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Biomedical subjects

O Kwon

Publications and source records attributed to O Kwon.

At least 37 records · Page 2Linked to original sources
PubMed1999

The CpxRA signal transduction system of Escherichia coli: growth-related autoactivation and control of unanticipated target operons.

In Escherichia coli, the CpxRA two-component signal transduction system senses and responds to aggregated and misfolded proteins in the bacterial envelope. We show that CpxR-P (the phosphorylated form of the cognate response regulator) activates cpxRA expression in conjunction with RpoS, suggesting an involvement of the Cpx system in stationary-phase survival. Engagement of the CpxRA system in functions beyond protein management is indicated by several putative targets identified after a genomic screening for the CpxR-P recognition consensus sequence. Direct negative control of the newly identified targets motABcheAW (specifying motility and chemotaxis) and tsr (encoding the serine chemoreceptor) by CpxR-P was shown by electrophoretic mobility shift analysis and Northern hybridization. The results suggest that the CpxRA system plays a core role in an extensive stress response network in which the coordination of protein turnover and energy conservation may be the unifying element.

Bacterial Proteins↗
PubMed1999

Regulation of adhE (encoding ethanol oxidoreductase) by the Fis protein in Escherichia coli.

The adhE gene of Escherichia coli encodes a multifunctional ethanol oxidoreductase whose expression is 10-fold higher under anaerobic than aerobic conditions. Transcription of the gene is under the negative control of the Cra (catabolite repressor-activator) protein, whereas translation of the adhE mRNA requires processing by RNase III. In this report, we show that the expression of adhE also depends on the Fis (factor for inversion stimulation) protein. A strain bearing a fis::kan null allele failed to grow anaerobically on glucose solely because of inadequate adhE transcription. However, fis expression itself is not under redox control. Sequence inspection of the adhE promoter revealed three potential Fis binding sites. Electrophoretic mobility shift analysis, using purified Fis protein and adhE promoter DNA, showed three different complexes.

Alcohol Dehydrogenase↗
PubMed1999

PAH extraction and estimation of plasma flow in human postischemic acute renal failure.

We determined the effect of postischemic injury to the human renal allograft on p-aminohippurate (PAH) extraction (E(PAH)) and renal blood flow. We evaluated renal function in 44 allograft recipients on two occasions: 1-3 h after reperfusion (day 0) and again on postoperative day 7. On day 0 subsets underwent intraoperative determination of renal blood flow (n = 35) by Doppler flow meter and E(PAH) (n = 25) by renal venous assay. Blood flow was also determined in another subset of 16 recipients on postoperative day 7 by phase contrast-cine-magnetic resonance imaging, and E(PAH) was computed from the simultaneous PAH clearance. Glomerular filtration rate (GFR) on day 7 was used to divide subjects into recovering (n = 23) and sustained (n = 21) acute renal failure (ARF) groups, respectively. Despite profound depression of GFR in the sustained ARF group, renal plasma flow was only slightly depressed, averaging 296 +/- 162 ml. min(-1). 1.73 m(-2) on day 0 and 202 +/- 72 ml. min(-1). 1.73 m(-2) on day 7, respectively. These values did not differ from corresponding values in the recovering ARF group: 252 +/- 133 and 280 +/- 109 ml. min(-1). 1.73 m(-2), respectively. E(PAH) was profoundly depressed on day 0, averaging 18 +/- 14 and 10 +/- 7% in recovering and sustained ARF groups, respectively, vs. 86 +/- 6% in normal controls (P < 0.001). Corresponding values on day 7 remained significantly depressed at 65 +/- 20 and 11 +/- 22%, respectively. We conclude that postischemic injury to the renal allograft results in profound impairment of E(PAH) that persists for at least 7 days, even after the onset of recovery. An ensuing reduction in urinary PAH clearance results in a gross underestimate of renal plasma flow, which is close to the normal range in the initiation, maintenance, and recovery stages of this injury.

Acute Kidney Injury↗
PubMed1998

Signal decay through a reverse phosphorelay in the Arc two-component signal transduction system.

Escherichia coli senses and signals anoxic or low redox conditions in its growth environment by the Arc two-component system. Under those conditions, the tripartite sensor kinase ArcB undergoes autophosphorylation at the expense of ATP and subsequently transphosphorylates its cognate response regulator ArcA through a His --> Asp --> His --> Asp phosphorelay pathway. In this study we used various combinations of wild-type and mutant ArcB domains to analyze in vitro the pathway for signal decay. The results indicate that ArcA-P dephosphorylation does not occur by direct hydrolysis but by transfer of the phosphoryl group to the secondary transmitter and subsequently to the receiver domain of ArcB. This reverse phosphorelay involves both the conserved His-717 of the secondary transmitter domain and the conserved Asp-576 of the receiver domain of ArcB but not the conserved His-292 of its primary transmitter domain. This novel pathway for signal decay may generally apply to signal transduction systems with tripartite sensor kinases.

Bacterial Outer Membrane Proteins↗
PubMed1998

Backleak, tight junctions, and cell- cell adhesion in postischemic injury to the renal allograft.

Postischemic injury in recipients of 3-7-d-old renal allografts was classified into sustained (n = 19) or recovering (n = 20) acute renal failure (ARF) according to the prevailing inulin clearance. Recipients of optimally functioning, long-standing allografts and living donors undergoing nephrectomy served as functional (n = 14) and structural controls (n = 10), respectively. Marked elevation above control of fractional clearance of dextrans of graded size was consistent with transtubular backleak of 57% of filtrate (inulin) in sustained ARF. No backleak was detected in recovering ARF. To explore a structural basis for backleak, allograft biopsies were taken intraoperatively, 1 h after reperfusion in all recipients, and again on day 7 after transplant in a subset (n = 10). Electron microscopy revealed disruption of both apical and basolateral membranes of proximal tubule cells in both sustained and recovering ARF, but cell exfoliation and tubule basement membrane denudation were negligible. Histochemical analysis of membrane-associated adhesion complexes confirmed an abnormality of proximal but not distal tubule cells, marked in sustained ARF but not in recovering ARF. Staining for the zonula occludens complex (ZO-1) and adherens complex (alpha, beta, and gamma catenins) revealed diminished intensity and redistribution of each cytoskeletal protein from the apico-lateral membrane boundary. We conclude that impaired integrity of tight junctions and cell-cell adhesion in the proximal tubule provides a paracellular pathway through which filtrate leaks back in sustained allograft ARF.

Acute Kidney Injury↗
PubMed1998

Nucleotide sequence heterogeneity in the small subunit ribosomal RNA gene variable (V4) region among and within geographic isolates of Theileria from cattle, elk and white-tailed deer.

The phylogenetic relationships among fourteen isolates of benign Theileria spp. infecting cattle, elk and white-tailed deer were studied by nucleotide sequence comparisons of the variable (V4) region (200 nucleotides) of the small subunit ribosomal RNA gene. Included were six Korean bovine, one Japanese bovine, three North American bovine, and four North American cervine isolates. The SSU rRNA gene from each isolate was amplified, cloned, and the V4 region fragment sequenced. Seven different nucleotide sequence patterns were obtained and classified. Type A was identical to T. buffeli SSU rRNA gene sequence (GenBank Accession No. Z15106) and was found in Korean, Japanese, and North American bovine isolates. Type B was found in bovine isolates from Korea, Japan and North America. Type C was found only in the Korean bovine isolate from Chungnam. Type D was found in a Korean and in a North American bovine isolate. Type E was found in a bovine isolate from Cheju Island of Korea and a North American cervine (elk) isolate. Types F and G were found only in North American cervine isolates (both white-tailed deer and elk) and appear to represent a species separate from the bovine isolates. The presence of several sequence types observed in most of the bovine Theileria isolates may indicate mixed species (or subspecies) populations and/or multiple genotypes within a single species.

Animals↗
PubMed1998

Distribution of cell membrane-associated proteins along the human nephron.

Cytoskeletal proteins associate with specific cell adhesion complexes and membrane proteins and influence the structural and functional organization of polarized epithelial cells in the kidney. Among such proteins that have been studied in cultured cell lines and in animals are the tight junction complex (ZO-1 and occludin), the adherens cell-cell adhesion complex (alpha-, beta-catenin and plakoglobin), and Na+,K+-ATPase, with its associated membrane skeleton proteins ankyrin and fodrin. Although abnormal distribution of these proteins has been implicated in the pathogenesis of various renal diseases, the relevance of these findings to corresponding disease of the human kidney remains to be established. As a first step towards elucidating a role for such proteins in human kidney disease, we undertook a histochemical analysis of the distribution of these proteins in biopsy specimens of human kidney taken from healthy kidney transplant donors. We found each protein to have a characteristic subcellular localization and an intensity of staining that varied among different segments of the nephron in a manner that is consistent with discrete, segmental nephron function.

Adult↗
PubMed1997

Ascorbate recycling in human neutrophils: induction by bacteria.

Ascorbate (vitamin C) recycling occurs when extracellular ascorbate is oxidized, transported as dehydroascorbic acid, and reduced intracellularly to ascorbate. We investigated microorganism induction of ascorbate recycling in human neutrophils and in microorganisms themselves. Ascorbate recycling was determined by measuring intracellular ascorbate accumulation. Ascorbate recycling in neutrophils was induced by both Gram-positive and Gram-negative pathogenic bacteria, and the fungal pathogen Candida albicans. Induction of recycling resulted in as high as a 30-fold increase in intracellular ascorbate compared with neutrophils not exposed to microorganisms. Recycling occurred at physiologic concentrations of extracellular ascorbate within 20 min, occurred over a 100-fold range of effector/target ratios, and depended on oxidation of extracellular ascorbate to dehydroascorbic acid. Ascorbate recycling did not occur in bacteria nor in C. albicans. Ascorbate did not enter microorganisms, and dehydroascorbic acid entry was less than could be accounted for by diffusion. Because microorganism lysates reduced dehydroascorbic acid to ascorbate, ascorbate recycling was absent because of negligible entry of the substrate dehydroascorbic acid. Because ascorbate recycling occurs in human neutrophils but not in microorganisms, it may represent a eukaryotic defense mechanism against oxidants with possible clinical implications.

Ascorbic Acid↗
PubMed1997

Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid.

Dehydroascorbic acid (DHA) is rapidly taken up by cells and reduced to ascorbic acid (AA). Using the Xenopus laevis oocyte expression system we examined transport of DHA and AA via glucose transporter isoforms GLUT1-5 and SGLT1. The apparent Km of DHA transport via GLUT1 and GLUT3 was 1.1 +/- 0.2 and 1.7 +/- 0.3 mM, respectively. High performance liquid chromatography analysis confirmed 100% reduction of DHA to AA within oocytes. GLUT4 transport of DHA was only 2-4-fold above control and transport kinetics could not be calculated. GLUT2, GLUT5, and SGLT1 did not transport DHA and none of the isoforms transported AA. Radiolabeled sugar transport confirmed transporter function and identity of all cDNA clones was confirmed by restriction fragment mapping. GLUT1 and GLUT3 cDNA were further verified by polymerase chain reaction. DHA transport activity in both GLUT1 and GLUT3 was inhibited by 2-deoxyglucose, D-glucose, and 3-O-methylglucose among other hexoses while fructose and L-glucose showed no inhibition. Inhibition by the endofacial inhibitor, cytochalasin B, was non-competitive and inhibition by the exofacial inhibitor, 4,6-O-ethylidene-alpha-glucose, was competitive. Expressed mutant constructs of GLUT1 and GLUT3 did not transport DHA. DHA and 2-deoxyglucose uptake by Chinese hamster ovary cells overexpressing either GLUT1 or GLUT3 was increased 2-8-fold over control cells. These studies suggest GLUT1 and GLUT3 isoforms are the specific glucose transporter isoforms which mediate DHA transport and subsequent accumulation of AA.

Animals↗
PubMed1997

Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli.

The first committed step in the biosynthesis of menaquinone (vitamin K2) is the conversion of chorismate to isochorismate, which is mediated by an isochorismate synthase encoded by the menF gene. This isochorismate synthase (MenF) is distinct from the entC-encoded isochorismate synthase (EntC) involved in enterobactin biosynthesis. MenF has been overexpressed under the influence of the T7 promoter and purified to homogeneity. The purified protein was found to have a molecular mass of 98 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 48 kDa. Thus, the enzyme is a homodimer. The purified enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 37 degrees C. The enzyme carries out the irreversible conversion of chorismate to isochorismate in the presence of Mg2+. The enzyme was found to have a Km of 195 +/- 23 microM and a k(cat) of 80 min(-1). In the presence of 30 mM beta-mercaptoethanol (BME), the k(cat) increased to 176 min(-1). The reducing agents BME and dithiothreitol stimulated the enzymatic activity more than twofold. Treatment of the enzyme with the cysteine-specific modifying reagent N-ethylmaleimide (NEM) resulted in the complete loss of activity. Preincubation of the enzyme with the substrate, chorismate, before NEM treatment resulted in complete protection of the enzyme from inactivation.

Cations, Divalent↗
PubMed1997

Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase.

o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity. The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-4) microM(-1) min(-1). The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine. Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out. Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity. A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis. Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+. Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity. When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold. Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to the enzyme.

Adenosine Triphosphate↗
PubMed1996

A new isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis encoded by the menF gene.

A new gene (menF) encoding an isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis has been cloned and sequenced. Overexpression of the encoded polypeptide under the influence of a T7 promoter showed an increase in specific activity of 2200-fold. Treatment with protamine sulfate resulted in another 3.5-fold increase in specific activity (7700-fold compared to the parent strain). The relative molecular mass of the overexpressed protein was M(r) 49 000, which is in full agreement with the DNA sequence predicted molecular mass of 48 777 Da. Purified enzyme converted chorismate to isochorismate with the product of the reaction shown to be isochorismate by its thermal conversion to salicylic acid. The fluorescence spectrum generated by the formed salicylic acid was identical to that of authentic salicylic acid. The 5' end of the flanking menD gene has also be redefined.

Amino Acid Sequence↗
PubMed1996

Anaerobic biosynthesis of enterobactin Escherichia coli: regulation of entC gene expression and evidence against its involvement in menaquinone (vitamin K2) biosynthesis.

In Escherichia coli, isochorismate is a common precursor for the biosynthesis of the siderophore enterobactin and menaquinone (vitamin K2). Isochorismate is formed by the shikimate pathway from chorismate by the enzyme isochorismate synthase encoded by the entC gene. Since enterobactin is involved in the aerobic assimilation of iron, and menaquinone is involved in anaerobic electron transport, we investigated the regulation of entC by iron and oxygen. An operon fusion between entC with its associated regulatory region and lacZ+ was constructed and introduced into the chromosome in a single copy. Expression of entC-lacZ was found to be regulated by the concentration of iron both aerobically and anaerobically. An established entC::kan mutant deficient in enterobactin biosynthesis was found to grow normally and synthesize wild-type levels of menaquinone under anaerobic conditions in iron-sufficient media. These results led to the demonstration of an alternate isochorismate synthase specifically involved in menaquinone synthesis encoded by the menF gene. Consistent with these findings, the entC+ strains were found to synthesize enterobactin anaerobically under iron-deficient conditions while the ent mutants failed to do so.

Anaerobiosis↗
PubMed1996

Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and properties of o-succinylbenzoyl-coenzyme A synthetase from Escherichia coli.

The coenzyme A (CoA)- and ATP-dependent conversion of o-succinylbenzoic acid [OSB; 4-(2'-carboxyphenyl)-4-oxobutyric acid], to o-succinylbenzoyl-CoA is carried out by the enzyme o-succinylbenzoyl-CoA synthetase. o-Succinylbenzoyl-CoA is a key intermediate in the biosynthesis of menaquinone (vitamin K2) in both gram-negative and gram-positive bacteria. The enzyme has been overexpressed and purified to homogeneity. The purified enzyme was found to have a native molecular mass of 185 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis established a subunit molecular mass of 49 kDa. Thus, the enzyme is a homotetramer. The enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 30 to 40 degrees C. The Km values for OSB, ATP, and CoA were 16, 73.5, and 360 microM, respectively. Of the various metal ions tested, Mg2+ was found to be the most effective in stimulating the enzyme activity. Studies with substrate analogs showed that neither benzoic acid nor benzoylpropionic acid (succinylbenzene) is a substrate for the enzyme. Thus, it appears that both the benzoyl carboxyl group and the succinyl side chain are required for activation of the aliphatic carboxyl group.

Benzoates↗
PubMed1995

Role of neutral endopeptidase in bronchial hyperresponsiveness to bradykinin induced by IL-1 beta.

Interleukin-1 beta (IL-1 beta) induces bronchial hyperresponsiveness (BHR) to bradykinin but not to acetylcholine. We examined whether this was mediated through the inhibition of neutral endopeptidase (NEP) activity and/or through the enhancement of airway microvascular leakage (AML) by IL-1 beta. We administered human recombinant IL-1 beta (500 U) or saline intratracheally and 24 h later measured the airway responses to bradykinin (1 mM; 45 breaths). IL-1 beta-treated rats showed a decrease of 18.5 and 21.1% of NEP activity in the lungs and tracheobronchial tree, respectively (P < 0.05), associated with an augmented response in total lung resistance to bradykinin but with no increase in Evans blue dye extravasation used as a marker of AML. Phosphoramidon (0.1 and 1 mM; 90 breaths), an NEP inhibitor, induced a dose-dependent increase in lung resistance to bradykinin without further enhancing BHR induced by IL-1 beta. Bradykinin-induced AML was not enhanced by phosphoramidon in either saline- or IL-1 beta-treated rats. Similarly, after captopril (1 mM; 90 breaths), an inhibitor of angiotensin-converting enzyme, there was no further enhancement of BHR to bradykinin induced by IL-1 beta. BHR to bradykinin induced by IL-1 beta may result from an inhibition of peptidase activity, such as NEP and angiotensin-converting enzyme, and is not associated with an enhancement of AML.

Administration, Inhalation↗
PubMed1994

Oxidative stress induces NF kappa B DNA binding and inducible NOS mRNA in human epithelial cells.

Free radicals generated by a partial reduction of O2 pose a serious threat to tissues and vital organs and cells. The major site of interaction between the lung and inhaled oxidants is the epithelium. We have examined the effect of pyrogallol, an O2- generator, on the ability of human epithelial cells to produce active DNA binding proteins and inducible nitric oxide synthase (iNOS) mRNA in cultured A549 epithelial cells. NF kappa B binding in the nuclei of these cells was determined by electrophoretic mobility shift assays. iNOS mRNA was measured using reverse transcription of PCR. There was a time- and concentration-dependent induction of NF kappa B binding, followed by a time and dose dependent increase in iNOS mRNA levels. These results suggest that in airways the initial response to oxidative stress may be to induce NF kappa B-responsive genes, such as iNOS, which may play an important role in defending the airway against oxidative stress.

Amino Acid Oxidoreductases↗