Right-sided aortic arch.
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Aorta, Thoracic↗
Biomedical subjects
K Duncan
Publications and source records attributed to K Duncan.
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↗
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↗
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↗
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↗
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↗
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↗
(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↗
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↗
Effects of music in patients who had chronic cancer pain.
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Adult↗
The Saccharomyces cerevisiae ARO1 gene. An example of the co-ordinate regulation of five enzymes on a single biosynthetic pathway.
The ARO1 gene of Saccharomyces cerevisiae encodes the arom multifunctional enzyme. Specific inhibitors of amino acid biosynthesis have been used to obtain evidence that expression of a cloned ARO1 gene is regulated in response to amino acid limitation. Northern blot analysis and sequence studies indicate that ARO1 is regulated by the well characterised S. cerevisiae 'general control' mechanism. This provides a very economical means of simultaneously tailoring the synthesis of five shikimate pathway enzymes to the needs of the cell.
Alcohol Oxidoreductases↗
ATP-dependent inactivation and slow binding inhibition of Salmonella typhimurium D-alanine:D-alanine ligase (ADP) by (aminoalkyl)phosphinate and aminophosphonate analogues of D-alanine.
In Salmonella typhimurium, D-alanine:D-alanine ligase (ADP) (EC 6.3.2.4) is the second enzyme in the three enzyme D-alanine branch pathway of peptidoglycan biosynthesis. The interaction of this enzyme with a possible transition-state analogue, the (aminoalkyl)phosphinate D-3-[(1-aminoethyl)phosphinyl]-2-heptylpropionic acid [Parsons et al. (1987) Abstracts of Papers, 193rd National Meeting of the American Chemical Society, Denver, CO, MEDI 63, American Chemical Society, Washington, DC], has been studied. This compound is a potent active site directed inhibitor and is competitive with D-alanine (Ki = 1.2 microM); it exhibits time-dependent inhibition in the presence of ATP. Kinetic analysis revealed a rapid onset of steady-state inhibition (kon = 1.35 X 10(4) M-1 s-1) followed by slow dissociation of inhibitory complex(es) with a half-life of 8.2 h. The inhibitory complex was shown to consist of E...I...ATP in equilibrium with E...I, Pi, and ADP. Similar time-dependent inhibition was also observed with D-(1-aminoethyl)phosphonic acid (D-Ala-P) (Ki = 0.5 mM; kon = 27 M-1 s-1; t1/2 for regain = 1.73 min) but not with D-(1-aminoethyl)phosphinic acid, which behaved as a simple competitive inhibitor (Ki = 0.4 mM). The mechanism of inhibition is discussed in the light of the precedents of glutamine synthase inhibition by methionine sulfoximine and phosphinothricin.
Alanine↗
A eukaryotic repressor protein, the qa-1S gene product of Neurospora crassa, is homologous to part of the arom multifunctional enzyme.
Little is known about the proteins involved in the control of gene expression in eukaryotes. Although some of these proteins have been sequenced, their biochemical functions are not well understood and the identification of homologies to proteins of known activities may give useful clues to the functions of these regulatory proteins. We report here that the qa-1S repressor protein of the fungus Neurospora crassa is strongly homologous to the pentafunctional arom enzyme found in many lower eukaryotes. We believe that this is the first known case of a repressor protein that is unequivocally homologous to metabolic enzymes. These findings allow us to propose likely functions for some regions of the qa-1S protein.
Amino Acid Sequence↗
The pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of monofunctional domains.
The nucleotide sequence of the Saccharomyces cerevisiae ARO1 gene which encodes the arom multifunctional enzyme has been determined. The protein sequence deduced for the pentafunctional arom polypeptide is 1588 amino acids in length and has a calculated Mr of 174555. Functional regions within the polypeptide chain have been identified by comparison with the sequences of the five monofunctional Escherichia coli enzymes whose activities correspond with those of the arom multifunctional enzyme. The observed homologies demonstrate that the arom polypeptide is a mosaic of functional domains and are consistent with the hypothesis that the ARO1 gene evolved by the linking of ancestral E. coli-like genes.
Alcohol Oxidoreductases↗
Molecular cloning and over-expression of the glyoxylate bypass operon from Escherichia coli ML308.
A recombinant plasmid carrying an 11 kb restriction-endonuclease-ClaI fragment of genomic DNA from Escherichia coli ML308 was constructed. This plasmid complements an aceA mutation. The plasmid encodes the structural genes of the glyoxylate bypass operon, namely malate synthase A (aceB), isocitrate lyase (aceA) and isocitrate dehydrogenase kinase/phosphatase (aceK), as judged by overexpression of enzyme activities and transcription/translation experiments in vitro. Subcloning confirmed that expression of the aceK gene is essential for growth on acetate.
Cloning, Molecular↗
The efficiency of folding of some proteins is increased by controlled rates of translation in vivo. A hypothesis.
We propose that the way in which some proteins fold is affected by the rates at which regions of their polypeptide chains are translated in vivo. Furthermore, we suggest that their gene sequences have evolved to control the rate of translational elongation such that the synthesis of defined portions of their polypeptide chains is separated temporally. We stress that many proteins are capable of folding efficiently into their native conformations without the help of differential translation rates. For these proteins the amino acid sequence does indeed contain all the information needed for the polypeptide chain to fold correctly (even in vitro, after denaturation). However, other proteins clearly do not fold efficiently into their native conformation in vitro. We argue that the efficiency of folding of these problematic proteins in vivo may be improved by controlled synthesis of the nascent polypeptide.
Codon↗
The anatomy of a multifunctional enzyme.
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3-Phosphoshikimate 1-Carboxyvinyltransferase↗
Automatic respiratory failure: sleep studies and Leigh's disease (case report).
A 34-year-old woman with a history of chronic nephropathy, kidney transplant rejections, and repeated hemodialysis developed symptoms of automatic respiratory failure during all states of sleep. The neuropathologic examination revealed symmetric brainstem lesions, explaining the sleep-related respiratory failure. Histology affirmed the diagnosis of Leigh's disease.
Adult↗