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Biomedical subjects

Y M Kim

Publications and source records attributed to Y M Kim.

At least 145 records · Page 8Linked to original sources
PubMed1999

Hemoglobin toxicity in experimental bacterial peritonitis is due to production of reactive oxygen species.

Hemoglobin (Hb) is a toxic molecule responsible for the extreme lethality associated with experimental Escherichia coli peritonitis, but the mechanism has yet to be elucidated. Hb, but not globin, showed toxic effects in a live E. coli model but not in a model using killed E. coli. Methemoglobin, hematin, and the well-known Fenton reagents iron and iron-EDTA demonstrated the same lethal effect in E. coli peritonitis as Hb, while the addition of the Fenton inhibitors desferrioxamine (DF) and diethylenetriamine pentaacetate removed most of the cytotoxic activity of iron. Administration of a combined dose of superoxide dismutase and catalase minimized the action of Hb and iron-EDTA, suggesting that both O(2)(.-) and H(2)O(2) are involved in the toxic action of Hb in this rat model. The combination of the antioxidative enzymes and DF further suppressed iron-mediated lethality. An electron spin resonance technique with the spin-trapping reagent 5, 5-dimethyl-1-pyroline-N-oxide (DMPO) showed O(2)(.-) generation in the peritoneal fluid of rats injected with E. coli alone or E. coli plus iron-DF, and (.)OH generation was detected in the peritoneal fluid of the rats injected with iron-EDTA. Hb did not show any spin adduct of oxygen radicals, suggesting that Hb produces non-spin-trapping radical ferryl ion, which decayed the spin adduct of DMPO. In the presence of Hb or iron-EDTA, O(2)(-)-generating activity and viability of phagocytes decreased, whereas lipid peroxidation of peritoneal phagocytes increased. Generation of oxygen radicals and lipid peroxidation did not differ in the live and dead bacterial models. Bacterial numbers in the peritoneal cavity and blood were markedly increased in the live bacterial model with Hb and iron-EDTA. The Fenton inhibitor iron-DF prevented the loss of phagocyte function, lipid peroxidation, and bacterial proliferation. These results led us to conclude that the lethal toxicity of Hb in bacterial peritonitis is associated with a Fenton-type reaction, the products of which decrease phagocyte viability, through the induction of lipid peroxidation, allowing bacterial proliferation and resulting in mortality.

Animals↗
PubMed1999

Cloning and molecular characterization of the genes for carbon monoxide dehydrogenase and localization of molybdopterin, flavin adenine dinucleotide, and iron-sulfur centers in the enzyme of Hydrogenophaga pseudoflava.

Carbon monoxide dehydrogenases (CO-DH) are the enzymes responsible for the oxidation of CO to carbon dioxide in carboxydobacteria and consist of three nonidentical subunits containing molybdopterin flavin adenine dinucleotide (FAD), and two different iron-sulfur clusters (O. Meyer, K. Frunzke, D. Gadkari, S. Jacobitz, I. Hugendieck, and M. Kraut, FEMS Microbiol. Rev. 87:253-260, 1990). The three structural genes of CO-DH in Hydrogenophaga pseudoflava were cloned and characterized. The genes were clustered on the chromosome in the transcriptional order cutM-cutS-cutL. The cloned cutM, cutS, and cutL genes had open reading frames of 864, 492, and 2,412 nucleotides, coding for proteins with calculated molecular weights of 30,694, 17,752, and 87,224, respectively. The overall identities in the nucleotide sequence of the genes and the amino acid sequence of the subunits with those of other carboxydobacteria were 64.5 to 74.3% and 62.8 to 72.3%, respectively. Primer extension analysis revealed that the transcriptional start site of the genes was the nucleotide G located 47 bp upstream of the cutM start codon. The deduced amino acid sequences of the three subunits of CO-DH implied the presence of molybdenum cofactor, FAD, and iron-sulfur centers in CutL, CutM, and CutS, respectively. Fluorometric analysis coupled with denaturing polyacrylamide gel electrophoresis of fractions from hydroxyapatite column chromatography in the presence of 8 M urea of active CO-DH and from gel filtration of spontaneously inactivated enzyme revealed that the large and medium subunits of CO-DH in H. pseudoflava bind molybdopterin and FAD cofactors, respectively. Iron-sulfur centers of the enzyme were identified to be present in the small subunit on the basis of the iron content in each subunit eluted from the denaturing polyacrylamide gels.

Aldehyde Oxidoreductases↗
PubMed1999

Client participation and provider communication in family planning counseling: transcript analysis in Kenya.

To examine how much and in what ways clients participate in family planning consultations, and how providers influence their behavior, investigators analyzed transcripts of 178 counseling sessions with female clients in Kenya and developed coding guides that focus on client participation. The results show that providers dominate most counseling sessions, and clients rarely take an active role. The most common way for clients to participate is by volunteering additional information when they respond to providers' questions. Providers may encourage clients to play a more active role by building a sense of rapport, by relating contraceptive information specific to each client's personal situation, and by rewarding clients' attempts to participate.

Journal Article↗
PubMed1998

Determination of free acetaldehyde in total blood for investigating the effect of aspartate on metabolism of alcohol in mice.

To explore the effect of sodium L-aspartate monohydrate (aspartate) as a NAD+ regenerating agent for acetaldehyde in alcohol metabolism, a simple HPLC method has been developed for the measurement of free acetaldehyde in total mice blood digested with alcohol and aspartate. The blood samples were collected in EDTA Vacutainer tubes, and treated with 2,4-dinitrophenylhydrazine (DNP hydrazine) reagent in total blood. Acetaldehyde DNP hydrazone was extracted from total blood and analyzed by HPLC using an Ultrasphere ODS column. The compounds were separated using acetonitrile-water (50:50, v/v) as mobile phase and detected at 356 nm. The detection limit for acetaldehyde DNP hydrazone was 0.1 ppm. A blank determination was carried out for each analysis and subtracted from the results. The amount of acetaldehyde in blood has been determined as a function of time lapse after sole alcohol administration and aspartate ingestion followed by alcohol administration, respectively. This comparative analysis demonstrates that the ingestion of aspartate before the administration of alcohol dramatically decreases the aldehyde level in blood, and aspartate may be utilized as a prospective antagonist for acceleration of ethanol metabolism and prevention of acetaldehyde toxicity.

Acetaldehyde↗
PubMed1998

Nitric oxide suppression of apoptosis occurs in association with an inhibition of Bcl-2 cleavage and cytochrome c release.

It is now known that caspase-3-like protease activation can promote Bcl-2 cleavage and mitochondrial cytochrome c release and that these events can lead to further downstream caspase activation. NO has been proposed as a potent, endogenous inhibitor of caspase-3-like protease activity. Experiments were carried out to determine whether NO could interrupt Bcl-2 cleavage or cytochrome c release by the inhibition of caspase activity linking these events. NO inhibited the capacity of purified caspase-3 to cleave recombinant Bcl-2. Both Bcl-2 cleavage and cytochrome c release were inhibited in tumor necrosis factor alpha- and actinomycin D-treated MCF-7 cells exposed to NO donors. The NO-mediated inhibition of Bcl-2 cleavage and cytochrome c release occurred in association with an inhibition of apoptosis and caspase-3-like activation. Thus, NO suppresses a key step in the positive feedback amplification of apoptotic signaling by preventing Bcl-2 cleavage and cytochrome c release.

Apoptosis↗
PubMed1998

Cloning and characterization of ribulose bisphosphate carboxylase gene of a carboxydobacterium, hydrogenophagea pseudoflava DSM 1084.

The ribulose bisphosphate carboxylase/oxygenase rbcL and rbcS genes of a carbon monoxide-oxidizing bacterium, Hydrogenophaga pseudoflava DSM 1084, were cloned and sequenced. The cloned rbcL and rbcS genes had open reading frames of 1422 and 351 nucleotides encoding RbcL and RbcS with calculated molecular masses of 52,689 and 13,541, respectively. The known active site residues in other RbcL proteins were conserved in the H. pseudoflava proteins. The H. pseudoflava RbcS protein lacked the 12-residue internal sequence found in the plant enzymes. The 2 genes were separated by a 134 bp intergenic region and cotranscribed as a 2.0 kb rbcLS mRNA. Novel two perfect 9 bp direct repeats overlapping with two dyad symmetries were found in the rbcLS promoter region.

Amino Acid Sequence↗
PubMed1998

Nitric oxide prevents IL-1beta and IFN-gamma-inducing factor (IL-18) release from macrophages by inhibiting caspase-1 (IL-1beta-converting enzyme).

Procytokine processing by caspase-1 is required for the maturation and release of IL-1beta and IFN-gamma-inducing factor (IGIF) (or IL-18) from activated macrophages (Mphi). Nitric oxide (NO) has emerged as a potent inhibitor of cysteine proteases. Here, we tested the hypothesis that NO regulates cytokine release by inhibiting IL-1beta-converting enzyme (ICE) or caspase-1 activity. Activated RAW264.7 cells released four to five times more IL-1beta, but not TNF-alpha, in the presence of the NO synthase inhibitor N(G)-monomethyl-L-arginine. Stimulated peritoneal Mphi from wild-type mice (inducible NO synthase (iNOS)+/+) also released more IL-1beta if exposed to N(G)-monomethyl-L-arginine, whereas Mphi from iNOS knockout mice (iNOS-/-) did not. Inhibition of NO synthesis in stimulated RAW264.7 cells also resulted in a threefold increase in intracellular caspase-1 activity. The NO donor S-nitroso-N-acetyl-DL-penicillamine inhibited caspase-1 activity in cells as well as the activity of purified recombinant caspase-1 and also prevented the cleavage of pro-IL-1beta and pro-IGIF by recombinant caspase-1. The inhibition of caspase-1 by NO was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. An in vivo role for the regulation of caspase-1 by NO was established in iNOS knockout animals, which exhibited significantly higher plasma levels of IL-1beta and IFN-gamma than their wild-type counterparts at 10 h following LPS injection. Taken together, these data indicate that NO suppresses IL-1beta and IGIF processing by inhibiting caspase-1 activity, providing evidence for a unique role for induced NO in regulating IL-1beta and IGIF release.

Animals↗
PubMed1998

Overexpression and characterization of the cDNA encoding the coat protein of cucumber mosaic virus (Strain ABI) isolated in Korea.

We constructed a recombinant CMVCP expression vector termed pMALCMV in which cDNA fragment encoding CMVCP is ligated into pMAL-c2, an E. coli expression vector. Overexpression of pMALCMV containing the entire open reading frame of CMV cDNA sequence and the maltose binding protein (MBP) leader gene was facilitated in E. coli TB1 cells, which resulted in the production of a fusion protein of MBP-CMVCP (Mr 67.7 kDa) that was immunoprecipitable with rabbit polyclonal antiserum specific for MBP. The CMVCP (Mr 24.5 kDa) was isolated through a preparative SDS polyacrylamide gel following digestion of the affinity ligand purified fusion protein with Factor Xa. The partial amino acid sequences of the cleaved proteins were confirmed at the amino terminus by peptide sequencing. The CMVCP antiserum was also prepared by intraperitoneal injection of this purified CP into a BALB/c mouse. Immunoblot analysis showed that the purified CMVCP from the Factor Xa cleavage reaction was an authentic overexpression product of the cloned CMVCP. Using an RNA mobility shift assay, it was demonstrated that CMVCP can bind to its own RNA transcript in a concentration dependent manner. However, the complex formed between CMVCP and its RNA was abolished by the addition of a polyclonal antibody that had been raised against CMVCP, confirming that the overexpressed CMVCP specifically interacts with its own RNA. Thus, our results can provide a basis for the development of a hybridoma cell line expressing the monoclonal antibody for CMVCP and molecular cloning of their genes, which may lead to the creation of CMV-resistant transgenic plants.

ATP-Binding Cassette Transporters↗
PubMed1998

Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts.

Recombinant humanized anti-HER2 antibody, rhuMAb HER2, inhibits the growth of breast cancer cells overexpressing HER2 and has clinical activity. We explored in preclinical models its capacity to enhance the tumoricidal effects of paclitaxel and doxorubicin. In cultures of naturally HER2-overexpressing cancer cells, rhuMAb HER2 inhibited growth and enhanced the cytotoxic effects of paclitaxel. Treatment of well established BT-474 breast cancer xenografts overexpressing HER2 in athymic mice with rhuMAb HER2 resulted in a dose-dependent antitumor activity. In combination studies, treatment with paclitaxel and rhuMAb HER2 or doxorubicin and rhuMAb HER2 resulted in greater inhibition of growth than that observed with any agent alone. The combination of paclitaxel and rhuMAb HER2 resulted in the highest tumor growth inhibition and had a significantly superior complete tumor regression rate when compared with either paclitaxel or rhuMAb HER2 alone. Clinical trials that are built on these results are under way.

Animals↗
PubMed1998

Generation of the short RNA transcript in Leishmaniavirus correlates with the growth of its parasite host, Leishmania.

Leishmaniavirus 1 is a double-stranded RNA virus that infects the New World kinetoplastid parasites, Leishmania braziliensis, and Leishmania guyanensis. The isolated virus particles contain an RNA-dependent RNA polymerase which exhibits both transcriptase activity for genome-length plus-strand synthesis and replicase activity for genome-length minus-strand synthesis. Recently, we identified a 320 nucleotide short RNA transcript of Leishmaniavirus 1-4, derived from the 5' end of the viral plus-strand, which is generated by the virus capsid via site-specific cleavage of the full-length positive single-stranded RNA. We have hypothesized that this short RNA transcript functions to regulate the virus life cycle during the growth of its parasite host, Leishmania guyanensis. To address this hypothesis, we measured the relative amount of short RNA transcripts and the absolute number of viral genomes per infected cell from log through stationary phase of the parasite growth cycle. In vitro assays of the viral polymerase showed an overall increase in viral polymerase activity from log growth into stationary phase which mirrored an in vivo increase in the quantity of double-stranded genome as measured by agarose gel electrophoresis. We have developed competitive reverse transcription-polymerase chain reaction (RT-PCR) assays to measure the relative amounts of viral transcripts in infected cells as well as the number of viral genomes per infected cell. The results of these assays show that the amount of full-length virus transcripts peaks in the parasite stationary phase (132 transcripts per cell), and that the short transcript is most abundant in the early stationary phase cells (24 transcripts per cell).

Animals↗
PubMed1998

Mutational analysis of disulfide bonds in the trypsin-reactive subdomain of a Bowman-Birk-type inhibitor of trypsin and chymotrypsin--cooperative versus autonomous refolding of subdomains.

It is widely believed that protein folding is a hierarchical process proceeding from secondary structure via subdomains and domains towards the complete tertiary structure. Accordingly, protein subdomains should behave as independent folding units. However, this prediction would underestimate the well-established structural significance of tertiary context and domain interfaces in proteins. The principal objective of this work was to distinguish between autonomous and cooperative refolding of protein subdomains by means of mutational analysis. The double-headed Bowman-Birk inhibitor of trypsin and chymotrypsin of known crystal structure was selected for study. The relative orientation of the two subdomains is stabilized by intramolecular and water-mediated hydrogen bonds and close ion pairs across a polar domain interface. The binary arrangement of a trypsin-reactive and a chymotrypsin-reactive subdomain facilitates the distinction of local and global irregularities in the mutants of this protein by means of functional assays. The functional consequences of five replacements in the S-S bond framework of the trypsin-reactive subdomain are analyzed in the present report. The mutants were subjected to refolding experiments in a refolding buffer and on trypsin-Sepharose as a template with complementary structure leading into a fully active state. The stability of the variants was assessed by means of subsequent equilibration experiments in solution. The mutants may be grouped into the following two classes: the class-I mutations located within beta-strand A are characterized by a breakdown of the trypsin- and the chymotrypsin-reactive subdomain upon refolding in solution and a complicated behavior in the equilibration experiments; by contrast, the Class-II mutations (beta-strand B) display rather local perturbations and a reversible return to the initial ratio of the two subdomains. This points to a significance of polar interactions connecting the beta-strand A of the trypsin-reactive with the chymotrypsin-reactive subdomain. In conclusion, the polar domain interface appears as a major refolding unit of the Bowman-Birk inhibitor.

Amino Acid Sequence↗
PubMed1998

Brazilin inhibits activities of protein kinase C and insulin receptor serine kinase in rat liver.

Hypoglycemic action of brazilin was found to be based on the improvement of peripheral glucose utility, and this action might be correlated with the insulin action pathway. In the present study we investigated the effect of brazilin on the insulin receptor autophosphorylation, protein kinase C (PKC), protein phosphatase and insulin receptor serine kinase in order to confirm whether the hypoglycemic mechanism is concerned with insulin action pathway. Brazilin was found to inhibit PKC and insulin receptor serine kinase, which are involved in the regulation of insulin signal pathway. But any significant effect was not shown on insulin receptor tyrosine kinase activity, autophosphorylation and phosphatase activity. These findings suggest that brazilin might enhance insulin receptor function by decreasing serine phosphorylation, which might mediate hypoglycemic effect of brazilin.

Animals↗
PubMed1998

Synthesis and evaluation of 5-aminosalicyl-glycine as a potential colon-specific prodrug of 5-aminosalicylic acid.

As a new colon-specific prodrug of 5-aminosalicylic acid (5-ASA), 5-aminosalicyl-glycine (5-ASA-Gly) was prepared by a simple synthetic route in good yield. Apparent partition coefficients of 5-ASA-Gly were lower than those of 5-ASA, which determined in CHCl3/pH 6.8 buffer or n-octanol/pH 6.8 buffer system. Stability of 5-ASA-Gly by peptidases was investigated by incubation of 5-ASA-Gly with the homogenates of tissue and contents of stomach, proximal small intestine or distal small intestine of rats at 37 degrees C. 5-ASA was not detected, indicating that the prodrug was stable in the upper intestine. The amount of 5-ASA liberated from incubation of the prodrug in cecal or colonic contents of rats was about 65% or 27% in 8 hrs, respectively, which indicated that the prodrug activation took place more readily in the rat cecum whose bacterial counts are high like human colon. Results from in vitro experiments suggested 5-ASA-Gly as a promising candidate of a colon-specific prodrug of 5-ASA.

Aminosalicylic Acids↗
PubMed1998

Synthesis and properties of dextran-5-aminosalicylic acid ester as a potential colon-specific prodrug of 5-aminosalicylic acid.

Dextran-5-aminosalicylic acid ester (dextran-5-ASA) was synthesized as a colon-specific prodrug of 5-aminosalicylic acid (5-ASA) which is active against inflammatory bowel diseases. Chemical stability of dextran-5-ASA in the bath of pH 1.2 or 6.8 was investigated at 37 degrees C for 6 hrs, and 5-ASA was not released on such conditions. Depolymerization (%) of dextran-5-ASA by dextranase with the degree of substitution (DS) of 18, 23, or 30 was 92, 62 or 45 in 8 hrs respectively, but was not affected by the MW of dextran (9,000, 40,600, 80,200 or 580,000). Distribution of 5-ASA in dextran, determined by gel filtration chromatography, appeared to be relatively uniform. Incubation of dextran-5-ASA (DS 18) in cecal contents of rats released 20% (28 g) and 35% (49 g) of 5-ASA in 8 hrs and 24 hrs, respectively, but no 5-ASA was liberated from small intestinal contents.

Aminosalicylic Acids↗
PubMed1998

Formation, properties and antimicrobial activities of cotton xanthate-Cu(II)-homosulfamine complex.

To develop a cotton derivatives with prolonged antimicrobial activities, homosulfamine (Hs) was coupled to cotton xanthate (CX) via chelate bond in the presence of Cu(II) ion by one- and two-bath processes. In one-bath process, CX was treated with Cu(II)-Hs solution. In two-bath process, CX was treated with Cu(II) ion solution to produce CX-Cu(II) complex, which was isolated and treated in turn with Hs solution. Effects of concentration, Cu(II)/Hs ratio, and pH on the binding of Hs were investigated at 10 degrees C. In one-bath process, binding of Hs took place readily with optimum pH around 5-6. The amount of binding increased to give a maximum within 5 min and decreased slowly to establish an equilibrium within an hour. In two-bath process, binding of Hs was much lower than that of one-bath process. Release of Hs from CX-Cu(II)-Hs was investigated by batch and flow method. Antimicrobial activities of CX-Cu(II)-Hs were tested against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli and it showed prolonged activity compared to that of free Hs.

Anti-Bacterial Agents↗
PubMed1998

Intramedullary anaplastic oligodendroglioma in a child.

Primary intramedullary anaplastic oligodendroglioma is a rare tumor, only four cases of which have been reported. The authors present the case of a 38-month-old boy with primary intramedullary anaplastic oligodendroglioma. He underwent partial removal of the tumor and spinal radiation therapy. The residual tumor disappeared 12 months after radiation, and 48 months after treatment there was no evidence of recurrence. This case shows that in primary intramedullary anaplastic oligodendroglioma, postoperative radiation therapy confined to the spinal cord can yield an optimal result.

Child, Preschool↗
PubMed1998

Rat liver postischemic lipid peroxidation and vasoconstriction depend on ischemia time.

In this investigation, we used chemiluminescence to study the ability of increasing durations of ischemia (1, 2, or 2.5 h) to induce enhanced generation of reactive oxygen species in a crystalloid perfused rat liver model. To evaluate the effect of reactive oxygen species generation upon the development of the postischemic hypoperfusion, hepatic vascular resistance was simultaneously monitored. One hour of ischemia did not produce sustained reactive oxygen species generation or development of no-reflow. Two hours of ischemia did not result in sustained reactive oxygen species generation but did produce no-reflow. Sustained reactive oxygen production was achieved after 2.5 h of ischemia and was accompanied by the development of no-reflow. We found that 2.5 h of ischemia is the threshold for sustained lipid peroxidation. Both lipid peroxidation and no-reflow could be mitigated through the administration of superoxide dismutase. Superoxide dismutase could reduce the amount of cell injury due to the enhanced lipid peroxidation induced by 2.5 h of ischemia. Limitation of reactive oxygen species generation to a critical threshold, either by restricting the duration of ischemia or by pharmacological intervention, may be an important means of preventing further cellular injury through no-reflow and lipid peroxidation.

Animals↗