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Biomedical subjects

K Kim

Publications and source records attributed to K Kim.

At least 433 records · Page 24Linked to original sources
PubMed1994

Inhibition of endothelium-dependent vasodilation by Escherichia coli endotoxemia.

To test the hypothesis that release of endothelium-derived relaxing factor/nitric oxide is inhibited by Gram-negative lipopolysaccharide (LPS; endotoxin), we examined endothelium-independent and endothelium-dependent vasodilator agents in aortic vascular smooth muscle isolated from guinea pigs 4 h after injection of saline (controls) or induction of Escherichia coli endotoxemia. LPS significantly inhibited vasodilator responses to the endothelium-dependent agonists acetylcholine (ACh; 10(-10)-10(-5) M) and ADP (10(-8)-10(-5) M). However, LPS did not affect vasodilator responses to the endothelium-independent agonist nitroprusside (10(-10)-10(-4) M). The nitric oxide synthase (NOS) inhibitor N gamma-nitro-L-arginine methyl ester (L-NAME) inhibited the vasodilator response to ACh; whereas, the cyclooxygenase inhibitor indomethacin (INDO) did not reduce vasodilator effects of ACh. Neither L-NAME nor INDO affected the vasodilator effects of nitroprusside in LPS or control vessels. In contrast, L-NAME converted the vasodilator action of ADP to a vasoconstrictor response that was blocked individually by INDO and the thromboxane synthase inhibitor dazoxiben, suggesting that ADP releases NO and also the vasoconstrictor and platelet aggregating eicosanoid thromboxane A2. These findings suggest that acute (4 h) endotoxemia inhibits function of the constitutive isoform of NOS in vascular endothelial cells. Since L-NAME unmasked a vasoconstrictor action of the endogenous purinoceptor agonist ADP, pharmacologic agents that inhibit NOS may exacerbate LPS-induced inhibition of endothelial NOS; this series of events could lead to diminution of vasodilator reserves and perhaps to augmentation of platelet aggregation during Gram-negative sepsis.

Acetylcholine↗
PubMed1994

Conformationally appropriate expression of the Toxoplasma antigen SAG1 (p30) in CHO cells.

The Toxoplasma gondii major surface antigen, called SAG1 or p30, is a highly immunogenic protein which has generated great interest as a diagnostic reagent, as a potential subunit vaccine, and for its role in invasion. Unfortunately, bacterial recombinant protein is grossly misfolded so that, for example, it is not effectively recognized by antibodies to native SAG1. To overcome this, we have turned to expression in CHO cells, using cotransfection of the SAG1 gene and the mouse dihydrofolate reductase (DHFR) gene into CHO cells that are DHFR-. SAG1 expression was amplified by methotrexate coselection of CHO cells in combination with fluorescence-activated cell sorting for SAG1 expression. The resulting population expressed recombinant SAG1 that is recognized by antiserum specific for natural, nonreduced SAG1, indicating that, unlike in bacteria, expression in CHO cells results in proper folding. Processing was at least partially correct in that, like natural SAG1, recombinant SAG1 was attached to the plasma membrane via a glycolipid anchor, although tunicamycin treatment was necessary to prevent N-glycosylation (SAG1 is not glycosylated in the parasite but does have a consensus N-linked site). Finally, purified recombinant SAG1 was recognized by human sera known to be reactive to toxoplasma proteins, indicating that this material has potential as a diagnostic reagent and possibly as a component of a subunit vaccine.

Animals↗
PubMed1994

Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair.

DNA damage frequently leads to the production of apurinic/apyrimidinic (AP) sites, which are presumed to be repaired through the base excision pathway. For detailed analyses of this repair mechanism, a synthetic analog of an AP site, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), has been employed in a model system. Tetrahydrofuran residues are efficiently repaired in a Xenopus laevis oocyte extract in which most repair events involve ATP-dependent incorporation of no more than four nucleotides (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989; Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 11:4441-4447, 1991). Using a series of column chromatography procedures to fractionate X. laevis ovarian extracts, we developed a reconstituted system of tetrahydrofuran repair with five fractions, three of which were purified to near homogeneity: proliferating cell nuclear antigen (PCNA), AP endonuclease, and DNA polymerase delta. This PCNA-dependent system repaired natural AP sites as well as tetrahydrofuran residues. DNA polymerase beta was able to replace DNA polymerase delta only for repair of natural AP sites in a reaction that did not require PCNA. DNA polymerase alpha did not support repair of either type of AP site. This result indicates that AP sites can be repaired by two distinct pathways, the PCNA-dependent pathway and the DNA polymerase beta-dependent pathway.

Animals↗
PubMed1994

Blockade of noradrenergic neurotransmission with diethyldithiocarbamic acid decreases the mRNA level of gonadotropin-releasing hormone in the hypothalamus of ovariectomized, steroid-treated prepubertal rats.

We have previously found that progesterone (P) augmented gonadotropin-releasing hormone (GnRH) mRNA levels in the hypothalamus of ovariectomized, estradiol-treated (OVX + E) prepubertal rats. In order to determine whether noradrenergic neurotransmission is involved in the stimulatory effect of P on GnRH gene expression, diethyldithiocarbamic acid (DDC, 500 mg/kg), a dopamine beta-hydroxylase inhibitor was administered i.p. 1 h before P (1 mg) injection into OVX + E treated rats, and the effect of DDC on the P-induced GnRH mRNA levels was examined. A single injection of P into OVX + E primed rats augmented norepinephrine (NE) content, while the administration of DDC effectively blocked the P-induced increase in NE content, along with the increase in dopamine content. Suppression of NE neurotransmission with DDC resulted in a marked decrease in the P-induced GnRH mRNA levels as well as GnRH release in vitro. These results clearly demonstrate that noradrenergic neurotransmission is involved in P-stimulated GnRH gene expression in the rat hypothalamus.

Animals↗
PubMed1994

The perfluorocarbon fluoromethyloadamantane offers cerebral protection in a model of isovolemic hemodilution in rabbits.

BACKGROUND AND PURPOSE: Perfluorocarbons (PFCs) are considered promising cerebral protection agents because they could combine the beneficial effects of decreased blood viscosity with enhanced oxygen-carrying capacity and oxygen tissue delivery, but trials of PFCs as hemodilutants have been very limited. We evaluated fluoromethyloadamantane (FMA), a new perfluorocarbon compound, as an isovolemic hemodilutant and compared it with low-molecular-weight dextran 40 (D40) and a control group. METHODS: Through a transorbital craniectomy, the internal carotid, anterior, and middle cerebral arteries were coagulated to create a cerebral infarction in anesthetized, mechanically ventilated rabbits. No other experimental procedure was performed in control animals. In the two other groups, hemodilution was commenced 30 minutes after the arterial occlusion with either D40 or FMA. Hemodynamic parameters and brain and systemic temperature were monitored throughout the experiments. All animals were killed 6 hours after the arterial occlusion. RESULTS: Hemodynamic and metabolic parameters and blood oxygen content were not affected by the infusion of either FMA or D40. Brain and systemic temperature remained constant. The ratio of infarct volume to the hemispheric volume was 19.6 +/- 3.7% in the FMA group (n = 17), 19.9 +/- 4.6% in the D40 group (n = 16), and 40.3 +/- 5.7% in the control group (n = 17). The difference in infarct volume of both FMA and D40 animals compared with controls was statistically significant (P < .01) when tested with Student's t test. There was no significant difference between FMA and D40 groups. CONCLUSIONS: These results suggest that FMA has cerebral protective properties and should be purified, optimized, and further tested experimentally to develop a stable, efficient, and safe oxygen carrier, potentially suitable for clinical trials.

Adamantane↗
PubMed1994

Cisplatin plus doxorubicin plus cyclophosphamide in metastatic or recurrent thymoma: final results of an intergroup trial. The Eastern Cooperative Oncology Group, Southwest Oncology Group, and Southeastern Cancer Study Group.

PURPOSE: The purpose of this study was to evaluate the impact of cisplatin, doxorubicin, and cyclophosphamide (PAC) in patients with advanced thymoma with respect to response rate, duration of remission, and overall survival. PATIENTS AND METHODS: Assessable patients with thymoma (n = 29) or thymic carcinoma (n = 1) with metastatic or locally progressive recurrent disease following radiotherapy were treated with intravenous cisplatin (50 mg/m2), doxorubicin (50 mg/m2), and cyclophosphamide (500 mg/m2) with normal saline hydration. Courses were repeated every 3 weeks for a maximum of eight cycles of therapy. RESULTS: Toxicity, which was primarily hematologic, was mild, with only one patient developing a fever associated with neutropenia. Three complete responses (CRs) and 12 partial responses (PRs) were observed (CR+PR rate, 50%; 95% confidence interval, 31.3% to 68.7%). Ten patients had stable disease. The median duration of response was 11.8 months (range, 0.9 to 70.5+), the time to treatment failure 18.4 months (range, 0.8 to 91.9+), and median survival time 37.7 months (range, 2 to 91.9+). CONCLUSION: This trial demonstrates that objective response rates and prolonged survival can be achieved in patients with advanced thymoma.

Adult↗
PubMed1994

Identification of the C-terminal amino acid amides by carboxypeptidase Y digestion and fast atom bombardment mass spectrometry.

The combination method of carboxypeptidase Y digestion and fast atom bombardment (FAB) mass spectrometry is described for the identification of C-terminal amino acid amides in peptides. Carboxypeptidase Y has amidase activity as well as exopeptidase activity in the same digestion buffer condition. Based on this concept, we develop a new technique which can definitively and easily identify the C-terminal amino acid amides. This method obviates the need for several complicated steps occurring in previous methods, but improves sensitivity, and enables exact identification of the amino acid amide by the difference of molecular mass. Analyses of carboxypeptidase Y digested peptides, not liberated free amino acid amides, were carried out by fast atom bombardment mass spectrometry. The use of truncated peptides by fast atom bombardment mass spectrometry in C-terminal amino acid amide determination gives several advantages over analyses of the liberated amino acid amides. The C-terminal amino acid amides of Allantostatin I (Leu-NH2), alpha-Melanocyte Stimulating Hormone (Val-NH2), and Ranatensin (Met-NH2) are unequivocally determined at a level of 0.90-2.3 nmol per peptide. This approach is based on entirely different principles than the previous approaches.

Amides↗
PubMed1994

Inhibition of metal-catalyzed oxidation systems by a yeast protector protein in the presence of thiol.

A protector protein from Saccharomyces cerevisiae prevented the inactivation of enzyme and oxidative damage to protein and DNA caused by a thiol/Fe3+/O2 metal-catalyzed oxidation (MCO) system but not when thiol was replaced by ascorbate. In the presence of a reduced thiol such as dithiothreitol and reduced glutathione, however, the protector protein prevented inactivation of E. coli glutamine synthetase against a MCO system comprised of ascorbate and Fe3+. The protector protein also inhibited the fragmentation of protein, incorporation of carbonyl groups into protein, strand breaks in pBluescript plasmid DNA, and the formation of 8-hydroxydeoxyguanosine in calf thymus DNA when induced by either the thiol/Fe3+ system or the ascorbate/Fe3+ system supplemented with dithiothreitol. These results suggest that antioxidant activity of protector protein against a MCO system requires thiol as a reducing equivalent to restore its catalytic activity.

Antioxidants↗
PubMed1994

Application of carboxypeptidase Y and fast atom bombardment mass spectrometry for C-terminal sequencing of small peptides.

The application of fast atom bombardment (FAB) mass spectrometry to the C-terminal amino acid sequence determination of peptides is reported. FAB mass spectrometric analysis of the peptides formed by carboxypeptidase Y (CPY) digestion conveniently provides information about C-terminal amino acid sequences. In these experiments, we accomplished the determination of C-terminal region amino acid sequence of Bradykinin and Angiotensin II. We describe advantages of the combination experiment of CPY and FAB mass spectrometry for C-terminal region amino acid studies of small peptides. The significant advantages of this method are the ability to study peptides without derivatization and the elimination of the separation step of liberated C-terminal amino acids and peptides. With this method, we could overcome several problems which conventionally happened in C-terminal sequence analysis.

Amino Acid Sequence↗
PubMed1993

Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker.

A system for stable transformation of Toxoplasma gondii tachyzoites was developed that exploited the susceptibility of Toxoplasma to chloramphenicol. Introduction of the chloramphenicol acetyltransferase (CAT) gene fused to Toxoplasma flanking sequences followed by chloramphenicol selection resulted in parasites stably expressing CAT. A construct incorporating the tandemly repeated gene, B1, targeted efficiently to its homologous chromosomal locus. Knockout of the single-copy gene, ROP1, was also successful. Stable transformation should permit the identification and analysis of Toxoplasma genes important in the interaction of this opportunistic parasite with its host.

Animals↗
PubMed1993

Alternative pathways and reactions of benzyl alcohol and benzaldehyde with horse liver alcohol dehydrogenase.

Liver alcohol dehydrogenase catalyzes the reaction of NAD+ and benzyl alcohol to form NADH and benzaldehyde by a predominantly ordered reaction. However, enzyme-alcohol binary and abortive ternary complexes form at high concentrations of benzyl alcohol, and benzaldehyde is slowly oxidized to benzoic acid. Steady-state and transient kinetic studies, equilibrium spectrophotometric measurements, product analysis, and kinetic simulations provide estimates of rate constants for a complete mechanism with the following reactions: (1) E<-->E-NAD+<-->E-NAD(+)-RCH2OH<-->E-NADH-RCHO<-->E-NADH<-->E ; (2) E-NADH<-->E-NADH-RCH2OH<-->E-RCH2OH<-->E; (3) E-NAD+<-->E-NAD(+)-RCHO-->E- NADH-RCOOH<-->E-NADH. The internal equilibrium constant for hydrogen transfer determined at 30 degrees C and pH 7 is about 5:1 in favor of E-NAD(+)-RCH2OH and has a complex pH dependence. Benzyl alcohol binds weakly to free enzyme (Kd = 7 mM) and significantly decreases the rates of binding of NAD+ and NADH. The reaction of NAD+ and benzyl alcohol is therefore kinetically ordered, not random. High concentrations of benzyl alcohol (> 1 mM) inhibit turnover by formation of the abortive E-NADH-RCH2-OH complex, which dissociates at 0.3 s-1 as compared to 6.3 s-1 for E-NADH. The oxidation of benzaldehyde by E-NAD+ (Km = 15 mM, V/E = 0.4 s-1) is inefficient relative to the oxidation of benzyl alcohol (Km = 28 microM, V/E = 3.1 s-1) and leads to a dismutation (2RCHO-->RCH2OH + RCOOH) as E-NADH reduces benzaldehyde. The results provide a description of final product distributions for the alternative reactions catalyzed by the multifunctional enzyme.

Alcohol Dehydrogenase↗
PubMed1993

Cloning, sequencing, and mutation of thiol-specific antioxidant gene of Saccharomyces cerevisiae.

We have previously shown that the yeast Saccharomyces cerevisiae contains an antioxidant enzyme that can provide protection against a thiol-containing oxidation system but not against an oxidation system without thiol. This 25-kDa enzyme was thus named thiol-specific antioxidant (TSA). We have now isolated and sequenced a yeast genomic DNA fragment that encodes TSA. Comparison of the predicted amino acid sequence of TSA with those of conventional antioxidant enzymes, including catalases, peroxidases, and superoxide dismutases, revealed no sequence homology. The 195-amino acid TSA sequence contains 2 cysteine residues. Southern blot analysis of petite yeast DNA, studies with protein synthesis inhibitors, and protein immunoblot analyses of cytosolic and mitochondrial proteins suggest that TSA is a cytosolic protein encoded by nuclear DNA (chromosome XIII). The yeast TSA gene was selectively disrupted by homologous recombination. The haploid tsa mutant was viable under air, suggesting that TSA is not essential for cell viability. The growth rates of the tsa mutant and wild-type strains were identical under anaerobic conditions. However, under aerobic conditions, especially in the presence of methyl viologen or a peroxide (t-butyl hydroperoxide or H2O2), the growth rate of the mutant was significantly less than that of wild-type cells. This result suggests that TSA is a physiologically important antioxidant.

Amino Acid Sequence↗
PubMed1993

Simultaneous analysis of 25 pesticides in crops using gas chromatography and their identification by gas chromatography-mass spectrometry.

The simultaneous analysis of 25 pesticides in soy beans and rices was performed by gas chromatography with dual electron-capture detection and nitrogen-phosphorus detection. The pesticides were extracted from the samples with solvent and the Bio-Beads S-X3 clean-up procedure was used. Recovery studies were performed at the 1-ppm level of pesticides added to each crop. Their recoveries ranged between 83 and 105% with coefficient of variations of 0.5-8.2%. The gas chromatographic properties of the 25 pesticides were also investigated. Conformation analysis was achieved by the retention time and characteristic fragment ions using the technique of gas chromatography-mass spectrometry-selected-ion monitoring.

Chromatography, Gas↗
PubMed1993

Unmasking of hydrogen tunneling in the horse liver alcohol dehydrogenase reaction by site-directed mutagenesis.

Primary and secondary kD/kT and kH/kT kinetic isotope effects have been studied as a probe of hydrogen tunneling in the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase (LADH). In the case of the wild-type enzyme, isotope effects at 25 degrees C do not clearly support hydrogen tunneling; this result is consistent with a reaction rate that is partially limited by the release of product benzaldehyde. The three-dimensional structure for LADH was used to design site-directed mutations in an effort to enhance the rate of the product release step and to "unmask" tunneling. Substitutions that increased the size of the alcohol binding pocket resulted in minor changes in isotope effects. By contrast, reduction in the size of the alcohol binding pocket through substitution at residues 57 and 93, which are in van der Waals contact with bound alcohol substrate, produced a clear demonstration of protium tunneling from the breakdown of the semiclassical relationship between kD/kT and kH/kT isotope effects. The temperature dependence of kD/kT isotope effects has also been pursued, leading to the conclusion that tunneling does, in fact, occur in the reaction catalyzed by wild-type LADH. Despite the unmasking of protium tunneling in site-directed mutants, substitutions that decrease the size of the alcohol pocket appear to result in less extensive tunneling in the hydride transfer. It is noteworthy that the mutant enzyme (Leu57-->Phe), which shows the greatest evidence of tunneling, has the same catalytic efficiency (Vmax/Km) as the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1993

Strand breaks in DNA induced by a thiol/Fe(III)/O2 mixed-function oxidase system and its protection by a yeast antioxidant protein.

Strand breaks can be produced in pBluescript plasmid DNA and calf thymus DNA by a mixed-function oxidase (MFO) system comprised of Fe3+, O2, and dithiothreitol as an electron donor. Superoxide dismutase does not block this damage whereas a 27-KDa yeast antioxidant protein specifically inhibits strand breaks in DNA induced by the dithiothreitol MFO system. In contrast, this protein does not inhibit strand breaks in DNA induced by an ascorbate MFO system although catalase inhibits damage in DNA caused by both MFO systems. Based on the specificity of this protein, we propose that the antioxidant protein functions as a sulfur radical scavenger.

Animals↗
PubMed1993

Removals of hydrogen peroxide and hydroxyl radical by thiol-specific antioxidant protein as a possible role in vivo.

Thiol-specific antioxidant protein (Protector Protein; PRP) from Saccharomyces cerevisiae was found to remove hydrogen peroxide and hydroxyl radical in the presence of dithiothreitol (DTT). Without DTT as a reducing equivalent, the antioxidant protein did not show the activities for destroying hydrogen peroxide and hydroxyl radical. N-ethylmaleimide (NEM) was observed to prevent the PRP from both removing hydrogen peroxide and protecting the cleavage of DNA. These observations suggest that the sulfhydryl of cysteine in PRP could function as a strong nucleophile to attack and destroy H2O2 and .OH.

Antioxidants↗