• The Korean Journal of Food And Nutrition
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• v.25 no.1
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• pp.123-131
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• 2012

The objective of the present study was to evaluate the potential antidiabetic and antioxidant effect of the ethanol extract from Chrysanthemum cornarium L. var. spatiosum(CSE) against alloxan-induced oxidative stress in pancreatic ${\beta}$-cells, HIT-T15. In this study, the antidiabetic effect of CSE was examined using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliu bromide(MTT) cell proliferation assay, lactate dehydrogenase(LDH) release assay, $NAD^+$/NADH ratio and insulin secretion. To further investigate whether CSE is involved in the antioxidant activity of alloxan-damaged HIT-T15 cells, its antioxidant effect against alloxan-induced oxidative stress was measured in HIT-T15 cells by determining the levels of antioxidant enzymes including superoxide dismutase(SOD), glutathione S-transferase(GST), glutathione reductase(GR) and glutathione peroxidase(GPx). The results of this analysis showed that alloxan significantly decreased cell viability, increased LDH leakage, and lowered $NAD^+$/NADH ratio and insulin secretion in HIT-T15 cells. However, CSE significantly increased the viability of alloxan-treated cells and lowered LDH leakage. The intracellular NAD+/NADH ratio and insulin secretion were also significantly increased by 1.7-fold and 1.3-fold, respectively, after treatment with 100 ${\mu}g/m{\ell}$ CSE. The HIT-T15 cells treated with alloxan showed significant decreases in the activities of antioxidant enzymes, while CSE significantly elevated the levels of antioxidant enzymes. These findings suggest that CSE could have a protective effect against cytotoxicity and dysfunction of pancreatic cells in the presence of alloxan-induced oxidative stress.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.7
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• pp.3253-3260
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• 2014

Background: A case-control study was conducted to evaluate the effect of household exposure, dietary habits, smoking and Glutathione S-Transferases M1, T1 polymorphisms on lung cancer among women in Mizoram, India. Materials and Methods: We selected 230 newly diagnosed primary lung cases and 460 controls from women in Mizoram. Multivariate logistic regression analysis was performed to estimate adjusted odds ratio (OR). Results: Exposure of cooking oil fumes (p<0.003), wood as heating source for cooking (p=0.004), kitchen inside living room (p=0.001), improper ventilated house (p=0.003), roasting of soda in kitchen (p=0.001), current smokers of tobacco (p=0.043), intake of smoked fish (p=0.006), smoked meat (p=0.001), Soda (p<0.001) and GSTM1 null genotype (p=0.003) were significantly associated with increased risk of lung cancer among women in Mizoram. Significantly protective effect was observed for intake of bamboo shoots (p=<0.001) and egg (p<0.001). A clear increase in dose response gradient was observed for total cooking dish years. Risk for lung cancer tends to increase with collegial effect of indoor environmental sources (p=0.022). Significant correlation was also observed for interaction of GST polymorphisms with some of dietary habits. Conclusions: We confirmed the important role of exposure of cooking oil emission and wood smoke, intake of smoked meat, smoked fish and soda (an alkali preparation used as food additives in Mizoram) and tobacco consumption for increase risk of lung cancer among Women in Mizoram.

• Korean Journal of Plant Resources
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• v.25 no.2
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• pp.184-192
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• 2012

The present study was designed to examine the potential antidiabetic and antioxidant effect of ethanol extract from $Prunus$ $mume$ fruit (PME) against alloxan-induced oxidative stress in pancreatic ${\beta}$-cells, HIT-T15. To evaluate the antidiabetic effect of PME, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliu bromide (MTT) cell proliferation assay, lactate dehydrogenase (LDH) release assay, $NAD^+$/NADH ratio and insulin secretion were assessed. We also measured its antioxidant effect against alloxan-induced oxidative stress in the cells by assessing the levels of the antioxidant enzymes including superoxide dismutase (SOD), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx). The results of this analysis showed that alloxan significantly decreased cell viability, increased LDH leakage, and lowered $NAD^+$ /NADH ratio and insulin secretion in HIT-T15 cells. However, PME significantly increased the viability of alloxan-treated cells and lowered LDH leakage. The intracellular $NAD^+$ /NADH ratio and insulin secretion were also increased by 1.5~1.9-fold and 1.4-fold, respectively, after treatment with the PME. The HIT-T15 cells treated with alloxan showed significant decreases in the activities of antioxidant enzymes, while PME significantly elevated the levels of antioxidant enzymes. Based on these results, we suggest that PME could have a protective effect against the cytotoxicity and dysfunction of pancreatic ${\beta}$-cells in the presence of alloxan-induced oxidative stress.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.66-66
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• 2003

In skeletal muscle cells, depolarization of the transverse tubules (T-tubules) results in Ca$\^$2+/ release from the sarcoplasmic reticulum (SR), leading to elevated cytoplasmic Ca$\^$2+/ and muscle contraction. This process has been known as excitation-contraction coupling (E-C coupling). Several proteins, such as the ryanodine receptor (RyR), triadin, junctin, and calsequestrin (CSQ), have been identified to be involved in the Ca$\^$2＋/ release process. However, the molecular interactions between the SR proteins have not been resolved. In the present study, the mechanisms of interaction between RyRl and triadin have been studied by in vitro protein binding and $\^$45/Ca$\^$2+/ overlay assays. Our data demonstrate that the intraluminal loop II of RyR1 binds to triadin in Ca$\^$2+/-independent manner. Moreover, we could not find any Ca$\^$2+/ binding sites in the loop II region. GST-pull down assay revealed that a KEKE motif of triadin, which was previously identified as a CSQ binding site (Kobayasi et al.,2000 JBC) was also a binding site for RyR1. Our results suggest that the intraluminal loop II of RyR could participate in the RyR-mediated Ca$\^$2+/ release process by offering a direct binding site to luminal triadin.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.279-285
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• 1993

Alterations of the p53 gene arc among the most frequent genetic changes in human cancer and often result in increased levels of p53 protein within the malignant cells. Detection of accumulated p53 protein can be a useful prognostic tool in human cancer. In order to make polyclonal antibodies for immunohistochemical screening. human p53 gene was expressed in E. coli in the form of GST (glutathione S-transfi.:rase) fusion proteins. Two p53 gene fragments. which were N('()I small fragment encoding amino acid residues of 1-151-: and Ncol large fragment of 159-393. were subeloned into the unique BamHI site present within the pGEX-2T vector using BamHI linker and recombinant plasmids pGTNS and pGTNL were constructed. respectively. The p53 cDNA fragment (from pC53-$SN_3$,) encoding amino acid 38-145 (proline at residue 72) was amplified by polymerase chain reaction(PCR). The amplified DNA was digested with BamHI and Prull and inserted into the BamHI-Smal sites of pG EX-2T and recombinant plasmid pGTBP was constructed. After IPTG induction of these plasmids for 4 hours. fusion proteins were purified from E. coli extracts with glutathione Sepharose beads. The bound proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and the molecular weights were 54 kDa. 53 kDa and 40 kDa. respectively. Approximately one milligram of fusion proteins were purified from 1 -liter cultures.

• Journal of Microbiology and Biotechnology
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• v.16 no.12
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• pp.1995-1999
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• 2006

Pseudomonas sp. K82 cultured in p-hydroxybenzoate induces protocatechuate 4,5-dioxygenase (PCD 4,5) for p-hydroxybenzoate degradation. In this study, a 6.0-kbp EcoR1 fragment containing p-hydroxybenzoate degradation genes was cloned from the genome of Pseudomonas sp. K82. Sequence analysis identified four genes, namely, pcaD, pcaA, pcaB, and pcaC genes known to be involved in p-hydroxybenzoate degradation. Two putative 4-hydroxyphenylpyruvate dioxygenases and one putative oxidoreductase were closely located by the p-hydroxybenzoate degradation genes. The gene arrangement and sequences of these p-hydroxybenzoate degradation genes were similar to those of Comamonas testosteroni and Pseudomonas ochraceae. PcaAB (PCD4,5) was overexpressed in the expression vector pGEX-4T-3, purified using a GST column, and confirmed to have protocatechuate 4,5-dioxygenase activity. The N-terminal amino acid sequences of overexpressed PCD4,5 were identical with those of purified PCD4,5 from Pseudomonas sp. K82.

• International Journal of Industrial Entomology and Biomaterials
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• v.10 no.1
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• pp.51-59
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• 2005

Genetic diversity within and between populations of Antheraea mylitta Drury was studied using thirty individuals from three ecoraces using 12 ISSR and 10 RAPD primers. Rally, Daba and Modal ecoraces were collected from Chattisgarh, Jharkhand and Orissa states of India respectively. The ISSR and RAPD primers generated $94.7\%$ and $95.6\%$ polymorphism among the 30 individuals. The cluster analysis grouped these individuals according to their ecorace. The intra-ecoracial heterozygosity estimated with ISSR markers were $0.123{\pm}0.18,\;0.169{\pm}0.17\;and\;0.214{\pm}0.17$ respectively for Modal, Raily and Daba ecoraces. Like wise, with RAPD markers the intraecoracial heterozygosity was $0.17{\pm}0.22$ in Modal, $0.229{\pm}0.17$ in Raily and $0.23{\pm}0.19$ in Daba ecoraces. However, the significantly low genetic differentiation (GST) (0.182 for ISSR and 0.161 for RAPD) and the high gene flow (Nm) (2.249 for ISSR and 2.60 for RAPD markers) among the ecoraces revealed that the amount of genetic diversity present among the ecoraces is not significant enough to make drastic genetic drifts among these ecoraces in the near future.

• International Journal of Industrial Entomology and Biomaterials
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• v.17 no.2
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• pp.223-228
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• 2008

A novel beetle antimicrobial protein from stimulated Copris tripartitus and the corresponding gene were isolated in parallel through differential display-PCR and expression in Escherichia coli. To find cDNA clones responsible for bacteria resistance, the suppression subtractive hybridization and GeneFishing differentially expressed genes system were employed in the dung beetle, Copris tripartitus immunized with lipopolysaccaride. One cDNA clone from eight subtracted clones was selected through dot blot analysis and confirmed by northern blot analysis. The 516-bp, selected cDNA clone was determined by 5' and 3' rapid amplication of cDNA ends and cloned into the GST fusion expression vector pGEX-4T-1 for expression of the protein. The expressed protein was predicted 14.7 kDa and inhibited the growth of gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. These results implied that the expressed protein is related to immune defense mechanism against microorganism.

• Journal of Fisheries and Marine Sciences Education
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• v.28 no.4
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• pp.903-912
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• 2016

Hagfish which belongs to the chordate contact cyclostomata, is important phylogenetic relationship between vertebrate and invertebrate. Cathepsins of the cysteine protease family have traditionally been thought to play a major role in intracellular protein degradation and turnover in lysosomes. In this study, Catepsin L was cloned from Inshore hagfish (Eptatretus burgeri), the cDNA encoding ORF of the Eptatretus burgeri Cathepsin L (EbCtL) is 978 bp. The cDNA encoding proEbCtL was expressed in Escherichia coli strain BL21(DE3) using the pGEX-4T-1 expression vector system. The recombinant proEbCtL protein was overexpressed as a approximately 55 kDa fusion protein. The overproduced soluble GST-fusion protein was then applied to glutathione-Sepharose 4B column chromatography; the sample harboring the fusion protein evidenced a high degree of purity when analyzed via SDS-PAGE and Western blot analysis. Its activity was quantied by cleaving the synthetic peptide Z-FR-AMC, Z-LLE-AMC, and Suc-AAF-AMC, and the optimal pH for the protease activity was 8, 9.5, and 9, respectively.

• Journal of Life Science
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• v.26 no.3
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• pp.282-288
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• 2016

Protein-protein interactions regulate the subcellular localization and function of receptors, enzymes, and cytoskeletal proteins. Proteins containing the postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain have potential to act as scaffolding proteins and play a pivotal role in various processes, such as synaptic plasticity, neural guidance, and development, as well as in the pathophysiology of many diseases. Multi-PDZ domain protein 1 (MUPP1), which has 13 PDZ domains, has a scaffolding function in the clustering of surface receptors, organization of signaling complexes, and coordination of cytoskeletal dynamics. However, the cellular function of MUPP1 has not been fully elucidated. In the present study, a yeast two-hybrid system was used to identify proteins that interacted with the N-terminal PDZ domain of MUPP1. The results revealed an interaction between MUPP1 and Wdpcp (formerly known as Fritz). Wdpcp was identified as a planar cell polarity (PCP) effector, which is known to have a role in collective cell migration and cilia formation. Wdpcp bound to the PDZ1 domain but not to other PDZ domains of MUPP1. The C-terminal end of Wdpcp was essential for the interaction with MUPP1 in the yeast two-hybrid assay. This interaction was further confirmed in a glutathione S-transferase (GST) pull-down assay. When coexpressed in HEK-293T cells, Wdpcp was coimmunoprecipitated with MUPP1. In addition, MUPP1 colocalized with Wdpcp at the same subcellular region in cells. Collectively, these results suggest that the MUPP1-Wdpcp interaction could modulate actin cytoskeleton dynamics and polarized cell migration.