Biomedical subjects

Eun Hee Cho

Publications and source records attributed to Eun Hee Cho.

13 recordsLinked to original sources

PubMed2006

Cloning and functional expression of the gene encoding an inhibitor against Aspergillus flavus alpha-amylase, a novel seed lectin from Lablab purpureus (Dolichos lablab).

Maize is one of the more important agricultural crops in the world and, under certain conditions, prone to attack from pathogenic fungi. One of these, Aspergillus flavus, produces toxic and carcinogenic metabolites, called aflatoxins, as byproducts of its infection of maize kernels. The alpha-amylase of A. flavus is known to promote aflatoxin production in the endosperm of these infected kernels, and a 36-kDa protein from the Lablab purpureus, denoted AILP, has been shown to inhibit alpha-amylase production and the growth of A. flavus. Here, we report the isolation of six full-length labAI genes encoding AILP and a detailed analysis of the activities of the encoded proteins. Each of the six labAI genes encoded sequences of 274 amino acids, with the deduced amino acid sequences showing approximately 95-99% identity. The sequences are similar to those of lectin members of a legume lectin-arcelin-alpha-amylase inhibitor family reported to function in plant resistance to insect pests. The labAI genes did not show any of the structures characteristic of conserved structures identified in alpha-amylase inhibitors to date. The recombinant proteins of labAI-1 and labAI-2 agglutinated human red blood cells and inhibited A. flavus alpha-amylase in a manner similar to that shown by AILP. These data indicate that labAI genes are a new class of lectin members in legume seeds and that their proteins have both lectin and alpha-amylase inhibitor activity. These results are a valuable contribution to our knowledge of plant-pathogen interactions and will be applicable for developing protocols aimed at controlling A. flavus infection.

Amino Acid Sequence↗

PubMed2005

Identification of ADP-ribosylation site in human glutamate dehydrogenase isozymes.

When the influence of ADP-ribosylation on the activities of the purified human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was measured in the presence of 100 microM NAD+ for 60 min, hGDH isozymes were inhibited by up to 75%. If incubations were performed for longer time periods up to 3 h, the inhibition of hGDH isozymes did not increased further. This phenomenon may be related to the reversibility of ADP-ribosylation in mitochondria. ADP-ribosylated hDGH isozymes were reactivated by Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase. The stoichiometry between incorporated ADP-ribose and GDH subunits shows a modification of one subunit per catalytically active homohexamer. Since ADP and GTP had no effects on the extent of modification, it would appear that the ADP-ribosylation is unlikely to occur in allosteric sites. It has been proposed that Cys residue may be involved in the ADP-ribosylation of GDH, although identification of the reactive Cys residue has not been reported. To identify the reactive Cys residue involved in the ADP-ribosylation, we performed cassette mutagenesis at three different positions (Cys59, Cys119, and Cys274) using synthetic genes of hGDH isozymes. Among the Cys residues tested, only Cys119 mutants showed a significant reduction in the ADP-ribosylation. These results suggest a possibility that the Cys119 residue has an important role in the regulation of hGDH isozymes by ADP-ribosylation.

Adenosine Diphosphate Ribose↗

PubMed2005

Critical role of the cysteine 323 residue in the catalytic activity of human glutamate dehydrogenase isozymes.

The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the Km of the enzyme for NADH and alpha-ketoglutarate increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency (kcat/Km) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in kcat values. There was a linear relationship between incorporation of [14C]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [14C] incorporated per mol of monomer for wild type hGDHs. No incorporation of [14C]p-chloromer-curibenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.

Adenosine Diphosphate Ribose↗

PubMed2005

Tissue-engineered semi-microporous segmented polyetherurethane vascular prostheses.

We investigated the biocompatibility of semi-microporous segmented polyetherurethane (SPEU) that was coated with human microvascular endothelial cells. Three types of prosthesis were examined: (1) non-porous (control), (2) SPEU without a collagen coating (PNC) and (3) collagen-coated SPEU (PC). The attachment and proliferation of endothelial cells on SPEU (PNC and PC prostheses) was twice as great as in the control prostheses and endothelial cell adhesion in the PC prostheses was 1.4-times greater than in the PNC prostheses. The degree of endothelial cell coverage in the control prostheses was significantly lower than in the PNC and PC prostheses (P < 0.05), and there was no significant difference between the PNC and PC prostheses in the degree of endothelial cell coverage. As regards the retention of endothelial cells under simulated blood-flow conditions, the PNC prostheses exhibited a greater decrease in endothelial cell coverage, as compared to the PC prostheses (P < 0.05). Similarly, the PNC prostheses exhibited less platelet adhesion than the PC prostheses (P < 0.05). In summary, the semi-microporous SPEU offered better surfaces to anchor and culture for endothelial cells, and had superior anti-coagulant properties, as compared to nonporous SPEU. The coating of SPEU with collagen influenced both the attachment of endothelial cells and blood coagulation on the SPEU.

Blood Coagulation↗

PubMed2004

Evaluation for safety of cultured corneal fibroblasts with cotreatment of alcohol and mitomycin C.

PURPOSE: To investigate the effects of alcohol and mitomycin C (MMC) on cultured corneal fibroblast of the rabbit to determine the safety of this compound for clinical use. METHOD: Corneal fibroblasts of New Zealand rabbits were cultured. Various concentrations (0%, 10%, 20%, 30%, 40%, and 60%) of ethanol were applied for 10, 20, 30, and 40 seconds to estimate dose- and time-dependent responses of cultured corneal fibroblasts. Cell viability was assessed using the MTT assay. Treated cells were additionally stained with Hoechst and annexin V for the identification of apoptosis. To investigate the coeffects of ethanol and MMC, dose and time dependency were evaluated after treatment with various concentrations of ethanol and MMC at different exposure times, and cell viability was established. To determine the latent effects of ethanol and MMC, cultured corneal fibroblasts were cotreated with various concentrations of ethanol and 0.02% MMC for various periods and washed out, and then one group was incubated for 24 hours and another group was not incubated. Cell viability was estimated, and Hoechst and annexin V staining were performed before and after incubation. To establish the pathway of cell death, caspase-3 activities were measured in cultured corneal fibroblasts treated with ethanol or MMC. RESULTS: Cell viability after ethanol treatment was dose and time dependent. After application of ethanol for 10 seconds, cell viability was significantly reduced with 20% ethanol (P = 0.001). At 20, 30, and 40 seconds of treatment with 10% ethanol, cell viability was significantly reduced (P < 0.01). Hoechst and annexin V staining revealed typical characteristics of apoptosis, such as bright fluorescent chromatin condensation, low fluorescence of nuclear fragmentation, and cell membrane shrinkage. Cell viability was more significantly reduced after cotreatment with alcohol and MMC, compared with treatment with alcohol alone. Moreover, cell viability was considerably decreased in the incubated group, compared with the nonincubated group. After 24 hours of incubation, cultured corneal fibroblasts cotreated with 10% ethanol and 0.02% MMC were stained with Hoechst and annexin V. Results were similar to data obtained with ethanol-treated cells. However, after application of 20% alcohol and MMC, a significant number of cells were not viable and were detached from the well walls. Caspase-3 activity significantly increased after treatment with 30% ethanol only and 30% ethanol in conjunction with 0.02% MMC. CONCLUSIONS: Alcohol and MMC reduced cell viability in cultured corneal fibroblasts in a dose- and time-dependent manner and had synergistic effects. This is related to the caspase-3 pathway, especially with concentrations of ethanol over 30%. Cotreatment with these reagents may significantly damage cultured corneal fibroblasts.

Animals↗

PubMed2004

Maternal serum levels of VCAM-1, ICAM-1 and E-selectin in preeclampsia.

Endothelial dysfunction is thought to be a central pathogenic feature in preeclampsia on the basis of elevated adhesion molecules. The aim of the present study was to compare the levels of soluble vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1) and E-selectin (sE-selectin) in sera of normal and preeclamptic pregnancies. We studied the serum levels of sVCAM-1, sICAM-1 and sE-selectin in normal pregnant women (n=63), mild preeclampsia (n=33) and severe preeclampsia (n=82). Concentrations of soluble adhesion molecules were determined with enzyme-linked immunoassay (ELISA). Serum concentrations of sVCAM-1 were significantly higher in both mild (p=0.004) and severe preeclampsia (p=0.000) than normal pregnancy. There were also significant differences in sVCAM- 1 levels between mild and severe preeclampsia (p=0.002). sICAM-1 levels of severe preeclampsia were statistically different from those of normal pregnancy (p=0.038). Levels of sE-selectin were elevated in both mild (p=0.011) and severe preeclampsia (p=0.000) compared to normal pregnancy, but no statistical difference between the mild and severe preeclampsia (p=0.345). These results suggest that all three soluble adhesion molecules are increased in severe preeclampsia, and sVCAM-1 among them may be useful in predicting the severity of preeclampsia.

Adult↗

PubMed2003

A novel flavin-containing monooxygenase from Methylophaga sp strain SK1 and its indigo synthesis in Escherichia coli.

We cloned a gene from Methylophaga sp. strain SK1. This gene was responsible for producing, the blue pigment, indigo. The complete open reading frame was 1371 bp long, which encodes a protein of 456 amino acids. The molecular mass of the encoded protein was 105 kDa, consisting of homodimer of 54 kDa with an isoelectric point of 5.14. The deduced amino acid sequence from the gene showed approximately 30% identities with flavin-containing monooxygenases (FMOs) of human (FMO1-FMO5), Arabidopsis, and yeast. It contained three characteristic sequence motifs of FMOs: FAD binding domain, FMO-identifying sequence motif, and NADPH binding domain. In addition, the biochemical properties such as substrate specificities and absorption spectra were similar to the eukaryotic FMO families. Thus, we assigned the enzyme to be a bacterial FMO. The recombinant Escherichia coli expressing the bacterial FMO produced up to 160 mg of indigo per liter in the tryptophan medium after 12h cultivation. This suggests that the recombinant strain has a potential to be applied in microbial indigo production.

Amino Acid Motifs↗

PubMed2003

The molecular basis of co-operative DNA binding between lambda integrase and excisionase.

Higher-order nucleoprotein complexes often stabilize catalytic proteins in appropriate conformations for optimal activity and contribute to regulation during reactions requiring association of proteins and DNA. Formation of such complexes, known as intasomes, is required for site-specific recombination catalysed by bacteriophage Lambda Integrase protein (Int). Int-catalysed recombination is regulated by a second bacteriophage-encoded protein, Excisionase (Xis), which both stimulates excision and inhibits integration. To exert its effect, Xis binds co-operatively with Int, thereby inducing and stabilizing a DNA bend that alters the intasome structures formed during recombination. A rare int mutant, int 2268 ts, was reported (Enquist, L.W. and Weisberg, R.A. (1984) Mol Gen Genet 195: 62-69) to be more defective for excision than integration. Here, we have determined that this mutant Int protein contains an E47K substitution, and that the resultant excision-specific defect is due, at least in part, to destabilized interactions between Int and Xis. Analysis of several engineered substitutions at Int position 47 showed that a negatively charged residue is required for co-operative DNA binding between Int and Xis, and suggest that the Int-E47 residue may contact Xis directly. Substitutions at Int position 47 also affect co-operative binding among Int proteins at arm-type DNA sites, and thereby reduce the efficiency of both integration and excision. Collectively, these results suggest that a single surface of the Int amino-terminal domain mediates two alternate types of co-operative binding interactions.

Attachment Sites, Microbiological↗

PubMed2002

Importance of glutamate 279 for the coenzyme binding of human glutamate dehydrogenase.

Although the structure of glutamate dehydrogenase (GDH) has been reported from various sources including mammalian GDH, there are conflicting views regarding the location and mechanism of actions of the coenzyme binding. We have expanded these speculations by photoaffinity labeling and cassette mutagenesis. Photoaffinity labeling with a specific probe, [(32)P]nicotinamide 2-azidoadenosine dinucleotide, was used to identify the NAD(+) binding site within human GDH encoded by the synthetic human GDH gene and expressed in Escherichia coli as a soluble protein. Photolabel-containing peptides generated with trypsin were isolated by immobilized boronate affinity chromatography. Photolabeling of these peptides was most effectively prevented by the presence of NAD(+) during photolysis, demonstrating a selectivity of the photoprobe for the NAD(+) binding site. Amino acid sequencing and compositional analysis identified Glu(279) as the site of photoinsertion into human GDH, suggesting that Glu(279) is located at or near the NAD(+) binding site. The importance of the Glu(279) residue in the binding of NAD(+) was further examined by cassette mutagenesis with mutant enzymes containing Arg, Gly, Leu, Met, or Tyr at position 279. The mutagenesis at Glu(279) has no effects on the expression or stability of the different mutants. The K(m) values for NAD(+) were 10-14-fold greater for the mutant GDHs than for wild-type GDH, whereas the V(max) values were similar for wild-type and mutant GDHs. The efficiency (k(cat)/K(m)) of the mutant GDH was reduced up to 18-fold. The decreased efficiency of the mutants results from the increase in K(m) values for NAD(+). In contrast to the K(m) values for NAD(+), wild-type and mutant GDHs show similar K(m) values for glutamate, indicating that substitution at position 279 had no appreciable effect on the affinity of enzyme for glutamate. There were no differences in sensitivities to ADP activation and GTP inhibition between wild-type and mutant GDH, suggesting that Glu(279) is not directly involved in allosteric regulation. The results with photoaffinity labeling and cassette mutagenesis studies suggest that Glu(279) plays an important role for efficient binding of NAD(+) to human GDH.

Adenosine Diphosphate↗

PubMed2002

Interactions between integrase and excisionase in the phage lambda excisive nucleoprotein complex.

Bacteriophage lambda site-specific recombination comprises two overall reactions, integration into and excision from the host chromosome. Lambda integrase (Int) carries out both reactions. During excision, excisionase (Xis) helps Int to bind DNA and introduces a bend in the DNA that facilitates formation of the proper excisive nucleoprotein complex. The carboxyl-terminal alpha-helix of Xis is thought to interact with Int through direct protein-protein interactions. In this study, we used gel mobility shift assays to show that the amino-terminal domain of Int maintained cooperative interactions with Xis. This finding indicates that the amino-terminal arm-type DNA binding domain of Int interacts with Xis.

Bacteriophage lambda↗

PubMed2002

Dipolarity, hydrogen-bond basicity and hydrogen-bond acidity of aqueous poly(ethylene glycol) solutions.

Poly(ethylene glycol) (PEG) in water is known to alter the structure and/or state of water to give a different polarity from that of pure water. We determined using the solvatochromic comparison method the dipolarity/polarizability (pi*), hydrogen bond (HB) accepting basicity (beta) and HB donating acidity (alpha) of aqueous solutions of PEGs of variegated molecular weights at different concentrations in order to understand the influence of the polymer on these properties of water. It was observed that PEG decreases alpha for water while it does not change pi* and beta appreciably in the range of the molecular weight and compositions studied.

Hydrogen Bonding↗

PubMed2002

The comparison between Behçet's disease and spondyloarthritides: does Behçet's disease belong to the spondyloarthropathy complex?

This study was to clarify whether Behcet's disease (BD) could be classified into the spondyloarthropathy (SpA) complex. It was undertaken on 58 patients with BD (BD group), 56 patients with SpA (SpA group), and 3 patients who concurrently satisfied the criteria for BD and SpA (BDSpA group). The clinical parameters and known susceptible HLA antigens were compared between BD group and SpA group. In addition, 3 patients in BDSpA group were reviewed. The prevalence of definitive sacroiliitis (SI) in BD group and SpA group was 46.4% and 5.2%, respectively. However, none had a definitive SI in healthy controls. Enthesitis was observed in 3.4% of BD group and in 50% of SpA group. The patterns of eye involvement were different between these two groups. HLA-B27 was negative in all 49 patients of BD group, whereas it was positive in 67.9% of SpA group. The prevalence of HLA-B51 was 51.7% in BD group, and that in SpA group was 21.4%. One patient in BDSpA group was considered to have concurrent BD and ankylosing spondylitis (AS). Another patient was closer to AS, and the third to BD. Conclusively, it seems that BD could not be classified into the SpA complex.

Adult↗

PubMedDate not supplied

Reactive amino acid residues involved in glutamate-binding of human glutamate dehydrogenase isozymes.

In the present study, the cassette mutagenesis at several putative positions (K94, G96, K118, K130, or D172) was performed to examine the residues involved in the glutamate-binding of the human glutamate dehydrogenase isozymes (hGDH1 and hGDH2). None of the mutations tested affected the expression or stability of the proteins. There was dramatic reduction in the catalytic efficiency in mutant proteins at K94, G96, K118, or K130 site, but not at D172 site. The K(M) values for glutamate were 4-10-fold greater for the mutants at K94, G96, or K118 site than for the wild-type hGDH1 and hGDH2, whereas no differences in the K(M) values for NAD(+) were detected between the mutant and wild-type enzymes. For K130Y mutant, the K(M) value for glutamate increased 1.6-fold, whereas the catalytic efficiency (k(cat)/K(M)) showed only 2-3% of the wild-type. Therefore, the decreased catalytic efficiency of the K130 mutant mainly results from the reduced k(cat) value, suggesting a possibility that the K130Y residue may be involved in the catalysis rather than in the glutamate-binding. The D172Y mutant did not show any changes in k(cat) value and K(M) values for glutamate and NAD(+), indicating that D172Y is not directly involved in catalysis and substrates binding of the hGDH isozymes. For sensitivity to ADP activation, only the D172Y mutant showed a reduced sensitivity to ADP activation. The reduction of ADP activation in D172Y mutant was more profoundly observed in hGDH2 than in hGDH1. There were no differences in their sensitivities to GTP inhibition between the wild-type and mutant GDHs at all positions tested. Our results suggest that K94, G96, and K118 residues play an important role, although at different degrees, in the binding of glutamate to hGDH isozymes.

Adenosine Diphosphate↗