Search PubMed⌕ Search

Biomedical subjects

K Duncan

Publications and source records attributed to K Duncan.

At least 55 records · Page 3Linked to original sources
PubMed1995

High-throughput screen for detecting antimycobacterial agents.

A simple, robust assay system which can be used to screen for inhibitors of mycobacterial growth has been developed. A strain of the rapidly growing saprophyte Mycobacterium aurum is used as the test organism. Inhibition of its growth is highly predictive of activity against Mycobacterium tuberculosis, which cannot itself be used in screening because of its growth characteristics and highly infectious nature. The viability of M. aurum in the presence of a test sample is monitored by measuring the uptake of radiolabelled uracil into the cells. In a microtiter plate format, the screen has the potential for testing several thousand samples per day.

Antitubercular Agents↗
PubMed1995

Biosynthetic origin of mycobacterial cell wall arabinosyl residues.

Designing new drugs that inhibit the biosynthesis of the D-arabinan moiety of the mycobacterial cell wall arabinogalactan is one important basic approach for treatment of mycobacterial diseases. However, the biosynthetic origin of the D-arabinosyl monosaccharide residues themselves is not known. To obtain information on this issue, mycobacteria growing in culture were fed glucose labeled with 14C or 3H in specific positions. The resulting radiolabeled cell walls were isolated and hydrolyzed, the arabinose and galactose were separated by high-pressure liquid chromatography, and the radioactivity in each sugar was determined. [U-14C]glucose, [6-3H]glucose, [6-14C]glucose, and [1-14C]glucose were all converted to cell wall arabinosyl residues with equal retention of radioactivity. The positions of the labeled atoms in the arabinose made from [1-14C]glucose and [6-3H]glucose were shown to be C-1 and H-5, respectively. These results demonstrated that the arabinose carbon skeleton is formed via the nonoxidative pentose shunt and not via hexose decarboxylation or via triose condensations. Since the pentose shunt product, ribulose-5-phosphate, is converted to arabinose-5-phosphate as the first step in 3-keto-D-manno-octulosonic acid biosynthesis by gram-negative bacteria, such a conversion was then searched for in mycobacteria. However, cell-free enzymatic analysis using both phosphorous nuclear magnetic resonance spectrometry and colorimetric methods failed to detect the conversion. Thus, the conversion of the pentose shunt intermediates to the D-arabino stereochemistry is not via the expected isomerase but rather must occur via novel metabolic transformations.

Arabinose↗
PubMed1995

Prospects for new interventions in the treatment and prevention of mycobacterial disease.

Mycobacterium tuberculosis claims more lives each year than any other single human pathogen. Despite the availability of effective drugs, the incidence of tuberculosis is increasing in much of the developing world and has recently reemerged as a public health problem in industrialized countries. In the first section of this chapter, current understanding of the fundamental biology of mycobacterial infection is reviewed from the perspective of development of new tools for disease control. A second section describes strategies for identification of novel antimycobacterial agents, with particular emphasis on recent progress in defining biosynthetic pathways for unique mycobacterial cell wall components. The third section focuses on current approaches to the development of new vaccine candidates consisting of live attenuated bacteria or individual antigenic subunits.

Anti-Bacterial Agents↗
PubMed1995

Synthesis and biological activity of novel cephalosporins containing a (Z)-vinyl dimethylphosphonate group.

A series of cephalosporins containing a novel 7-[2-(Z)-(2-amino-thiazol-4- yl)-3-(dimethoxy-phosphoryl)-acryloylamino] group were prepared and their antibacterial activity measured against a range of pathogens. In general the compounds displayed a broad spectrum of activity against both Gram positive and Gram negative organisms, except Pseudomonas aeruginosa. Activity against the latter could be achieved by introducing a catechol moiety at the 3 position of the cephalosporin. The methyl phosphonates in general were stable to a wide range of beta-lactamases, including the TEM enzymes and the Enterobacter cloacae P99 chromosomal enzyme. In addition, they showed the advantage of being highly water soluble.

Animals↗
PubMed1993

Injuries of the ulnar collateral ligament of the thumb metacarpophalangeal joint. Biomechanical and prospective clinical studies on the usefulness of valgus stress testing.

In an effort to determine whether valgus stress testing of the thumb metacarpophalangeal joint is predictive of a torn and displaced ulnar collateral ligament, anatomic and prospective clinical studies were performed and the results correlated. In the anatomic study on autopsy specimens, dividing the proper collateral ligament resulted in a significant increase in valgus instability of the flexed metacarpophalangeal joint. Significantly less laxity was noted when the joint was tested in extension. When the accessory collateral ligament/palmar plate complex was also divided, valgus instability in extension increased to the extent that it no longer differed significantly from the values obtained when the joint was tested in 30 degrees flexion. In the clinical study, valgus instability of greater than 35 degrees when the joint was positioned in extension and then stressed consistently indicated the presence of tears of the proper and accessory collateral ligaments: A Stener's lesion was present in 15 of 17 such cases (87%). Values on valgus stress testing of the metacarpophalangeal joint in extension and 30 degrees flexion are highly predictive of both disruption and displacement of the ulnar collateral ligament of the thumb.

Biomechanical Phenomena↗
PubMed1991

Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases.

The lysine residues involved in Schiff-base formation at the active sites of both the 3-dehydroquinase component of the pentafunctional arom enzyme of Neurospora crassa and of the monofunctional 3-dehydroquinase of Escherichia coli were labelled by treatment with 3-dehydroquinate in the presence of NaB3H4. Radioactive peptides were isolated by h.p.l.c. following digestion with CNBr (and in one case after further digestion with trypsin). The sequence established for the N. crassa peptide was ALQHGDVVKLVVGAR, and that for the E. coli peptide was QSFDADIPKIA. An amended nucleotide sequence for the E. coli gene (aroD) that encode 3-dehydroquinase is also presented, along with a revised alignment of the deduced amino acid sequences for the biosynthetic enzymes.

Amino Acid Sequence↗
PubMed1991

Molecular biology of human renin and its gene.

This article describes investigations of several aspects of the molecular biology of the human renin gene and the three-dimensional structure of renin and its precursor, prorenin. Because of the importance of the RAS in hypertension, heart failure, renal failure, and possibly other disorders such as atherosclerosis, it is critical to understand the detailed control of this system. This control involves regulation at the transcriptional level, folding of prorenin, sorting of prorenin to a regulated pathway where it is proteolytically cleaved to renin and released in response to secretogogues, constitutive release of uncleaved prorenin, and nonproteolytic activation of prorenin. Currently there is great interest not only in the control of renin in the kidney, the sole source of circulating renin, but also at extrarenal sites where RAS activity may regulate cardiovascular functions. The renin gene was found to be expressed significantly in the renal juxtaglomerular cells and several other cell types. Most tissue culture cells did not express the gene; exceptions were cultured SK-LMS-1 cells and cAMP-stimulated human lung fibroblasts. Cultured human uterine-placental cells expressed the human renin gene at levels higher than in other cell types assessed. Renin mRNA had the same start site in the placental cells as the kidney and was regulated by calcium ionophores and cAMP. Thus, these cells provide primary nontransformed human cells to study the homologous human promoter. Transfected renin promoters showed cell type-specific expression and cAMP responsiveness in these cells in constructs containing as few as 102 bp of 5'-flanking DNA. DNA upstream from this appears to contain an inhibitory element(s) that may have some tissue specificity in its distribution. The cAMP response is not due to cAMP induction of a transcription factor that secondarily affects the renin promoter. A novel element may be involved, since the promoter does not contain a CRE element that mediates many cAMP responses, and the cells do not appear to respond to another known cAMP-responsive transcription factor, AP-2. Studies with transfected vectors expressing a mutant cAMP-responsive protein kinase A regulatory subunit suggest that cAMP is not responsible for basal renin promoter activity in the placental cells. By contrast, cAMP induces in essence gene activation in WI26VA4 transformed human lung fibroblasts in which renin mRNA levels increase by up to 150-fold in response to forskolin. Thus, cAMP may activate renin gene expression under certain circumstances and tissue-specific renin gene expression may be directed by more than one mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Purification and characterization of the D-alanyl-D-alanine-adding enzyme from Escherichia coli.

The Escherichia coli D-alanyl-D-alanine-adding enzyme, which catalyzes the final cytoplasmic step in the biosynthesis of the bacterial peptidoglycan precursor UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelyl-D-Ala-D- Ala, has been purified to homogeneity from an E. coli strain that harbors a recombinant plasmid bearing the structural gene for this enzyme, murF. The enzyme is a monomer of molecular weight 49,000, and it has a turnover number of 784 min-1 for ATP-driven amide bond formation. Experiments monitoring the fate of radiolabeled UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-2,6-diaminopimelate and D-trifluoroalanine proved that the preceding enzyme in the D-alanine branch pathway, D-alanine:D-alanine ligase (ADP), is capable of synthesizing fluorinated dipeptides, which the D-Ala-D-Ala-adding enzyme can then incorporate to form UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-2,6-diaminopimelyl-D-++ +trifluoroAla-D- trifluoroAla.

Amino Acid Sequence↗
PubMed1990

Renin gene expression in nephroblastoma.

Most cases of nephroblastoma have high plasma levels of prorenin which is biologically inactive. Plasma prorenin levels fall to normal following nephrectomy. In order to ascertain whether renin synthesis occurs in nephroblastomas we decided to search for renin-specific mRNA using a cDNA probe and Northern blot analyses on total RNA purified from snap-frozen human tumour tissue obtained at nephrectomy. We demonstrated renin-specific mRNA in 5/11 (45 per cent) nephroblastomas. It was 1.6 Kb in length, similar to the mRNA detected in normal kidney tissue and in kidneys with renal artery stenosis. In one of the cases of nephroblastoma, in which we could detect no normal renin mRNA at 1.6 Kb, the cDNA probe hybridized with a higher molecular weight mRNA 3 Kb in length. We conclude that some nephroblastomas synthesize renin.

Gene Expression↗
PubMed1990

High performance isoelectric focusing using capillary electrophoresis instrumentation.

High performance isoelectric focusing in capillaries provides rapid, high resolution separation of proteins based on their isoelectric points. Results can be obtained in a matter of minutes with little or no sample preparation. The technique requires the use of coated capillaries to reduce electroendosmosis so that stable, focused zones can be attained. Once focused, protein zones may be mobilized by the addition of salt to the catholyte or anolyte buffer. On-tube UV monitoring enables direct detection of sample components during mobilization with mass sensitivity equal to that of silver staining. The linear relationship between mobilization time and isoelectric point allows the technique to be used for estimation of protein pI. Demonstrated applications include separation of proteins in biological fluids with possible clinical applications, and characterization of biopharmaceutical proteins and monoclonal antibodies.

Capillary Action↗
PubMed1990

Carpal tunnel syndrome. Evaluation of a quantitative provocational diagnostic test.

A diagnostic test combining the sensitivity of the Semmes-Weinstein monofilament measurement and the specificity of the wrist flexion provocational test has been evaluated in a group of 21 patients (33 hands) with electrodiagnostically verified carpal tunnel syndrome and 30 asymptomatic hands (controls). Semmes-Weinstein monofilament testing consisted of several sensory threshold measurements obtained by the application of force-calibrated Semmes-Weinstein monofilaments to each digit in the hand with the wrist in neutral position. The quantitative provocational diagnostic test employed Semmes-Weinstein measurements obtained with the wrist both in the neutral and flexed positions. The sensitivity (82%) and specificity (86%) of the combined test were calculated. It was more sensitive and specific than the wrist flexion test alone and more specific than the Semmes-Weinstein sensibility test. The combined test is recommended as the most accurate and sensitive quantitative clinical test for median nerve compression evaluated by the authors to date.

Adult↗
PubMed1989

(1-Aminoethyl)boronic acid: a novel inhibitor for Bacillus stearothermophilus alanine racemase and Salmonella typhimurium D-alanine:D-alanine ligase (ADP-forming).

(1-Aminoethyl)boronic acid (Ala-B), an analogue of alanine in which a boronic acid group replaces the carboxyl group, has been synthesized and found to inhibit the first two enzymes, alanine racemase (from Bacillus stearothermophilus, EC 5.1.1.1) and D-alanine:D-alanine ligase (ADP-forming) (from Salmonella typhimurium, EC 6.3.2.4), of the D-alanine branch of bacterial peptidoglycan biosynthesis. In both cases, time-dependent, slow binding inhibition is observed due to the generation of long-lived, slowly dissociating complexes. Ala-B inhibits alanine racemase with a Ki of 20 mM and a kappa inact of 0.15-0.35 min-1. Time-dependent loss of activity is paralleled by conversion of the 420-nm chromophore of initial bound PLP aldimine to a 324-nm absorbing species. On dilution of Ala-B, racemase activity is regained with a t1/2 of ca. 1 h. The D-Ala-D-Ala ligase also shows progressive inhibition by Ala-B provided ATP (but not AMP-PNP or AMP-PCP) is present. The presence of D-alanine along with ATP also leads to Ala-B-induced inactivation. Kinetic analysis suggests Ala-B can compete with D-alanine at either of the two D-alanine binding sites, and on inactivation with Ala-B, labeled D-alanine, and labeled ATP, the inactive enzyme has stoichiometric amounts of D-alanine, ADP, Pi, and Ala-B bound. The half-life of inactive enzyme complexes varied from approximately 2 h (without D-alanine) to 4.5 days (with D-alanine). No D-Ala-D-Ala-B dipeptide was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1989

Nucleotide sequence of a cluster of Escherichia coli enterobactin biosynthesis genes: identification of entA and purification of its product 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase.

The nucleotide sequence of a region of the Escherichia coli chromosome encoding part of a cluster of genes involved in the biosynthesis of the iron chelator enterobactin has been determined. Four closely linked open reading frames, corresponding to the coding regions of entE (carboxy-terminal 144 amino acids), entB (32,554 daltons), entA (26,249 daltons), and an unidentified gene (P15) encoding a 14,970-dalton protein, were found. The lack of intergenic sequences and promoterlike elements suggests that these genes form part of the same transcription unit. We report the purification to homogeneity of the entA product, 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase. It is an octamer of native molecular weight 210,000; the amino-terminal amino acid sequence confirmed the entA coding region. No isochorismate synthase activity was associated with this polypeptide. This finding leads to the conclusion that the recent suggestion (M. S. Nahlik, T. P. Fleming, and M. A. McIntosh, J. Bacteriol. 169:4163-4170, 1987) that 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase and isochorismate synthase activities reside on a single 26,000-dalton bifunctional enzyme is incorrect, even though the entA and entC mutations map to the same genetic locus.

Amino Acid Sequence↗