Generation of Anti-HLA-DR4 Specific Antibodies by Immunization of the Recombinantly Expressed Allelic Subtype-Specific Region of the HLA-DRB1*0405 Molecules

Kim,Kil Lyong,Park,Jung-Hyun,Cho,Eun-Wie,Lee,Yun-Jung,Hahm,Kyung-Soo,Chung,Jin The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.2

HLA-DR4 is the dominant allele of MHC class II genes in Koreans. In particular, the DRB1*0405 subtype has been reported to be almost exclusively expressed in Far East Asians, and has also been observed to be strongly associated with rheumatoid arthritis in Koreans and the Japanese. Identification of this specific allele has been mainly performed by PCR- based methods, which is often time consuming, costly, and involves tedious procedures such as the isolation of genomic DNA,PCR, and gel electrophoresis. To develop a more convenient tool for screening vast amounts of samples as well as to generate reagents which might also be used in other applications, in this study, antibodies were produced against this specific HLA subtype. By PCR,an allele-specific region covering the β1 domain of DRB1*0405 was amplified and recombinantly expressed in E.coli. Immunization of Lewis rats with the purified protein yielded an allele specific antiserum. Western blot analysis showed the selective detection of the HLA-DR β-chain. Using this antiserum, established cell lines and peripheral blood lymphocytes were analyzed on their HLA haplotype by fluorescence activated flowcytometry. These novel antibodies will provide a powerful tool in the detection and investigation of DR4 alleles.

Novel Vectors for the Convenient Cloning and Expression of In Vivo Biotinylated Proteins in Escherichia coli

Kim, Kil Lyong,Park. Jung Hyun,Cho, Eun Wie,Na, Shin Young 생화학분자생물학회 2001 BMB Reports Vol.32 No.5

Biotinylation of recombinant proteins is a powerful tool for the detection and analysis of proteins of interest in a large variety of assay systems. The recent development of in vivo biotinylation techniques in E, coli has opened new possibilities for the production of site-specifically biotinylated proteins without the need for further manipulation after the isolation of the recombinantly expressed proteins. In the present study, a novel vector set was generated which allows the convenient cloning and expression of proteins of interest fused with an N-terminal in vivo biotinylated thioredoxin (TRX) protein. These vectors were derived from the previously reported pBIOTRX vector into which was incorporated part of the pBluescript II+ phagemid multiple cloning site (MCS), amplified by PCR using a pair of sophisticated oligonucleotide primers. The functionality of these novel vectors was examined in this system by recombinant expression of rat transforming growth factor-β. Western-blot analysis using TRX-specific antibodies or peroxidaseconjugated streptavidin confirmed the successful induction of the fusion protein and the in vivo conjugation of biotin molecules, respectively. The convenience of molecular subcloning provided by the MCS and the effective in vivo biotinylation of proteins of interest makes this novel vector set an interesting alternative for the production of biotinylated proteins.

Asymmetric Polymerase Chain Reaction-Single-Strand Conformation Polymorphism (Asymmetric PCR-SSCP) as a Simple Method for Allele Typing of HLA-DRB

Kim, Kil Lyong,Kang, Joo Hyun,Maeng, Cheol Young,Kim, Kyeong Hee 생화학분자생물학회 2001 BMB Reports Vol.32 No.6

Asymmetric PCR and single-strand conformation polymorphism (SSCP) methods were combined to analyze human leukocyte antigen (HLA)-DRB allele polymorphism. Asymmetric PCR amplification was applied to generate single-stranded DNA (ssDNA) using the nonradioactive oligonucleotide primers designed for the polymorphic exon 2 region. The conformational differences of ssDNAs, depending on the allele type, were analyzed by nondenaturing polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The ssDNAs were clearly separated from double-stranded DNA without interference and obviously migrated depending on their allele type. This method was applied to the genomic DNA either from homozygous or from heterozygous cell lines containing the DR4 allele as template DNA using DR4-specific primers, and satisfying results were obtained. Compared to the standard PCR-SSCP method, this asymmetric PCR-SSCP method has advantages of increased speed, reproducibility, and convenience. Along with PCR-SSP or sequence-based typing, this method will be useful in routine typing of HLA-DRB allele.

Molecular Cloning and Recombinant Expression of the Long Form of Leptin Receptor ( Ob - Rb ) cDNA as Isolated from Rat Spleen

Kim, Kil Lyong,Lee, Myung Kyu,You, Kwan Hee,Park, Jung Hyun,Na, Shin Young,Ju, Sung Kyu 생화학분자생물학회 1970 BMB Reports Vol.34 No.2

Leptin is a circulating non-glycosylated protein that is mainly produced in adipocytes. Leptin acts in the brain to regulate food intake and energy expenditure. Previously we reported our success in the isolation of a partial cDNA of the long form of the leptin receptor, OB-Rb, from rat spleen, and showed that leptin might also play a role in peripheral immune organs. In the present study, for the first time, the complete coding regian of OB-Rb cDNA was cloned from rat splenocytes, and its nucleotide sequence was determined. The cDNA was then further expressed in E. coli and mammalian cells, thereby confirming the functional integrity of this receptor. Prokaryotically overexpressed OB-R protein was then used as an immunizing antigen in BALE/c mice to produce leptin receptor-specific antibodies. By using them, we confirmed the cell surface expression of OB-Rb in transfected CHO cells. It is our belief that the reagents, as produced in this study, will be of great use in further studies of the biological role of rat leptin.

Generation of Anti - HLA - DR4 Specific Antibodies by Immunization of the Recombinantly Expressed Allelic Subtype - Specific Region of the HLA - DRB1*0405 Molecules

Kim, Kil Lyong,Hahm, Kyung Soo,Lee, Yun Jung,Park, Jung Hyun,Cho, Eun Wie,Chung, Jin 생화학분자생물학회 1999 BMB Reports Vol.31 No.1

HLA-DR4 is the dominant allele of MHC class II genes in Koreans. In particular, the DRB1*0405 subtype has been reported to be almost exclusively expressed in Far East Asians, and has also been observed to be strongly associated with rheumatoid arthritis in Koreans and the Japanese. Identification of this specific allele has been mainly performed by PCR-based methods, which is often time consuming, costly, and involves tedious procedures such as the isolation of genomic DNA, PCR, and gel electrophoresis. To develop a more convenient tool for screening vast amounts of samples as well as to generate reagents which might also be used in other applications, in this study, antibodies were produced against this specific HLA subtype. By PCR, an allelespecific region covering the β1 domain of DRB1*0405 was amplified and recombinantly expressed in E.coli. Immunization of Lewis rats with the purified protein yielded an allele specific antiserum. Western blot analysis showed the selective detection of the HLA-DR β-chain. Using this antiserum, established cell lines and peripheral blood lymphocytes were analyzed on their HLA haplotype by fluorescence activated flow cytometry. These novel antibodies will provide a powerful tool in the detection and investigation of DR4 alleles.

Asymmetric Polymerase Chain Reaction-Single-Strand Conformation Polymorphism (Asymmetric PCR-SSCP) as a Simple Method for Allele Typing of HLA-DRB

Kim,Kil Lyong,Maeng,Cheol-Young,Kim,Kyeong Hee,Kang,Joo Hyun The Korea Science and Technology Center 1999 BMB Reports Vol.32 No.6

Asymmetric PCR and single-strand conformation polymorphism (SSCP) methods were combined to analyze human leukocyte antigen (HLA)-DRB allele polymorphism. Asymmetric PCR amplification was applied to generate single-stranded DNA (ssDNA) using the nonradioactive oligonucleotide primers desinged for the polymorphic exon 2 region. The conformational differences of ssDNAs, depending on the allele type, were analyzed by nondenaturing polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The ssDNAs were clearly separated from double-stranded DNA without interference and obviously migrated depending on their allele type. This method was applied to the genomic DNA either from homozygous or from heterozygous cell lines containing the DR4 allele as template DNA using DR4-specific primers, and satisfying results were obtained. Compared to the standard PCR-SSCP method, this asymmetric PCR-SSCP method has advantages of increased speed, reproducibility, and convenience. Along with PCR-SSP or sequence-based typing, this method will be useful in routine typing of HLA-DRB allele.

Novel Vectors for the Convenient Cloning and Expression of In Vivo Biotinylated Proteins in Escherichia coli

Kim, Kil-Lyong,Cho, Eun-Wie,Park, Jung-Hyun,Na, Shin-Young The Korea Science and Technology Center 1999 BMB Reports Vol.32 No.5

Biotinylation of recombinant proteins is a powerful tool for the detection and analysis of proteins of interest in a large variety of assay systems. The recent development of in vivo biotinylation techniques in E. coli has opened new possibilities for the production of site-specifically biotinylated proteins without the need for further manipulation after the isolation of the recombinantly expressed proteins. In the present study, a novel vector set was generated which allows the convenient cloning and expression of proteins of interest fused with an N-terminal in vivo biotinylated thioredoxin (TRX) protein. These vectors were derived from the previously reported pBIOTRX vector into which was incorporated part of the pBluescript II+phagemid multiple cloning site (MCS), amplified by PCR using a pair of sophisticated oligonucleotide primers. The functionality of these novel vectors was examined in this system by recombinant expression of rat transforming growth factor-β. Western-blot analysis using TRX-specific antibodies or peroxidase-conjugated streptavidin confirmed the successful induction of the fusion protein and the in vivo conjugation of biotin molecules, respectively. The convenience of molecular subcloning provided by the MCS and the effective in vivo viotinylation of proteins of interest makes this novel vector set an interesting alternative for the production of biotinylated proteins.

Generation of Anti - HLA - DR4 Specific Antibodies by Immunization of the Recombinantly Expressed Allelic Subtype - Specific Region of the HLA - DRB1 * 0405 Molecules

Kim, Kil Lyong,Hahm, Kyung Soo,Lee, Yun Jung,Park, Jung Hyun,Cho, Eun Wie,Chung, Jin 생화학분자생물학회 1999 BMB Reports Vol.31 No.2

HLA-DR4 is the dominant allele of MHC class II genes in Koreans. In particular, the DRBl*0405 subtype has been reported to be almost exclusively expressed in Far East Asians, and has also been observed to be strongly associated with rheumatoid arthritis in Koreans and the Japanese. Identification of this specific allele has been mainly performed by PCR-based methods, which is often time consuming, costly, and involves tedious procedures such as the isolation of genomic DNA, PCR, and gel electrophoresis. To develop a more convenient tool for screening vast amounts of samples as well as to generate reagents which might also be used in other applications, in this study, antibodies were produced against this specific HLA subtype. By PCR, an allele specific region covering the β1 domain of DRB1*0405 was amplified and recombinantly expressed in E.coli. Immunization of Lewis rats with the purified protein yielded an allele specific antiserum. Western blot analysis showed the selective detection of the HLA-DR β-chain. Using this antiserum, established cell lines and peripheral blood lymphocytes were analyzed on their HLA haplotype by fluorescence activated flow cytometry. These novel antibodies will provide a powerful tool in the detection and investigation of DR4 alleles.

Decreased hippocampal cholinergic neurostimulating peptide precursor protein associated with stress exposure in rat brain by proteomic analysis

Kim, Hong Gi,Kim, Kil Lyong Wiley Subscription Services, Inc., A Wiley Company 2007 Journal of neuroscience research Vol.85 No.13

<P>The stress response alters behavior, autonomic function, and secretion of multiple hormones, including corticotropin-releasing factor, adrenocorticotropin hormone, and cortisol, through the hypothalamic-pituitary-adrenal axis. Constitutive stress responses lead to a number of psychiatric disorders, including depression, posttraumatic stress disorder, Alzheimer's disease (AD), and other anxiety disorders through increased stress hormones and other unknown factors. Here, we performed a proteomic analysis of rat brain exposed to restraint stress compared with a nonstress group by using 2D-DIGE and MALDI-TOF analysis. Several proteins were identified by peptide mass fingerprint (PMF), including down-regulated hippocampal cholinergic neurostimulating peptide precursor protein (HCNP-pp). The current study demonstrates that HCNP-pp mRNA and protein expression are decreased in rat hippocampus after stress exposure. The level of HCNP-pp in H19-7, a rat hippocampal cell line, significantly decreases with dexamethasone treatment, a synthetic glucocorticoid. Thus, this finding suggests that HCNP-pp expression may decrease in response to stress exposure. Decreased HCNP-pp from stress exposure may result in lower levels of HCNP that might contribute to a loss of acetylcholine production. © 2007 Wiley-Liss, Inc.</P>

말초정맥로를 통한 Fentanyl의 용량에 따른 기침반사반응

김종훈,홍정연,길혜금,김원옥,이승룡 대한마취과학회 1997 Korean Journal of Anesthesiology Vol.33 No.1

Background : We observed fentanyl known as centrally-acting antitussive agents provoke a cough response in some patients at induction of anesthesia. This may be of clinical importance. Method : 121 patients(ASA class I) were assigned randomly to 4 groups. Each group was given different doses of fentanyl〔Group 1(n=30); 0.5ug/kg, Group 2(n=30); 1ug/kg, Group 3(n=33); 2ug/kg, Group 4(n=28); 4 g/kg〕, within 1 second through a peripheral venous cannula before induction of anesthesia. All patients were observed carefully in order to detect a cough response and any side effects. Result : The incidences of FCR(Fentanyl Cough Response) were 0% in Group 1, 10.0% in Group 2, 30.3% in Group 3, and 39.3% in Group 4. The ED50 of FCR was 4.25ug/kg. The mean onset-time from the end of fentanyl administration to the beginning of coughing was 12.5 seconds. FCR was decreased with aging, but not affected by weight, height, or smoking. Other serious side effects were not accompanied. Conclusion : Fentanyl can evoke the pulmona chemoreflex dose-dependently and the ED50 was 4.25 g/kg. (Korean J Anesthesiol 1997; 33: 59∼62)