatment bone marrow CD34+ cell content determines PBPC mobilization capacity and may help select hematopoietic rescue strategies.

Adult↗
PubMed1982

Bone marrow transplantation with major ABO blood group incompatibility using erythrocyte depletion of marrow prior to infusion.

Twenty-five patients with major ABO blood group incompatibility between donor and recipient underwent allogeneic bone marrow transplantation using erythrocyte depletion of the bone marrow infusate prior to administration. Over 95% of the original erythrocyte content of the marrows was removed, while retaining 75% of the mononuclear cell content and 57% of the granulocyte-monocyte colony-forming units. Recipients, well hydrated and premedicated with corticosteroids, diphenhydramine, and mannitol, tolerated infusions well. The frequency of engraftment, rate of recovery of peripheral blood leukocytes, granulocytes, and platelets, and the incidence of graft-versus-host disease was similar to that observed following ABO blood group compatible bone marrow transplantation. Erythroid development following ABO blood group incompatible transplantation was significantly impaired until hemagglutinins fell to 1:4 or lower, at which time recovery of erythrocytes was detected in the peripheral blood. The erythrocyte hypoplasia associated with incompatible hemagglutinins was temporary. Erythrocyte purging is a safe and effective technique to perform bone marrow transplantation across major ABO blood group incompatibilities.

ABO Blood-Group System↗
PubMed1981

Sampling procedure and determination of lead in canned foods.

A method is presented for improving within-can homogeneity and simplifying the determination of lead in canned foods. The entire content of a canned food product is blended with 2N HNO3 and allowed to stand 16 h; then the sample is again blended and a subsample is taken. The subsample is digested by wet ashing using nitric acid-perchloric acid or nitric acid-sulfuric acid-hydrogen peroxide. The pH of the sample is adjusted with ammonium hydroxide, and the lead is extracted into butyl acetate as the pyrrolidinecarbodithioate complex. Lead concentration is determined by flame atomic absorption spectrometry. Recoveries of lead ranged from 92 to 104% for added 100-mesh lead particulate. Within-sample variability for green beans was reduced from 37% obtained with blending alone to 3.7% using the proposed procedure at the 3.1 micrograms/g added lead level. Variability was 7.6% at the 0.68 microgram/g level and 16.4% at the 0.20 microgram/g level of added lead. The use of nitric acid-sulfuric acid-hydrogen peroxide for digestion has the advantage of not forming a precipitate during the neutralization step, as occurs when nitric acid-perchloric acid is used for dissolution.

Food Contamination↗
PubMedDate not supplied

Trace element levels in tissues from cattle fed a sewage sludge-amended diet.

The levels of 20 elements (Al, Ca, Cd, Cl, Co, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Pb, Rb, Sb, Se, V, and Zn) are reported for kidney, liver, muscle, spleen, and brain tissues taken from two groups of six steers per group in a feeding study conducted at Colorado State University. The control group was fed a normal feedlot cattle ration and the test group was fed the same ration amended with 12% (by weight) air-dried municipal sewage sludge. Elemental levels are also reported for the control and test diets, control and test feces, and sewage sludge added to the diet. All samples were analyzed by inductively coupled plasma emission spectroscopy and neutron activation analysis. Brief descriptions of the analytical methods are included. The levels of all metals determined were elevated in the test diet (as much as 19-fold for Cd) compared with the control diet. The levels of Pb and Cd in kidney and of Pb, Cd, and Cu in liver in the test animals were high enough to cause concern from a toxicological standpoint if these tissues were consumed regularly by humans. None of the levels of any of the other elements in the control and test animal tissues were high enough to cause similar concern with respect to human consumption.

Activation Analysis↗