Biomedical subjects

S M Byun

Publications and source records attributed to S M Byun.

At least 19 recordsLinked to original sources

PubMed2001

Binge drinking in Chinese, Korean, and White college students: genetic and ethnic group differences.

Studies of Asian college students have found that rates of binge drinking are associated with variation in the aldehyde dehydrogenase (ALDH2) gene. Chinese and Koreans have different prevalence rates of the ALDH2*2 allele, alcohol use, and alcoholism. The association of ALDH2 status and ethnic group with binge drinking was examined in 328 Chinese, Korean, and White college students. Ethnic group differences were found, with Whites having the highest rate of binge drinking, followed by Koreans and then Chinese. Among Asian participants, ALDH2 status and ethnicity related to binge drinking in an additive manner. Possessing an ALDH2*2 allele and being Chinese were protective factors, and being White and being Korean without an ALDH2*2 allele were risk factors for binge drinking. These results suggest that ALDH2 status, as well as other factors that differ in Koreans and Chinese, but do not interact with ALDH2, are associated with binge drinking among Asians.

Adult↗

PubMed2001

Expression of bisecting N-acetylglucosaminyltransferase-III in human hepatocarcinoma tissues, fetal liver tissues, and hepatoma cell lines of Hep3B and HepG2.

In this paper, uridine diphosphate (UDP)-N-acetylglucosamine/beta-D-mannoside beta-1,4 N-acetylglucosaminyltransferase III (GlcNAc-transferase-III C 2.4.1.144) activity was determined in human hepatoma cell lines of Hep3B and HepG2, and also compared with those of normal liver tissues and primary hepatocytes. GlcNAc-transferase-III enzymes of Hep3B and HepG2 were mainly detected in the membrane fraction. When GlcN,GlcN-biant-PA and UDP-GlcNAc were used as substrates, the Km values (4.7 mM for UDP-GlcNAc and 1.1 mM for GlcN, GlcN-biant-PA) of Hep3B GlcNAc-transferase-III were distinguishable from those of HepG2 GlcNAc-transferase-III (6.8 mM for UDP-GlcNAc and 3.4 mM for GlcN,GlcN-biant-PA). Furthermore, Hep3B enzyme in membrane fraction showed about 1.5-fold higher specific activity (1423 pmol/hr/mg) than that of HepG2 (1066 pmol/hr/mg). Normal liver cells and primary adult hepatocytes are characterized by a very low level of GlcNAc-transferase-III activity, whereas human hepatoma cells exhibited high activities. These data were supported by reverse transcription-polymerase chain reaction results, showing that expression of the GlcNAc-transferase-III mRNA increased in proportion to the enzymatic activities. Although the mechanism underlying the induction of this enzyme is unknown, lectin blot analysis showed that oligosaccharides in many glycoproteins were observed in hepatoma cells. By treating hepatocarcinoma cultures that express GlcNAc-transferase-III with inhibitors (tunicamycin, deoxymannojirimycin, and swainsonine) of different steps of the glycosylation, we provide evidence that expression of GlcNAc-transferase-III mRNA is dependent on glycosylation of cellular proteins.

Adult↗

PubMed2000

Asp41-His48 region of streptokinase is important in binding to a substrate plasminogen.

Streptokinase is a plasminogen activator protein produced by several strains of beta-hemolytic streptococci. Random mutagenesis of streptokinase was carried out for the determination of critical amino acid residues in plasminogen activation. We selected and sequenced 14 streptokinase mutants with no plasminogen activation activity on skim milk-plasminogen overlay plate. Specific activities of the selected streptokinase mutants were determined with chromogenic assay. Eight mutants (V19F, V35E, E85D, L292R, D325P, D341E, I345N, and M369L) resulted in greatly decreased amidolytic activities. However, unexpectedly, six mutants (D41C, S44K, S44P, R45P, H48T, and D220G) showed substantial amidolytic activities comparable to that of wild type. Moreover, five-point mutations were concentrated on the Asp41-His48 region. These data indicate that the Asp41-His48 region in a streptokinase-plasminogen binary complex plays an important role in binding to a substrate plasminogen.

Amidohydrolases↗

PubMed2000

Modulation of the peptide-binding specificity of a single-chain class II major histocompatibility complex.

We designed and expressed a single-chain class II major histocompatibility complex molecule capable of forming a stable complex with an antigenic peptide. The peptide-binding preference of the single-chain (sc) human leukocyte antigen derived from DRB5(*)0101 (DR51) was determined to be similar to that of the authentic one, which requires a bulky hydrophobic residue at position-1 (P1) as a primary anchor. For modulation of the peptide-binding affinity, we modified binding pocket 1 of sc DR51 by site-directed mutagenesis. The relative binding affinity of the engineered sc DR51 for several P1-substituted peptides was measured by competition assaying with a fluorescence labeled peptide. The sc DR51 molecule showed high affinity to the self-peptide derived from myelin basic protein, 87-98 with Phe as the P1 residue (F90F). While reduction of pocket 1 volume (betaG86V) decreased the affinity of F90F, it rather increased the affinity of the Ala-substituted peptide as to the P1 residue (F90A). Through more extensive engineering in the peptide-binding groove of the sc DR51 molecule, it is expected that we can construct sc DR51 variants with various peptide ligand motifs.

Binding Sites↗

PubMed2000

Enhancement of secretion and extracellular stability of staphylokinase in Bacillus subtilis by wprA gene disruption.

Staphylokinase (SAK), a polypeptide secreted by Staphylococcus aureus, is a plasminogen activator with a therapeutic potential in thrombosis diseases. A Bacillus subtilis strain which is multiply deficient in exoproteases was transformed by an expression plasmid carrying a promoter and a signal sequence of subtilisin fused in frame with the sak open reading frame. However, the amount of SAK secretion was marginal (45 mg/liter). In contrast, disruption of the wprA gene, which encodes a subtilisin-type protease, strongly promoted the production of SAK in the stationary phase (181 mg/liter). In addition, the extracellular stability of mature SAK was dramatically enhanced. These data indicate a significant role of the wprA gene product in degrading foreign proteins, both during secretion and in the extracellular milieu.

Bacillus subtilis↗

PubMed2000

Identification of H-2K(b)-restricted T-cell epitopes within the nucleocapsid protein of Hantaan virus and establishment of cytotoxic T-cell clones.

Although neutralizing antibodies against Hantaan virus (HTV) can protect hosts from viral infection, T-cell responses to HTV are also important in host defense against HTV. However, much less is known about cytotoxic T lymphocyte (CTL) responses to HTV. To identify CTL epitopes in the HTV nucleocapsid protein (NP), we selected 7 H-2K(b)-motif-fitting peptides. Of these peptides, 3 peptides (NP3, NP4, and NP7) were recognized by CTL responses derived from HTV-immunized mouse splenocytes. NP3 and NP4 peptides were also recognized by HTV-immunized splenocytes after secondary in vitro stimulation with the relevant peptide, but NP7 could not be recognized after in vitro stimulation. These results agree well with peptide immunization studies showing that peptide-specific CTL responses could be induced with NP3 and NP4 but not with NP7 peptide. Furthermore, CTL activity assay using targets, prepared to express the antigen (NP) endogenously, demonstrated that NP3 and NP4 peptides could be presented endogenously. CTL elicited with NP4 peptide retained some cross-reactivity and was difficult to long-term culture. However, NP3-elicited CTL was very specific for NP3 peptide and was stable enough to be cloned. Among many CTL lines elicited with HTV or HTV NP peptides, 6 NP3-specific CTL clones were established and have been maintained more than 2 years. All 6 CTL clones were characterized to be CD3+, CD4-, CD8+, CD25+, CD62L-, and NK1.1-, and to use TCR Vbeta6. This preferential usage of TCR Vbeta6 indicates that TCR Vbeta6 regions are important for recognition of the HTV NP3 epitope (NP221-228, SVIGFLAL) on H-2K(b) molecule. Our data demonstrate the definition of mouse CTL epitopes in HTV and the generation of HTV-specific mouse CTL clones.

Animals↗

PubMed1998

The Leu-3 residue of Serratia marcescens metalloprotease inhibitor is important in inhibitory activity and binding with Serratia marcescens metalloprotease.

Serratia marcescens metalloprotease inhibitor (SmaPI) is a proteinase inhibitor toward Serratia marcescens metalloprotease (SMP). In sequential deletion analysis of the N-terminal region of the SmaPI, SmaPIs starting at Ser-2 and Leu-3 residues, respectively, had nearly a full inhibitory activity toward SMP. However, SmaPI starting at Ala-4 residue showed severely decreased inhibitory activity. Furthermore, kinetic analysis demonstrated that SmaPI starting at the Ala-4 residue had an inhibition constant for SMP approximately fourfold higher than that of wild-type SmaPI. The interactions of Leu-3 with SMP contribute 0.73 kcal mol-1 to the overall stability of the SMP-SmaPI complex (8.44 kcal mol-1). To elucidate the detailed role of the Leu-3 residue in inhibitory activity of SmaPI, several site-directed mutations were introduced. The inhibitory activities of Leu-3 mutants in which the Leu-3 has been converted to Ala, Asp, Gly, Ile, Lys, Phe, or Pro were correlated with the hydrophobicities of substituted amino acids. About 0.3 kcal mol-1 is attributable to the side chain of the Leu-3 residue in the binding with SMP. From these results, it is suggested that (i) in contrast with the Erwinia chrysanthemi inhibitor, Gly-1 and Ser-2 of SmaPI are not critical and (ii) the hydrophobicity of Leu-3 may be important in its inhibitory activity and binding with SMP.

Amino Acid Sequence↗

PubMed1998

Dimeric beta-cyclodextrin-based supramolecular ligands and their copper(II) complexes as metalloenzyme models.

New supramolecular ligands possessing linear 13- and 15-membered pyridine diamidetriamine chelators between the primary sides of two beta-cyclodextrin cavities were synthesized, and characterized by MALDI-MS, NMR, IR and UV-Visible spectroscopy. Fluorescence and pH-metric titration were carried out in order to ascertain their behavior as bifunctional hosts for fluorescent guests and Cu(II) ion. The pKa value for the Cu(II) promoted deprotonation of amide ligands was determined to be 6.2 from pH-absorbance profile. Above pH 8.0, two deprotonated amides and three amino groups chelated Cu(II) ion, and yielded penta-coordinated Cu(II) complexes. The Cu(II) complexes catalyzed the hydrolysis of p-nitrophenyl acetate, adamantate and amino acids. Especially, the complex containing 13-membered chelator is an artificial metalloesterase with catalytic rate constant kcat = 3.8 x 10(-3) min-1 and Michaelis constant K(m) = 3.5 x 10(-4) M for the hydrolysis of p-nitrophenyl adamantate via metal-hydroxide mechanism.

Carbohydrate Sequence↗

PubMed1998

Streptokinase secretion by Serratia marcescens signaled by the C-terminal 41 amino acid segment of metalloprotease.

In order to investigate the secretion signal of Serratia marcescens metalloprotease (SMP) and examine the ability of the secretion signal to secrete foreign proteins, hybrid genes encoding the passenger-SMP C-terminal segments were constructed. As a passenger protein, streptokinase (SK) deprived of its signal peptide was used. Three kinds of SMP C-terminal segments containing 41, 80, or 220 amino acid residues were fused to the C-terminus of SK as secretion signals. The SK-SMP chimeric proteins containing 41 or 220 amino acid segments of the SMP C-terminus were secreted into the culture medium by the SMP transporter of S. marcescens. This result suggests that cytoplasmic SK is secreted into the external medium by the C-terminal segments of SMP and also shows that the smallest, 41 amino acid segment of the SMP C-terminus functions as a secretion signal of foreign proteins as well as SMP.

Biological Transport↗

PubMed1997

Cloning and expression of the gene for xylose isomerase from Thermus flavus AT62 in Escherichia coli.

The gene encoding xylose isomerase (xylA) was cloned from Thermus flavus AT62 and the DNA sequence was determined. The xylA gene encodes the enzyme xylose isomerase (XI or xylA) consisting of 387 amino acids (calculated Mr of 44,941). Also, there was a partial xylulose kinase gene that was 4 bp overlapped in the end of XI gene. The XI gene was stably expressed in E. coli under the control of tac promoter. XI produced in E. coli was simply purified by heat treatment at 90 degrees C for 10 min and column chromatography of DEAE-Sephacel. The Mr of the purified enzyme was estimated to be 45 kDa on SDS-polyacrylamide gel electrophoresis. However, Mr of the cloned XI was 185 kDa on native condition, indicating that the XI consists of homomeric tetramer. The enzyme has an optimum temperature at 90 degrees C. Thermostability tests revealed that half life at 85 degrees C was 2 mo and 2 h at 95 degrees C. The optimum pH is around 7.0, close to where by-product formation is minimal. The isomerization yield of the cloned XI was about 55% from glucose, indicating that the yield is higher than those of reported enzymes. The K(m) values for various sugar substrates were calculated as 106 mM for glucose. Divalent cations such as Mn2+, Co2+, and Mg2+ are required for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity.

Aldose-Ketose Isomerases↗

PubMed1997

Identification of the functional importance of valine-19 residue in streptokinase by N-terminal deletion and site-directed mutagenesis.

Streptokinase (SK) is a bacterial plasminogen activator of multi-domain structure. In deletion analysis of the N-terminal region of SK, the deletion of 20 amino acids (SK delta N20) resulted in the dramatic reduction of plasminogen activator activity compared to deletion of 7 (SK delta N7) and 13 amino acids (SK delta N13). The incubation time to reach maximal active site generation in an equimolar mixture of SK delta N20 and plasminogen was the same as that for wild-type SK. To identify the functional residues important in plasminogen activation, several site-directed mutations were introduced at the region spanning Ser16-Val20 of SK. The results showed that Val19 residue is important for the activity of the SK-plasminogen complex.

Amino Acid Sequence↗

PubMed1997

Effect on product specificity of cyclodextrin glycosyltransferase by site-directed mutagenesis.

Cyclodextrin glycosyltransferase (CGTase; EC 2.4.1.19) catalyzes the degradation of starch into cyclodextrins through an intramolecular transglycosylation reaction. Tyr-89, Asn-94, and Tyr-100 are located near the putative active center. To analyze their roles in product specificity, Tyr-89, Asn-94, and Tyr-100 of CGTase from alkalophilic Bacillus sp. I-5 were replaced with different amino acids. Among the mutants, the N94S mutant protein produced about two times more alpha-cyclodextrin than the wild-type at all incubation times. The Y89F and Y100F mutant proteins were changed to more beta-specific enzymes. From these results it is suggested that the changing of the residues located at the near active site can change the product specificity of CGTase.

Amino Acid Sequence↗

PubMed1996

Transforming growth factor-beta1 inhibits human keratinocyte proliferation by upregulation of a receptor-type tyrosine phosphatase R-PTP-kappa gene expression.

We studied regulation of a receptor-type tyrosine phosphatase R-PTP-kappa gene expression in a human keratinocyte cell line, HaCaT. Addition of TGF-beta 1 to the HaCaT cells markedly induced the expression of R-PTP-kappa mRNA in a time- and dose-dependent manner. The induction of R-PTP-kappa mRNA expression was observed at a dose as low as 0.02 ng/ml TGF-beta1 and reached a peak at 2 ng/ml TGF-beta 1 after 6 h treatment. The TGF-beta 1-induced R-PTP-kappa mRNA expression was suppressed by sodium orthovanadate, a tyrosine phosphatase inhibitor, and H7, a serine/threonine kinase inhibitor, but not by genistein, a tyrosine kinase inhibitor. In addition, the inducing effect is not dependent on de novo protein synthesis. Taken together, these results suggest that TGF-beta 1 inhibits the human keratinocyte proliferation in vitro, possibly through induction of R-PTP-kappa gene expression.

Cell Division↗

PubMed1996

RcsC-mediated induction of colanic acid by secretion of streptokinase in Escherichia coli K-12.

The introduction of a plasmid containing skc (streptokinase-coding gene) fused with ompA signal sequence into Escherichia coli K-12 strains, rendered the bacteria mucoid. Measurement of the synthesis of beta-galactosidase from a cps-lacZ fusion (lacZ fusion to a gene necessary for capsule synthesis) showed that the mucoid phenotype was due to induction of the capsular polysaccharide colanic acid synthesis. The introduction of a plasmid carrying skc fused with malE (gene encoding maltose-binding protein) also induced cps-lacZ expression, but intracellular expression of streptokinase in E. coli did not. The cps expression by secretion of streptokinase was diminished to the basal level in a cps-lacZ strain carrying a rcsC mutation. These results show that the secretion of streptokinase in E. coli induces colanic acid synthesis through the RcsC-dependent pathway.

Bacterial Proteins↗

PubMed1996

Secretion of Bacillus alpha-amylase from yeast directed by glucoamylase I signal sequence of Saccharomyces diastaticus.

For the secretion of Bacillus stearothermophilus alpha-amylase from yeast, a recombinant plasmid pGAT17 was constructed by fusing B. stearothermophilus alpha-amylase structural gene in frame to the promoter and signal sequence of Saccharomyces diastaticus glucoamylase I gene (STA1). The secretion of the heterologous alpha-amylase from S. diastaticus transformed with pGAT17 was confirmed by the halo formation around colonies on selective starch agar medium. About 80% of the total alpha-amylase activity was detected in the extracellular culture medium. The secreted alpha-amylase was glycosylated and its molecular weight increased from 61 kDa to 75 kDa. The thermostability of the the glycosylated alpha-amylase was markedly enhanced, compared with that of the non-glycosylated enzyme from E. coli.

Amino Acid Sequence↗

PubMed1996

C-terminal peptide of streptokinase, Met369-Pro373, is important in plasminogen activation.

Streptokinase(SK), a plasminogen activator, is known to have multi-domain structure. The function of the C-terminal region of streptokinase was investigated with SK mutants constructed by truncating 26, 33, 37, 40, 41, 46, 47, 70 or 97 amino acid residues from the C-terminus. The truncated SKs were expressed in E. coli and purified. The 41 residue deletion (SKP373) from the C-terminus had not effect on the plasminogen activation activity. However, the deletion of 46 amino acid residues (SKP368) resulted in the dramatic reduction of the plasminogen activation efficiency. The result suggests that the C-terminal peptide from Met369 to Pro373 of SK may play an important role on the plasminogen activation.

Cloning, Molecular↗

PubMed1996

Molecular cloning and expression of a cDNA encoding a protein tyrosine phosphatase from mouse brain.

A mouse brain cDNA library was screened with a radioisotope-labeled probe of human placental protein tyrosine phosphatase. The isolated clone (MBPTP1B) was found to contain an open reading frame of 1,296 nucleotides as well as 5' (709 nucleotides) and 3' (341 nucleotides) non-coding regions. This cDNA encodes a PTP of 432 amino acids having a mass of 49,563 daltons and exhibiting 83% and 93.5% sequence identity to that of human PTP1B and rat PTP1, respectively. The expression of the cDNA in yeast was identified by western blot analysis and PTP activity assay.

Amino Acid Sequence↗

PubMed1996

Interleukin 4-induced proliferation in normal human keratinocytes is associated with c-myc gene expression and inhibited by genistein.

We studied the effect of IL-4 on the proliferation of cultured normal human keratinocytes. Keratinocyte proliferation was stimulated by IL-4 and inhibited by anti-IL-4 antibody in a concentration-dependent manner. Anti-IL-6 antibody did not inhibit normal human keratinocyte proliferation, suggesting that the IL-4 could directly induce proliferation of these cells. IL-4 significantly induced cell cycle G0/G1 to S phase progression. The keratinocyte proliferation by IL-4 was mediated through one of the growth control genes, c-myc protooncogene. The expression of c-myc mRNA was significantly increased after IL-4 treatment of the keratinocytes, suggesting that c-myc plays a key role in the control of proliferation. The signal transduction pathways induced by IL-4 in the keratinocytes were studied with inhibitors of signal transduction. Genistein, a tyrosine kinase inhibitor, suppressed the level of the induced c-myc mRNA expression, but H7, a serine/threonine kinase inhibitor, and okadaic acid, a protein phosphatase 1 and 2A inhibitor, did not block the induced c-myc gene expression. Taken together, these results suggest that IL-4 stimulates the proliferation of keratinocytes in vitro by promoting a transition from G0/G1 to S phase of the cell cycle. Induction of c-myc after IL-4 treatment could indicate an important role for c-myc in the proliferation of keratinocytes. Our observations also suggest that tyrosine kinases may be involved in IL-4-induced proliferation.

Base Sequence↗