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Biomedical subjects

K Duncan

Publications and source records attributed to K Duncan.

At least 37 records · Page 2Linked to original sources
PubMed1998

The impact of genomics on the search for novel tuberculosis drugs.

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has highlighted the need for new drugs to treat tuberculosis. Drugs that either shorten the overall duration of therapy or that simplify the regimen would significantly improve compliance and hence reduce treatment failure rates. The drug development process begins with identification and validation of specific targets. These may be relevant for inhibiting growth of the bacterium in vitro, and hence yield novel bactericidal agents, or they may be required at other stages of growth, such as survival in host macrophages. With the availability of the complete genome sequence of M. tuberculosis, the primary sequence of every drug target in the pathogen is known. A combination of approaches is being employed to exploit the information contained in the genome and thereafter to identify lead compounds that may yield new drugs.

Acetamides↗
PubMed1998

Cutaneous T-cell lymphoma: centuries of controversy.

Two hundred years have passed since the description of mycosis fungoides by Alibert. During this time, the disease has been the focus of intense controversy and research, with these two intimately intertwined. In this article, the major components of the controversies surrounding cutaneous T-cell lymphoma are examined. The next millenium will see the resolution of these controversies and the fruition of continued research into this condition.

Contraindications↗
PubMed1998

The provision of enabling services to higher-risk pregnant women and children in Medicaid managed care.

As managed care organizations become the major payer model for the higher-need, higher-risk population of Aid to Families with Dependent Children women and children, they would benefit from looking at lessons learned during the past decade as policy makers and providers struggled to identify less costly, but effective strategies to reduce morbidity and mortality associated with poor birth outcomes. This article discusses the experience of one community in providing enabling services, the opportunity for new partnerships, measuring the outcomes, and the financing of enabling services in a managed care model.

Child Health Services↗
PubMed1998

Radiopharmaceuticals in PET imaging.

OBJECTIVE: PET isotopes are being used more commonly in nuclear medicine. This paper introduces the nuclear medicine technologist to the use of PET imaging. The major focus of this article is on the isotopes that are used in this modality and how they differ from traditional isotopes. The nuclear medicine technologist will become familiar with PET isotopes by reading about PET history, the production and availability of PET isotopes, and the use of this modality in a traditional hospital setting.

Cyclotrons↗
PubMed1997

Running exercise may reduce risk for lung and liver cancer by inducing activity of antioxidant and phase II enzymes.

The effects of exercise, ethanol, and exercise plus ethanol-treatments on activity of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) and UDP-glucuronosyl transferase (UDP-GT) in lung and liver were investigated. All treatments induced SOD and CAT activity in the lung while CAT activity was enhanced only by the combined treatments in the liver. Ethanol reduced hepatic SOD activity, while with the combined treatment SOD was normal. Exercise enhanced UDP-GT activity in liver and lung while ethanol had no effect and GST activity was induced in the liver by the combined treatment. Thus exercise may reduce risk for lung and hepatic cancer and prevent an ethanol-induced increase in risk for hepatic cancer by enhancing activity of antioxidant and phase II enzymes.

Animals↗
PubMed1997

Biosynthetic origin of mycobacterial cell wall galactofuranosyl residues.

SETTING: Mycobacterial galactofuran is essential to the linking of the peptidoglycan and mycolic acid cell wall layers. Galactofuran biosynthesis should thus be essential for viability. OBJECTIVE: The objective was to determine the pathway of galactofuranosyl biosynthesis and to clone a gene encoding an essential enzyme necessary for its formation. DESIGN: Specific enzymatic conversions involved in formation of galactopyranose and galactofuranose residues in other bacteria were tested for in Mycobacterium smegmatis. M. tuberculosis deoxyribonucleic acid (DNA) was identified by homology. RESULTS: It was shown that the de novo synthesis of the galactose carbon skeleton occurred in M. smegmatis by the transformation of UDP-glucopyranose to UDP-galactopyranose via the enzyme UDP-glucose 4-epimerase (E.C. 5.1.3.2). The N-terminal sequence of this enzyme was obtained after purification. The galactose salvage pathway enzyme, UDP-glucose-galactose-1-phosphate uridylyltransferase (E.C. 2.7.7.12), was also shown to be present. The critical biosynthetic transformation of the galactopyranose to galactofuranose ring form was shown to occur at the sugar nucleotide level via the enzyme UDP-galactopyranose mutase (E.C. 5.4.99.9). The M. tuberculosis DNA encoding this enzyme was sequenced, the gene expressed in Escherichia coli, and the expected enzymatic activity demonstrated. CONCLUSION: Galactofuranose biosynthesis can now be pursued as a potential drug target in M. tuberculosis.

Amino Acid Sequence↗
PubMed1996

Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, an enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-N-acetylglucosamine and the drug fosfomycin.

BACKGROUND: UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), catalyses the first committed step of bacterial cell wall biosynthesis and is a target for the antibiotic fosfomycin. The only other known enolpyruvyl transferase is 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, an enzyme involved in the shikimic acid pathway and the target for the herbicide glyphosate. Inhibitors of enolpyruvyl transferases are of biotechnological interest as MurA and EPSP synthase are found exclusively in plants and microbes. RESULTS: The crystal structure of Escherichia coli MurA complexed with UDP-N-acetylglucosamine (UDP-GlcNAc) and fosfomycin has been determined at 1.8 A resolution. The structure consists of two domains with the active site located between them. The domains have a very similar secondary structure, and the overall protein architecture is similar to that of EPSP synthase. The fosfomycin molecule is covalently bound to the cysteine residue Cys115, whereas UDP-GlcNAc makes several hydrogen-bonding interactions with residues from both domains. CONCLUSIONS: The present structure reveals the mode of binding of the natural substrate UDP-GlcNAc and of the drug fosfomycin, and provides information on the residues involved in catalysis. These results should aid the design of inhibitors which would interfere with enzyme-catalyzed reactions in the early stage of the bacterial cell wall biosynthesis. Furthermore, the crystal structure of MurA provides a model for predicting active-site residues in EPSP synthase that may be involved in catalysis and substrate binding.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1996

Enzymatic synthesis of UDP-galactofuranose and an assay for UDP-galactopyranose mutase based on high-performance liquid chromatography.

A method to prepare UDP-galactofuranose (UDP-Galf) free of UDP-galactopyranose (UDP-Galp) is described. The UDP-Galf is synthesized enzymatically from UDP-Galp using the enzyme UDP-galactopyranose mutase. Treatment of UDP-Galp with the enzyme yields an equilibrium mixture of UDP-Galp and UDP-Galf in which UDP-Galf is approximately 7%. In spite of its low yield, the UDP-Galf is readily purified from starting UDP-Galp using a Dionex PA-100 ion exchange HPLC column. The purified UDP-Galf was characterized by chemical degradations, by electrospray mass spectrometry, and by several nuclear magnetic resonance techniques. In addition, an HPLC assay for the enzyme UDP-galactopyranose mutase is presented that requires 0.5 microgram of UDP-Galf per assay and can be used for both qualitative and quantitative measurements of the enzyme activity. These procedures should thus aid in the characterization of the enzymes involved in galactofuranosyl biosynthesis for the cell walls of Mycobacteria, for the lipophosphoglycan of Leishmania, and for other microorganisms where galactofuranosyl residues are found.

Bacterial Proteins↗
PubMed1996

Characterization of a Cys115 to Asp substitution in the Escherichia coli cell wall biosynthetic enzyme UDP-GlcNAc enolpyruvyl transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycin.

The antibiotic fosfomycin inhibits bacterial cell wall biosynthesis by inactivation of UDP-GlcNAc enolpyruvyl tranferase (MurA). Prior work has established that Cys115 of Escherichia coli and Enterobacter cloacae MurA is the active site nucleophile alkylated by fosfomycin and implicated this residue in the formation of a covalent phospholactyl-enzyme adduct derived from substrate, phosphoenolpyruvate (PEP). On the basis of sequencing information from putative MurA homolog from Mycobacterium tuberculosis, we generated a C115D mutant of E. coli MurA that was highly active but fully resistant to time-dependent inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the observed reversible competitive inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the observed reversible competitive inhibition vs PEP. In contrast to the broad pH-independent behavior of wild-type (WT) MurA, C115D mutant activity titrated across the pH range examined (pH 5.5-9) with an apparent pKa approximately 6, with kcatC115D ranging from approximately 10kcatWT at pH 5.5 to <0.1kcatWT at pH9.0. Km(PEP)115D was relatively constant in the pH range examined and increased approximately 100-fold relative to Km(PEP)WT. A fosfomycin-resistant C115E mutant with -1% activity of the C115D mutant was found to follow a pH dependence similar to that observed for C115D MurA. The contrasting pH dependences of WT and C115D MurA was also observed in the reaction with the pseudosubstrate, (Z)-3-fluorophosphoenolpyruvate, strongly suggesting a role for Cys/Asp115 as the general acid in the protonation of C-3 of PEP during MurA-catalyzed enol ether transfer. The difference in nucleophilicity between the carboxylate side chains of Asp115 and Glu115 and the thiolate group of Cys115 suggests that covalent enzyme adduct formation is not required for catalytic turnover and, furthermore, provides a chemical rationale for the resistance of the C115D and C115E mutants to fosfomycin inactivation.

Alkyl and Aryl Transferases↗
PubMed1996

Mode of action of GR122222X, a novel inhibitor of bacterial DNA gyrase.

GR122222X is a potent inhibitor of the supercoiling reaction of bacterial DNA gyrase. We show that this compound binds stoichiometrically to inactivate the ATPase activity of a 43-kDa N-terminal fragment of the B subunit and competitively inhibits the binding of a radiolabelled coumarin drug to N-terminal fragments of GyrB. These and other data suggest that GR122222X has a mode of action similar, but not identical, to that of coumarin antibiotics.

DNA Topoisomerases, Type II↗
PubMed1996

Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase.

We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region. The genes were expressed in E. coli under control of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies. Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (the conversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment. The crude orf6 gene product converted UDP-[alpha-D-U-14C]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of synthetic alpha-galactofuranose-1-phosphate. No mutase activity was detected in extracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells. The orf6 gene product was purified by anion-exchange chromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form. The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed. These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDP-galactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 +/- 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide.

Amino Acid Sequence↗
PubMed1995

Distinguishing depressed from nondepressed individuals using energy and psychosocial variables.

This study was conducted to determine whether energy level can be used to distinguish depressed from nondepressed individuals and to assess the relative importance of energy level and psychosocial variables in making this distinction. Fifty-seven participants experiencing a current episode of major depression and a matched sample of nondepressed participants completed a self-report questionnaire containing measures of energy level and psychosocial variables. Discriminant analysis revealed that energy level correctly classified 93% as depressed or nondepressed, whereas psychosocial variables correctly classified 87%. Combining the energy and psychosocial variables did not increase the accuracy of classification over that achieved by using only the energy measures. The measure of exhaustion provided the greatest relative contribution to the overall discriminant function.

Arousal↗