• KSBB Journal
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• v.20 no.1 s.90
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• pp.21-25
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• 2005

Paraquat, a widely used herbicide, has been suggested as a potential risk factor for Parkinson's disease. Heme oxygenase-1(HO-1), a marker for oxidative stress and endoplasmic reticulum(ER) stress, is known to catalyze heme to biliverdin, carbon monoxide and free iron in response to various stimuli. Here we show that paraquat activates HO-1 expression in a time-and dose-dependent manner in substantia nigra(SN) dopaminergic neuronal cells. Activation of Ho-1 by paraquat was regulated primarily at the level of gene transcription. Deletion analysis of the promoter and the 5' distal enhancers, E1 and E2, of the HO-1 gene revealed that the E2 enhancer is a potent inducer of the paraquat-dependent Ho-1 gene expression in dopamninergic neuronal cells. Mutational analysis of the E2 enhacer further demonstrated that the transcription factor activator protein-1(AP-1) plays an important role in mediating paraquat-induced HO-1 gene transcription. Moreover, using specific inhibitors of the mitogen-activated protein kinases(MAPKs), we investigated the role of paraquat and MAPKs for HO-1 gene regulation in dopaminergic cells. The c-Jun N-terminal kinase(JNK) inhibitor SP600125 significantly suppressed the expression of HO-1 by paraquat. All these results demonstrate that induction of HO-1 by paraquat requies the activation of the AP-1 and JNK pathway.

• Journal of Microbiology and Biotechnology
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• v.20 no.8
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• pp.1196-1203
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• 2010

In the present work, methanethiol and dimethyldisulfide were investigated as sulfur sources for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis predicted a high methionine yield for these reduced compounds, provided that they could be utilized. Wild-type cells were able to grow on both methanethiol and dimethyldisulfide as sole sulfur sources. Isotope labeling studies with mutant strains, exhibiting targeted modification of methionine biosynthesis, gave detailed insight into the underlying pathways involved in the assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as an entire molecule, adding the terminal S-$CH_3$ group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as the enzyme catalyzing the reaction. The deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur sources. Plasmid-based overexpression of metY in the ${\Delta}$metY background restored the capacity to grow on methanethiol or dimethyldisulfide as sole sulfur sources. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1,780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased 2-fold in the engineered strain. This positive effect was limited by a depletion of the metY substrate O-acetylhomoserine, suggesting a need for further metabolic engineering targets towards competitive production strains.

• Applied Biological Chemistry
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• v.36 no.6
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• pp.525-532
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• 1993

To characterize the prolamines in rice cultivars, the complete coding sequences of 17 prolamine genes from the database were analyzed. According to the phylogenic analysis of the sequences, these genes could be classified into 4 groups, Group I to IV. The multiple alignment of the deduced amino acid sequences revealed that the four groups differ from one another in chain length caused by deletion of short internal amino acids or carboxyl terminal fragments. Each group was also found to have different amino acid composition with 1, 4, 10 and 30% of sulfur containing amino acids (methionine and cysteine) in Group I to IV prolamines, respectively. Also the isoelectric points of these groups showed the different values of 9.2, 8.2, 6.7 and 7.4. Finally, from the analysis of codon usage pattern of prolamine genes, the codon usage for arginine, serine, threonine, isoleucine, asparagine, aspartic acid, glutamic acid and cysteine were higly biased. In the analysis of the codon usage pattern, the relation of the fraction of G/C ending codons to effective codon numbers suggests the different translational efficiency in the expression of the prolamine multigenes.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.3
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• pp.277-286
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• 2020

Polycystic kidney disease 2-like-1 (PKD2L1), also known as polycystin-L or TRPP3, is a non-selective cation channel that regulates intracellular calcium concentration. Calmodulin (CaM) is a calcium binding protein, consisting of N-lobe and C-lobe with two calcium binding EF-hands in each lobe. In previous study, we confirmed that CaM is associated with desensitization of PKD2L1 and that CaM N-lobe and PKD2L1 EF-hand specifically are involved. However, the CaM-binding domain (CaMBD) and its inhibitory mechanism of PKD2L1 have not been identified. In order to identify CaM-binding anchor residue of PKD2L1, single mutants of putative CaMBD and EF-hand deletion mutants were generated. The current changes of the mutants were recorded with whole-cell patch clamp. The calmidazolium (CMZ), a calmodulin inhibitor, was used under different concentrations of intracellular. Among the mutants that showed similar or higher basal currents with that of the PKD2L1 wild type, L593A showed little change in current induced by CMZ. Co-expression of L593A with CaM attenuated the inhibitory effect of PKD2L1 by CaM. In the previous study it was inferred that CaM C-lobe inhibits channels by binding to PKD2L1 at 16 nM calcium concentration and CaM N-lobe at 100 nM. Based on the results at 16 nM calcium concentration condition, this study suggests that CaM C-lobe binds to Leu-593, which can be a CaM C-lobe anchor residue, to regulate channel activity. Taken together, our results provide a model for the regulation of PKD2L1 channel activity by CaM.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.304-313
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• 1998

Sixteen Korean field transmissible gastroenteritis viruses (TGEVs) were isolated using swine testicular cell (STC) and the genomic diversity of them was analyzed. All TGEV isolates produced a typical cytopathic effect in STC and were confirmed as TGEV by immunofluorescence assay using monoclonal antibody against TGEV and PCR using TGEV specific primers. RNAs from TGEV field isolates and vaccine TGEV were extracted and amplified by RT and PCR. The RT-PCR products were digested with selected restriction enzymes and analyzed RFLP patterns. The N-terminal end region of S gene and ORF 3 and 3-1 genes of TGEV amplified by TGEV specific primer pairs seemed to be conserved. Most specific variations were detected in S gene amplified by TGEV 4/6 primer pairs which includes antigenic sites A and D. When the PCR products were treated with Sau3AI and Ssp I, Bvac(vaccine strain), field isolates 133 and 347 were differentiated from Miller and Purdue types. In the case of D5 field isolates, it was classified into Purdue type by Sau 3AI but classified into independent TGEV by Ssp I. Two different TGEV strains from D2 sample were confirmed by plaque purification and RT-PCR-RFLP analysis. To investigate the change occurring in TGEV genome after serial passage, the TGEV P44 strain was passaged through STC. There were specific changes in S gene and a large deletion was observed in ORF 3 and 3-1 genes. These studies showed that a distinct difference in genome exists among TGEV field isolates.

• BMB Reports
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• v.53 no.10
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• pp.521-526
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• 2020

Endogenous retroviruses (ERVs) are retrotransposons present in various metazoan genomes and have been implicated in metazoan evolution as well as in nematodes and humans. The long terminal repeat (LTR) retrotransposons contain several regulatory sequences including promoters and enhancers that regulate endogenous gene expression and thereby control organismal development and response to environmental change. ERVs including the LTR retrotransposons constitute 8% of the human genome and less than 0.6% of the Caenorhabditis elegans (C. elegans) genome, a nematode genetic model system. To investigate the evolutionarily conserved mechanism behind the transcriptional activity of retrotransposons, we generated a transgenic worm model driving green fluorescent protein (GFP) expression using Human endogenous retroviruses (HERV)-K LTR as a promoter. The promoter activity of HERV-K LTR was robust and fluorescence was observed in various tissues throughout the developmental process. Interestingly, persistent GFP expression was specifically detected in the adult vulva muscle. Using deletion constructs, we found that the region from positions 675 to 868 containing the TATA box was necessary for promoter activity driving gene expression in the vulva. Interestingly, we found that the promoter activity of the LTR was dependent on che-1 transcription factor, a sensory neuron driver, and lin-15b, a negative regulator of RNAi and germline gene expression. These results suggest evolutionary conservation of the LTR retrotransposon activity in transcriptional regulation as well as the possibility of che-1 function in non-neuronal tissues.

• Molecules and Cells
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• v.28 no.6
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• pp.553-558
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• 2009

In the periphery, a galectin-1 receptor, CD7, plays crucial roles in galectin-1-mediated apoptosis of activated T-cells as well as progression of T-lymphoma. Previously, we demonstrated that $NF-{\kappa}B$ downregulated CD7 gene expression through the p38 MAPK pathway in developing immature thymocytes. However, its regulatory pathway is not well understood in functional mature T-cells. Here, we show that CD7 expression was downregulated by Twist2 in Jurkat cells, a human acute T-cell lymphoma cell line, and in EL4 cells, a mature murine T-cell lymphoma cell line. Furthermore, ectopic expression of Twist2 in Jurkat cells reduced galectin-1-induced apoptosis. While full-length Twist2 decreased CD7 promoter activity, a C-terminal deletion form of Twist2 reversed its inhibition, suggesting an important role of the C-terminus in CD7 regulation. In addition, CD7 expression was enhanced by histone deacetylase inhibitors such as trichostatin A and sodium butyrate, which indicates that Twist2 might be one of candidate factors involved in histone deacetylation. Based on these results, we conclude that upregulation of Twist2 increases the resistance to galectin-1-mediated-apoptosis, which may have significant implications for the progression of some T-cells into tumors such as Sezary cells.

• Archives of Pharmacal Research
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• v.25 no.3
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• pp.364-369
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• 2002

Hepatitis C virus (HCV) is remarkably efficient at establishing chronic infection. One of the reasons for this appears to be the suppression of the accessory cell function of professional antigen presenting cells. In the present study, the immunosuppressive activity of HCV protein was examined on dendritic cells (DCs) generated from mouse bone marrow progenitor cells in vitro. We found that the DCs forced to express HCV protein have defective allostimulatory ability. DCs expressing HCV protein were phenotypically indistinguishable from normal DCs. However, they were unable to produce IL-12 effectively when stimulated with lipopolysaccharide. The functional domain of the HCV protein essential for immunosuppression was determined using a series of ${NH_2}-and$ C-terminal deletion mutants of HCV core protein. We found that amino acid residues residing between the 21 st and the 40th residues from the ${NH_2}-terminus$ of HCV core protein are required for immunosuppression. These findings suggest that HCV core protein suppresses the elicitation of protective Th1 responses by the inhibition of IL-12 production by DCs.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.11a
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• pp.69-80
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• 1994

Although ureases play important roles in microbial nitrogen metabolism and in the pathogenesis of several human diseases, little is known of the mechanism of metallocenter biosynthesis in this Ni-Containing enzyme. Klebsiella aerogenes urease apo-protein was purified from cells grown in the absence of Ni. The purified apo-enzyme showed the same native molecular weight, charge, and subunit stoichiometry as the holo-enzyme. Chemical modification studies were consistent with histidinyl ligation of Ni. Apo-enzyme could not be activated by simple addition of Ni ions suggesting a requirement for a cellular factor. Deletion analysis showed that four accessory genes (ureD, ureE, ureF, and ureG) are necessary for the functional incorporation of the urease metallocenter. Whereas the $\Delta$ureD, $\Delta$ureF, and $\Delta$ureG mutants are inactive and their ureases lack Ni, the $\Delta$ureE mutants retain partial activity and their ureases possess corresponding lower levels of Ni. UreE and UreG peptides were identified by SDS-polyacrylamide gel comparisons of mutant and wild type cells and by N-terminal sequencing. UreD and UreF peptides, which are synthesized at ve교 low levels, were identified by using in vitro transcription/translation methods. Cotransformation of E. coli cells with the complementing plasmids confirmed that ureD and ureF gene products act in trans. UreE was purified and characterized. immunogold electron microscopic studies were used to localize UreE to the cytoplasm. Equilibrium dialysis studies of purified UreE with $^{63}$ NiC1$_2$ showed that it binds ～6 Ni in a specific manner with a $K_{d}$ of 9.6 $\pm$1.3 $\mu$M. Results from spectroscopic studies demonstrated that Ni ions are ligated by 5 histidinyl residues and a sixth N or O atom, consistent with participation of the polyhistidine tail at the carboxyl termini of the dimeric UreE in Ni binding. With these results and other known features of the urease-related gene products, a model for urease metallocenter biosynthesis is proposed in which UreE binds Ni and acts as a Ni donor to the urease apo-protein while UreG binds ATP and couples its Hydrolysis to the Ni incorporation process.ouples its Hydrolysis to the Ni incorporation process.s.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.4
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• pp.234-241
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• 2000

The Saccharomyces cerevisiae PMR1 gene encodes a Ca2+-ATPase localized in the Golgi. We have investigated the effects of PMR1 disruption in S. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human ${\alpha}$1-antitrypsin (${\alpha}$1-AT), human antithrombin III (ATHIII), and Aspergillus niger glucose oxidase (GOD). The pmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of the pmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from the pmr1 mutant compared to that of the wild-type strain. The pmr1 mutant strain secreted ${\alpha}$1-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in the pmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in the mnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-${\alpha}$1,3-mannose antibody revealed that GOD secreted in the pmr1 mutant did not have terminal ${\alpha}$1,3-linked mannose unlike those secreted in the mnn9 mutant and the wild type strains. The present results indicate that the pmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.