Biomedical subjects

I S Choe

Publications and source records attributed to I S Choe.

At least 19 recordsLinked to original sources

PubMed2001

Molecular cloning and characterization of Mycobacterium bovis BCG pcp gene encoding pyrrolidone carboxyl peptidase.

The Mycobacterium bovis bacilli Calmette-Guerin (BCG) pcp gene that encodes the pyrrolidone carboxyl peptidase (Pcp) was cloned from a lambdagtll genomic library and sequenced. The nucleotide sequence contains a 669 bp open reading frame coding for a protein of 222 amino acid residues with a calculated molecular mass of 23,209 Da. The deduced amino acid sequence is highly homologous to the Pcps from Bacillus amyloliquefaciens, Pseudomonas fluorescens, Bacillus subtilis, Streptococcus pyogenes, and Staphylococcus aureus. A multiple sequence alignment revealed highly conserved domains. The BCG pcp gene was overexpressed in Escherichia coli. The Pcp was purified to homogeneity. The recombinant protein was further confirmed by an enzymatic assay.

Amino Acid Sequence↗

PubMed2001

Up-Regulation of ninjurin expression in human hepatocellular carcinoma associated with cirrhosis and chronic viral hepatitis.

The cDNA clones, which were highly expressed in liver tissues of hepatocellular carcinoma (HCC) patients, were identified using dot hybridization and reconfirmed by Northern blot analysis. One of the clones, ninjurin (nerve injury induced protein), showed a much higher expression level in the liver tissue of HCC patients than in normal liver tissue. Interestingly, the presence of ninjurin mRNA transcripts was detected with high intensity in HCC tissues when combined with viral infection and cirrhosis, but not with a normal liver or HCC tissue unrelated with viral infection and cirrhosis. We produced a N-terminal part of recombinant ninjurin protein, as well as a monoclonal antibody specific to this polypeptide. The intensity of immunohistochemical staining of the liver tumor tissue, and regenerating tissue for the ninjurin protein, was stronger than that of normal liver tissue. These results suggest that ninjurin may play an important role in the development of HCC combined with cirrhosis and viral infection.

Animals↗

PubMed2001

Dendritic cell-tumor coculturing vaccine can induce antitumor immunity through both NK and CTL interaction.

Immunization of dendritic cells (DC) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL) that are responsible for protection and regression. We show here that immunization with bone marrow-derived DC cocultured with tumor cells can induce a protective immunity against challenges to viable tumor cells. In this study, we further investigated the mechanism by which the antitumor activity was induced. Immunization of mice with DC cocultured with murine colon carcinoma. CT-26 cells, augmented CTL activity against the tumor cells. Concomitantly, an increase in natural killer (NK) cell activity was also detected in the same mice. When DC were fixed with paraformaldehyde prior to coculturing with tumor cells, most of the CTL and NK cell activity diminished, indicating that DC are involved in the process of presenting the tumor antigen(s) to CTL. NK cell depletion in vivo produced markedly low tumor-specific CTL activity responsible for tumor prevention. In addition, RT-PCR analysis confirmed the high expression of INF-gamma mRNA in splenocytes after vaccination with DC cocultured with tumors, but low expression in splenocytes from NK-depleted mice. Most importantly, the tumor protective effect rendered to DC by the coculturing with CT-26 cells was not observed in NK-depleted mice, which suggests that DC can induce an antitumor immune response by enhancing NK cell-dependent CTL activation. Collectively, our results indicate that NK cells are required during the priming of cytotoxic T-cell response by DC-based tumor vaccine and seem to delineate a mechanism by which DC vaccine can provide the desired immunity.

Adenocarcinoma↗

PubMed2001

Genomic DNA sequence and transcription factor binding sites of mouse ninjurin.

The complete genomic DNA sequence of mouse ninjurin gene has been cloned and sequenced by screening a bacterial artificial chromosome (BAC) library of mouse 129/SvJ genomic DNA. The mouse ninjurin gene comprises four exons and the translatable sequences are included in the first three exons. The putative promoter region of the mouse ninjurin gene lacks the consensus "CAAT" or "TATA" sequence. Nonetheless, it has demonstrated the promoter activity in transient transfection experiment using the construct containing putative promoter sequence of mouse ninjurin and reporter gene. The nucleotide sequence of the putative promoter region shows 83% homology with the corresponding DNA sequence of human ninjurin gene that had been previously reported, and reveals a high degree of conservation between the two species. Analysis of the DNA sequence identified the putative promoters and the binding sites for a variety of transcription factors of mouse ninjurin.

Amino Acid Sequence↗

PubMed2001

Inhibition of glucocorticoid-mediated, caspase-independent dendritic cell death by CD40 activation.

Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive agents that act on a variety of immune cells, including T cells, monocytes/macrophages, osteoclasts, and dendritic cells (DC). However, the mechanism(s) by which GC exert anti-inflammatory effects is still largely unknown. It is already well known that GC treatment inhibits DC maturation and interleukin (IL)-12 production by DC. In this study, we investigated the apoptosis induction of DC by a synthetic GC, dexamethasone (Dex). The stimulation with Dex resulted in DC apoptosis in a dose- and time-dependent manner as it was measured by determining annexin V-positive cells and mitochondrial potential. In contrast, monocytes that are precursor cells of DC are resistant to Dex-mediated apoptosis. The Dex-induced apoptosis of DC was independent of caspase activation because it was not inhibited by the broad caspase inhibitor, Z-VAD-fmk. It is interesting that agonistic CD40 antibody completely inhibited Dex-induced cell death, whereas other inflammatory stimuli did not show the same effect, suggesting that CD40 signaling may selectively modulate GC-mediated DC apoptosis. Taken together, our findings revealed an important role of GC and CD40 signaling in the regulation of immune responses in which DC play a key role in the inflammatory process of various immunomediated diseases.

Anti-Inflammatory Agents↗

PubMed2000

Effect of high blood flow on the expression of endothelial constitutive nitric oxide synthase in rats with femoral arteriovenous shunts.

The effect of high blood flow on the expression of endothelial nitric oxide synthase has been investigated in the femoral arteriovenous shunt (AVS) rats created by inserting U-shaped polyurethane tubes in the left femoral arteries and veins. Three days after inserting the femoral AVS, the mean aortic blood flow rate in the abdominal aorta of the AVS rats was about 2.0 times higher than that in the control rats (110.0 +/- 8.4 ml/min vs 52.7 +/- 2.7 ml/min, p < 0.001). The competitive reverse transcriptase-polymerase chain reaction (RT-PCR) data revealed that the mRNA expression level of the endothelial constitutive nitric oxide synthase (ecNOS) was increased in the aortas of the femoral AVS rats compared to that in the control rats. Western blot analysis using a monoclonal antibody against ecNOS revealed that the ecNOS protein levels were markedly increased in the aortas of femoral AVS rats, but ecNOS protein levels in aortas without endothelium were not significantly increased. Inducible nitric oxide synthase (iNOS) protein was not expressed in the aortic tissues with and without endothelium in the control rats. This iNOS expression was not increased by the high blood flow in the femoral AVS rats. These findings suggest that high blood flow could up-regulate the expression levels of ecNOS mRNA and proteins in femoral arteriovenous shunt rats.

Animals↗

PubMed1999

Protective antitumor activity through dendritic cell immunization is mediated by NK cell as well as CTL activation.

Dendritic cells (DCs) are potent professional antigen-presenting cells (APC) capable of inducing the primary T cell response to antigen. Although tumor cells express target antigens, they are incapable of stimulating a tumor-specific immune response due to a defect in the costimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach using tumor-DC coculture to improve the antigen presenting capacity of tumor cells, which does not require a source of tumor-associated antigen. Immunization of a weakly immunogenic and progressive tumor cocultured with bone marrow-derived DCs generated an effective tumor vaccine. Immunization with the cocultured DCs was able to induce complete protective immunity against tumor challenges and was effective for the induction of tumor-specific CTL (cytotoxic T lymphocyte) activity. Furthermore, high NK cell activity was observed in mice in which tumors were rejected. In addition, immunization with tumor-pulsed DCs induced delayed tumor growth, but not tumor eradication in tumor-bearing mice. Our results demonstrate that coculture of DCs with tumors generated antitumor immunity due to the NK cell activation as well as tumor-specific T cell. This approach would be useful for designing tumor vaccines using DCs when the information about tumor antigens is limited.

Animals↗

PubMed1999

Enhanced antigen-presenting activity and tumour necrosis factor-alpha-independent activation of dendritic cells following treatment with Mycobacterium bovis bacillus Calmette-Guérin.

Dendritic cells (DCs) are most potent among the antigen-presenting cells and are believed to be crucial for the initiation of a primary T-cell response to foreign antigens. Mycobacterial infection within macrophages is controlled by cell-mediated immunity. To elucidate the stimulation of immune response by Mycobacterium bovis bacillus Calmette-Guérin (BCG), we purified DCs from precursor cells in human peripheral blood mononuclear cells (PBMC) by culturing them with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) and characterized their surface antigen expression. The interaction of cultured DCs with BCG resulted in increased surface expression of several DC-related marker antigens. BCG also induced reduction of endocytosis, enhancement of CD83 expression as well as B7 costimulatory molecules and IL-12 production, suggesting that BCG treatment directly induces DCs to mature. BCG-treated DCs were much more potent antigen-presenting cells in allogeneic immune response than untreated DCs. Moreover, while the neutralization of tumour necrosis factor-alpha (TNF-alpha) significantly blocked the DC maturation induced by lipopolysaccharide (LPS), it could not inhibit the induction of DC maturation by the BCG treatment, indicating that TNF-alpha production plays a minor role in the BCG-induced DC maturation. However, the neutralization of TNF-alpha resulted in decreased IL-12 production by activated DCs. These results suggest that infection with BCG might evoke direct activation and maturation of DC and the general immune stimulant effect of BCG might be related with the activation of DCs.

Antigen Presentation↗

PubMed1999

Cloning of the genomic sequence encoding a processed adenylate kinase 2 pseudogene.

A chromosomal DNA sequence harboring a processed AK2B pseudogene was isolated from a human genomic library. It was a variant of the AK2B gene sequence including several point mutations, deletions, and insertions. The nucleotide sequence of the ORF of the AK2B pseudogene predicted a truncated form of the AK2B mutant suggesting that the processed pseudogene is nonfunctional. A repetitive sequence, AAAAGAGAG, found in the 5' and 3' flanking regions of the pseudogene and the poly(A) tract in the 3' end junction suggest that a mRNA of AK2B may have been converted to the processed pseudogene by retrotransposition events. Previously, it was suggested that an adenylate kinase (AK) 2 related gene on chromosome 2, confirmed by Southern analysis using somatic cell hybrid cell lines, may be a processed pseudogene. It is proposed that the processed pseudogene isolated in this study may be the AK2 related nonfunctional gene localized on human chromosomes 2.

Adenylate Kinase↗

PubMed1999

Isolation and characterization of cDNA clone for human liver 10-formyltetrahydrofolate dehydrogenase.

A cDNA clone encoding 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) was isolated from a human fetal liver cDNA library. It contained the open reading frame of 2,709 base pairs and predicted a protein comprising 902 amino acids with a calculated molecular weight of 98,700 Da. The deduced protein showed about 93.6% homology (90.5% identity, 3.1% favored substitutions) when compared with rat 10-FTHFDH. The distribution of 10-FTHFDH transcript in various human tissues was studied by Northern blot analysis using poly(A+) RNAs from different tissues. The 10-FTHFDH transcript with an approximate size of 2.7 kb was mainly expressed in human kidney, skeletal muscle, and liver and rarely expressed in other tissues.

Amino Acid Sequence↗

PubMed1999

Cloning and expression of human cDNA encoding human homologue of pituitary tumor transforming gene.

Recently, a potent transforming gene which was exclusively expressed in rat pituitary tumor but not in normal pituitary had been isolated and named as pituitary tumor transforming gene (PTTG). A cDNA clone encoding human homologue of rat PTTG was isolated from human fetal liver cDNA library. It contained an open reading frame of 603 base pairs predicting a protein composed of 201 amino acids with a calculated molecular weight of 26 kDa. The deduced protein showed about 85% homology (78% identity, 7% favored substitution) with the rat PTTG. Northern blot analysis showed that the cDNA hybridized to 1.0 kb mRNA species which was expressed in fetal liver and several cancer cell lines. These results suggest that the presence of the human homologue of rat PTTG gene may not be restricted to pituitary tumor.

Amino Acid Sequence↗

PubMed1998

Isolation of cDNA clones encoding human histone macroH2A1 subtypes.

cDNA clones encoding two different subtypes of histone macroH2A1, macroH2A1.1 and macroH2A1.2, have been isolated from human liver tissue. The open reading frames in the isolated clones predicted proteins comprising 368 and 371 amino acids respectively. Estimated molecular masses of the two proteins were 39.0 kDa and 39.4 kDa. Human histone macroH2A1.1 and macroH2A1.2 showed about 98% identity with their counterparts isolated from rat. The features of the nucleotide sequences of the two macroH2A1 subtypes in human were the same as in the rat system. Northern blot analysis showed that the macroH2A1.1 and macroH2A1.2 subtypes were expressed as mRNA species with a size of 1.5 and 4.4 kb, respectively. They were expressed in all human tissues examined.

Amino Acid Sequence↗

PubMed1998

House dust mite-induced sensitivity in mice.

Sensitivity induced by house dust mite (HDM) extract in mice was investigated in this study. Sensitized B10.RIII mice (H-2r background) had T-cell proliferative responses to HDM extract in vitro and an HDM-specific IgE response. When mice were immunized by injection and intranasal inhalation with HDM extract, a histologic study showed eosinophils and mononuclear cell infiltration in the lung tissue and bronchial wall. Tcr alphabeta-positive cells were also found in the cell infiltration area of the lung lesions. In the control mice that were immunized by injection or intranasal inhalation (but not both), we did not observe cell accumulation in the lung tissue or in the bronchial wall. Epitope studies suggest that T cells recognize multiple epitopes. Molecular analysis of these HDM-specific T-cell hybridoma clones suggest that T-cell receptor use is restricted to members of the V alpha 8 and Vbeta 6 subfamilies.

Allergens↗

PubMed1998

Increment of efficiency in the identification of noble genes by colony hybridization assay. (Screening of low redundant clones from human fetal cDNA library).

For the rapid identification of noble genes in a specific tissue by computer analysis from the cDNA sequences determined by single-pass cDNA sequencing, clone redundancy was one of the major obstacles. To facilitate the efficiency in identification of noble genes, it was necessary to reduce the number of clones to be sequenced by eliminating the redundant clones for a rapid analysis. In order to increase the probability of isolating noble sequences from the cDNA clones of human fetal liver tissue origin, colony hybridization assay was adopted and redundant clones were efficiently removed. Four cDNA clones highly redundant in the human fetal liver cDNA libraries including alpha-globin, gamma-globin, serum albumin and H19 RNA sequences were selected as the probes. Two hundreds and sixty two cDNA clones were randomly selected and tested with the probes for hybridization properties. The identity of each cDNA clone giving positive or negative signals in the hybridization assay was determined by DNA homology search with the nucleic acid databases. Among the 76 clones giving positive signals, 57 clones (75%) were found to be identical to the probe sequences and could be eliminated by colony hybridization assay before neucleotide sequencing.

Cloning, Molecular↗

PubMed1998

Cloning and expression of human adenylate kinase 2 isozymes: differential expression of adenylate kinase 1 and 2 in human muscle tissues.

A cDNA clone coding for adenylate kinase 2B was isolated from fetal liver, and the expression of AK2 was investigated in human tissues. The ORF in the cDNA clone for human AK2B predicted a protein comprising 232 amino acids (25.6 kDa). The features of AK2A and AK2B sequences in human were the same as those in the bovine system. Each of the recombinant proteins, AK2A and AK2B, was expressed in Escherichia coli cells, and the purified recombinant proteins were enzymatically active. The distribution of AK2 transcripts in various human tissues was examined by Northern analysis. Unlike in the bovine system, it was found that the AK2A transcript was the major form of AK2 mRNA species in all human tissues. The transcripts of AK2 isozymes were relatively abundant in heart, liver, and also in skeletal muscle, where the expression level of AK2 was known to be low. Western blot analysis of AK isozymes in human heart and skeletal muscle revealed that AK2 protein was found only in heart, whereas AK1 was detected in both tissues. These tissue-specific expressions of the AK isozymes in human might suggest the presence of organ-specific regulation of the AK2 gene including a post-transcriptional control in skeletal muscle.

Adenylate Kinase↗

PubMed1997

Cloning and expression of human cDNA encoding Na+, K(+)-ATPase gamma-subunit.

A cDNA clone encoding the Na+, K(+)-Atpase gamma-subunit polypeptide was cloned from a human fetal liver cDNA library. The deduced amino acid sequence of the protein comprised 58 amino acids with a calculated molecular weight of 6400 Da, and showed about 86% homology when compared with those of the bovine, rat and mouse Na+, K(+)-ATPase gamma-subunits published elsewhere. Northern blot analysis showed that the cDNA hybridized to a 0.7 kb mRNA which was expressed in human kidney, pancreas and fetal liver.

Amino Acid Sequence↗

PubMed1997

Peptide-specific CTL induction in HBV-seropositive PBMC by stimulation with peptides in vitro: novel epitopes identified from chronic carriers.

Cytotoxic T lymphocytes (CTL) recognize and destroy virus-infected cells, and it has been established that epitope-based peptides could induce such CTL in vivo as well as in vitro. In this study attempts were made to define the epitopes that are recognized by the CTL, and thus a series of 9- to 10-mer peptides derived from the amino acid sequences of hepatitis B virus (HBV) proteins were synthesized on the basis of the previously described HLA-A2 peptide binding motif. The binding assay of the synthetic peptides using transporter-associated with antigen processing (TAP)-deficient human cell line, T2, showed that eight out of 11 peptides tested enhanced the expression of HLA-A2 molecules on the T2 cell surface. Some of these peptides triggered activation of CTL in peripheral blood mononuclear cells of HBV-seropositive chronic carriers. The activated CTL in turn recognized and killed the T2 cells pulsed with the same peptides. This study shows that novel HLA-A2-restricted epitopes exist in the natural repertoire of immunity against HBV. These findings can be useful in developing peptide-based therapeutics against viral infections.

Carrier State↗

PubMed1997

Molecular cloning of the leuB genes from Mycobacterium bovis BCG and Mycobacterium tuberculosis.

A gene responsible for the biosynthesis of leucine has been cloned by the complementation of the Escherichia coli leuB6 auxotroph mutant after transformation with the Mycobacterium bovis BCG genomic DNA library, which was constructed by ligating the partially digested BCG DNA with Sau3A1 into the pUC19 digested with BamHI. Sequencing of the leuB gene of BCG revealed an ORF (open reading frame) of 1.011 bp encoding isopropylmalate dehydrogenase with a calculated molecular weight of 42 kDa. The leuB gene of Mycobacterium tuberculosis isolated from Korean tuberculosis patient is shown to be identical to that of BCG except one bp.

3-Isopropylmalate Dehydrogenase↗