• Proceedings of the Botanical Society of Korea Conference
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• 2002.04a
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• pp.62-72
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• 2002

This study has investigated the biosynthesis and function of the heavy metal binding peptides, the phytochelatins, in plants. PCs are synthesised enzymatically from glutathione by the enzyme PC synthase in the presence of heavy metal ions. Using Arabidopsis thaliana as a model organism cadmium-sensitive, phytochelatin-deficient mutants have been isolated and characterised in previous studies. The cadl mutants have wildtype levels of glutathione, are PC deficient and lack PC synthase activity. Thus, the CADl gene has been proposed to encode PC synthase. The CADl gene was isolated by a positional cloning strategy The gene was mapped and a candidate identified. Each of four cadl mutants had a single base pair change in the candidate gene and the cadmium-sensitive, cadl phenotype was complemented by the candidate gene. This demonstrated the CADl gene had been cloned. A homologous gene in the fission yeast, Schizosaccharomyces pombe was identified through database searches. A targeted-deletion mutation of this gene was constructed and the mutant, like cadl mutants of Arabidopsis, was cadmium-sensitive and PC-deficient. A comparison of the redicted amino acid sequences reveals a highly conserved N-terminal region Presumed to be the catalytic domain and a variable C-terminal region containing multiple Cys residues proposed to be involved in activation of the enzyme by metal ions. Similar genes were also identified in animal species. The Arabidopsis CADl/AtPCSl and S. pombe SpbPCS genes were expressed in E. coli and were shown to be sufficient for glutathione-dependent, heavy metal activate PC synthesis in vitro, thus demonstrating these genes encode PC synthase enzymes. Using RT-PCR, AtPCSl expression appeared to be independent of Cd exposure. However, at higher levels of Cd exposure a AtPCSl-CUS reporter gene construct appeared to be more highly expressed. Using the reporter gene construct, AtPCSl was expressed most tissues. Expression appeared to be greater in younger tissues and same higher levels of expression was observed in some regions, including carpels and the base of siliques. AtPCS2 was a functional gene encoding an active PC synthase. However, its Pattern of expression and the phenotype of a mutant (or antisense line) have not been determined. Assuming the gene is functional then it has clearly been maintained through evolution and must provide some selective advantage. This implies that, at least in some cells or tissue, it is likely to be the dominant PC synthase expressed. This remains to be determined

• BMB Reports
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• v.53 no.6
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• pp.323-328
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• 2020

Matrix metalloproteinase 1 (MMP-1), a calcium-dependent zinccontaining collagenase, is involved in the initial degradation of native fibrillar collagen. Tissue necrosis factor-alpha (TNFα) is a pro-inflammatory cytokine that is rapidly produced by dermal fibroblasts, monocytes/macrophages, and keratinocytes and regulates inflammation and damaged-tissue remodeling. MMP-1 is induced by TNFα and plays a critical role in tissue remodeling and skin aging processes. However, the regulation of the MMP1 gene by TNFα is not fully understood. We aimed to find additional cis-acting elements involved in the regulation of TNFα-induced MMP1 gene transcription in addition to the nuclear factor-kappa B (NF-κB) and activator protein 1 (AP1) sites. Assessments of the 5'-regulatory region of the MMP1 gene, using a series of deletion constructs, revealed the requirement of the early growth response protein 1 (EGR-1)-binding sequence (EBS) in the proximal region for proper transcription by TNFα. Ectopic expression of EGR-1, a zinc-finger transcription factor that binds to G-C rich sequences, stimulated MMP1 promoter activity. The silencing of EGR-1 by RNA interference reduced TNFα-induced MMP-1 expression. EGR-1 directly binds to the proximal region and transactivates the MMP1 gene promoter. Mutation of the EBS within the MMP1 promoter abolished EGR-1-mediated MMP-1 promoter activation. These data suggest that EGR-1 is required for TNFα-induced MMP1 transcriptional activation. In addition, we found that all three MAPKs, ERK1/2, JNK, and p38 kinase, mediate TNFα-induced MMP-1 expression via EGR-1 upregulation. These results suggest that EGR-1 may represent a good target for the development of pharmaceutical agents to reduce inflammation-induced MMP-1 expression.

• Journal of Life Science
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• v.15 no.4 s.71
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• pp.513-521
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• 2005

A bacterium producing five fibrinolytic isozymes was isolated from black bean chung kuk. The bacterium was identified as Bacillus subtilis BB-1 by 16s rDNA sequence homology search. A gene out of five fibrinolytic genes in the Bacillus subtilis BB-1 was cloned by shot-gun method. A Cla I DNA fragment of B. subtilis BB-1 chromosome was cloned in to pBluescript II SK(-) and showed the fibrinolytic activity to bacterial cells. The Cla I DNA fragment was sequenced and the sequences did not show homology with gene for protease or fibrinolytic enzyme genes in other organisms. The Cla I DNA fragment was reduced to 2,142 bp by activity-guided PCR cloning method. The optimum pH and temperature of the enzyme were 5.0 and $35^{\circ}C$, respectively. Substrate specificity of the fibrinolytic enzyme was detected in skim milk, casein, gelatin and blood agar plates. The activity of the enzyme was not detected with these substrates. Taken together, this enzyme is a new fibrinolytic enzyme and may be used to prevent thrombosis and arteriosclerosis.

• Journal of Life Science
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• v.13 no.3
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• pp.291-297
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• 2003

A human endogenous retroviral family (HERV-W) has recently been described that is related to multiple sclerosis-associated retrovirus (MSRV) sequences that have been identified in particles recovered from monocyte cultures from patients with multiple sclerosis. Two pol fragments (HWP-FB10 and HWP-FBl2) of HERV-W family were identified and analysed by the PCR approach with cDNA library of human fetal brain. They showed 89 percent nucleotide sequence similarity with that of the HERV-W (accession no. AF009668). Deletion/insertion or point mutation in the coding region of the pol fragments from human fetal brain resulted in amino acid frameshift that induced a mutated protein. Phylogenetic analysis of the HERV-W family from GenBank database indicates that the HWP-FB10 is very closely related to the AC000064 derived from human chromosome 7q21-q22. Further studies on the genetic relationship with neighbouring genes and functional role of these new HERV-W pol sequences are indicated.

• Journal of Life Science
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• v.31 no.4
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• pp.400-409
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• 2021

Sanguinarine, a natural benzophenanthridine alkaloid, has been considered a potential therapeutic target for the treatment of cancer because it can induce apoptosis in human cancer cells; however, the underlying mechanisms of action still remain unclear. Tumor suppressor p53 deletion or mutation is an important reason for the resistance of colorectal cancer cells to anticancer agents. Therefore, in the present study, the role of p53 during apoptosis induced by sanguinarine was investigated in p53wild type (WT, p53+/+) and p53null (p53+/+) HCT116 colon carcinoma cells. Sanguinarine significantly caused greater reductions in cell viability in HCT116 (p53+/+) cells than in HCT116 (p53-/-) cells. Consistently, sanguinarine promoted more DNA damage and apoptosis in HCT116 (p53+/+) cells than in HCT116 (p53-/-) cells while increasing the expression of p53 and cyclin-dependent kinase inhibitor p21^WAF1/CIP1. Sanguinarine increased the activity of caspase-8 and caspase-9, which are involved in the initiation of extrinsic and intrinsic apoptosis pathways, respectively, and it activated caspase-3, a typical effect caspase, in HCT116 (p53+/+) cells. Sanguinarine also increased the generation of reactive oxygen species (ROS), and the Bax/Bcl-2 ratio, while destroying the integrity of mitochondria in HCT116 (p53+/+) cells, but not in HCT116 (p53-/-) cells. Overall, the results indicate that sanguinarine induced p53-dependent apoptosis through ROS-mediated activation of extrinsic and intrinsic apoptotic pathways in HCT116 colorectal cancer cells.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.389-397
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• 2001

Cocaine-amphetamine regulated transcript (CART), a satiety factor regulated by leptin, is associated with food intake and motor behavior. In knock out studies, Leu34Phe mutation of human CART gene resulted in obese phenotype but mice carrying a targeted deletion of the CART gene exhibited no dramatic increase of body weight on normal fat diet. To establish a new transgenic mouse model for determining the function of CART on feeding behavior in vivo, we constructed the fusion gene, CART gene under the control of neurofilament light chain promoter, which regulates gene expression at the stage of neuronal differentiation. Transgenic mice were generated by microinjection method and screened by PCR and Southern blot analyses. In these transgenic mice, overexpression of CART was detected by in situ hybridization in spinal cords and brains at 13.5 days post-coitum embryos. At six weeks of age, RT-PCR analysis showed that exogenous CART mRNA was expressed strongly in brains and spinal cords, but not much in other tissues. Our results suggest that these transgenic mice provide a new model to investigate the function of CART gene in neuronal network associated with feeding behavior.

• Clinical and Experimental Pediatrics
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• v.45 no.3
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• pp.302-310
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• 2002

Purpose : X-linked agammaglobulinemia(XLA) is an immunodeficiency caused by abnormalities in Bruton's tyrosine kinase(Btk), and is characterized by a deficiency of peripheral blood B cells. We studied cytoplasmic expression of Btk protein and analyzed the Btk gene in peripheral blood mononuclear cells(PBMC) from three XLA families in Korea. Methods : Heparinized venous blood samples were collected from four XLA patients and additional family members in three unrelated XLA families. Mononuclear cells were separated from their blood and the intracellular Btk protein was characterized by a flow cytometry. The mutation analysis was performed using direct sequencing. Results : Cytoplasmic expression of Btk protein in monocytes was not detected in the patients with XLA. We observed a novel deletion and two point mutations within introns(intron 1 and intron 18) resulting in alternative splicings. In XLA family 2, a 980 bp deletion(from intron 9+191 T to intron 10-215 C) including exon 10 was found in patient P2. He was the only sporadic case in this study, because his mother and brother showed a normal Btk expression by flow cytometry. Conclusion : These identified genetic alterations support the molecular heterogeneity of Btk gene in XLA disease. Additionally, by means of flow cytometric analysis, we diagnosed three hypogammaglobulinemia patients as XLA. Advancements in diagnostic methods has facilitated a prompt and definite diagnosis of this disease.

• Journal of Animal Science and Technology
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• v.53 no.2
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• pp.107-111
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• 2011

This study was undertaken to reveal the relationship between genetic variations and the basic coat color classification system in Jeju horses. Genetic variations of the melanocortinreceptor 1 (MC1R) and agouti signaling protein (ASIP) genes were investigated using pyrosequencing technique. A nucleotide substitution mutation for MC1R g.901C>T and an ASIP 11-bp deletion mutation were screened. Black horses had MC1R $E^+$/- ($E^+/E^+$ or $E^+/E^e$) and ASIP $A^a/A^a$ genotypes. In contrast, chestnut horse genotypes were MC1R $E^e/E^e$ and ASIP -/-. Thus, black and bay horses have at least one dominant MC1R allele, $E^+$, whereas chestnut horses have homozygous recessive alleles $E^e/E^e$. This suggests that the MC1R genotypes determine chestnut or black/bay coat color, regardless of the genotype distribution of ASIP. In addition, the horses with MC1R $E^+$/- and a dominant ASIP $A^A$/- allele showed bay coat color, but not black, suggesting that the ASIP $A^A$ allele represses black coat color development in the hairs of the body, but not in the mane and all four legs. Pedigree analysis showed a consistent relationship between the genotype distribution of the MC1R and ASIP genes and basic coat color patterns, even in the $F_1$ progeny. The results of this study revealed the relationship between the coat color phenotype and genetic background and suggested that useful information may be provided for molecular breeding of Jeju horses.

• Clinical and Experimental Pediatrics
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• v.51 no.1
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• pp.73-77
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• 2008

Purpose : p16 gene, mapped to the 9p21 chromosomal region, has emerged as a candidate tumor suppressor gene in human neoplasm. It is an inhibitor of cyclin-dependent kinase and inhibits Rb phosphorylation. In a variety of tumors including childhood acute lymphoblastic leukemia (ALL), deletion and/or mutation of the p16 gene has been found. Despite their high frequency, the prognostic importance of p16 alterations is still controversial in ALL and has been reported to be either unfavorable or similar to that of other patients. We studied the correlation between loss of p16 protein confirmed by immunohistochemical staining and clinical outcomes of patients diagnosed as ALL. Methods : We performed an immunohistochemical staining for p16 protein in 74 cases of bone marrow biopsy slide initially diagnosed as ALL between January 1998 and December 2006. We reviewed the clinical manifestations, laboratory findings, treatment outcomes retrospectively. Results : Of 74 slides, 12 were negative for p16 protein. Seven were males and 5 were females with a median age at diagnosis was 5.8 (1.3-18.8) years. Initial WBC were 17,225 $(500-403,300)/{\mu}L$. By immunologic surface marker analysis, 7 patients were early pre-B CALLA (+) and 5 patients were T-cell ALL. Two patients of intermediate risk group had relapsed and died. Three patients had family history of breast cancer. Four patients died and overall survival rates were $53.5{\pm}18.7%$. Conclusion : Loss of p16 protein is supposed to be an independent risk factor of childhood ALL associated with poor outcomes. In clinical setting, the clinician must take into account p16 status, not only at the genomic but also at the protein level. Further clinical experience on thoroughly investigated cases will help a better understanding between p16 status and clinical outcomes.

• Journal of Life Science
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• v.26 no.4
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• pp.414-418
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• 2016

This study tested the association between genetic polymorphisms of the isocitrate dehydrogenase 3, beta subunit (IDH3B) gene and economic traits in an F₂ crossbred population of Landrace × Jeju (South Korea) Black pigs. A 304-bp insertion/deletion mutation in promoter region was screened for determining genotypes of the IDH3B gene in a total of 1,105 F₂ pigs. Three genotypes (AA, AB, and BB) were identified in the founder, F₁, and F₂ populations. Association analysis showed significant differences in carcass weights (CW), backfat thicknesses in three positions of the body (4^th-5^th ribs, BF5; 11^th-12^th ribs, BF12; 13^th rib-1^st lumbar, BFL), and carcass lengths (CL) (p<0.05), but not in meat color (MC), eye muscle area (EMA), or marbling scores (MARB) (p>0.05). The F₂ IDH3B BB homozygotes showed heavier CW (80.790±0.725 kg) and shorter CL (101.875±0.336 cm) than the other genotypes (p<0.05). In addition, the BF levels between the 4^th - 5^th and 11^th - 12^th vertebrae were thicker in the carcasses of pigs with the IDH3B BB genotype than with the other genotypes (p<0.05). These results suggested that genetic variations in the IDH3B gene may serve as molecular genetic markers for improving the Landrace × Jeju Black pig crossbreeding systems.