• Korean Journal of Clinical Laboratory Science
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• v.51 no.1
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• pp.78-85
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• 2019

Membrane proteins are involved in many common diseases, including heart disease and cancer. In various disease states, such as cancer, abnormal signaling pathways that are related to the membrane proteins cause the cells to divide out of control and the expression of membrane proteins can be altered. Membrane proteins have the hydrophobic environment of a lipid bilayer, which makes an analysis of the membrane proteins notoriously difficult. Therefore, this study evaluated the efficacy of two different methods for optimal membrane protein extraction. High-speed centrifuge and reagent-based method with a -/+ filter aided sample preparation (FASP) were compared. As a result, the high-speed centrifuge method is quite effective in analyzing the mitochondrial inner membranes, while the reagent-based method is useful for endoplasmic reticulum membrane analysis. In addition, the function of the membrane proteins extracted from the two methods were analyzed using GeneGo software. GO processes showed that the endoplasmic reticulum-related responses had higher significance in the reagent-based method. An analysis of the process networks showed that one cluster in the high-speed centrifuge method and four clusters in the reagent-based method were visualized. In conclusion, the two methods are useful for the analysis of different subcellular membrane proteins, and are expected to assist in selecting the membrane protein extraction method by considering the target subcellular membrane proteins for study.

• Korean Journal of Ecology and Environment
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• v.52 no.3
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• pp.221-230
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• 2019

Barbel steed (Hemibarbus labeo) is a small freshwater fish species as semi-bottom dwellers distributed in eastern Asia. We carried out characterization of the cytochrome c oxidase subunit I (COI) gene from the mitochondrial DNA of H. labeo in the Sumjin River to identify the phylogenetic location of H. labeo in the genus Hemibarbus and Cyprinidae. Multiple alignment of the 577 bp COI sequence revealed high sequence homology (99~100%) between Seomjin River H. labeo. The nucleotide sequence similarity between H. labeo (HD1) and H. mylodon was 88.91% and that of H. longirostis was 88.81% among the three species found in Korea. In addition, the nucleotide sequence similarities of H. maculatus, H. meditus, H. umbrifer and H. barbus showed 98.97%, 97.20%, 96.87% and 98.85%, respectively. Phylogenetic analysis on seven species of the genus Hemibarbus showed that the H. labeo collected in this study formed two clades. One of which consisted of Hadong, Imsil, Kangjin. The other one formed a step with HD2, HD8 and HD9 of Hadong and the H. labeo reported in Busan, Asan and Seoul, Korea. Phylogenetic position of the H. labeo among Cyprinidae showed 0.143 for the evolutionary distance from Zacco platypus and 0.006 for the H. maculatus. In addition, the genetic position of the H. labeo among 28 species of Cyprinidae was found to be located in Group I, including Gobioninae fishes. The results of this study will provide key genetic information for the taxonomic comparison in Cyprinidae and study of model fish for pollution monitoring in freshwater environments.

• Journal of Life Science
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• v.31 no.8
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• pp.778-785
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• 2021

The germination of cucumber seeds begins with the degradation of reserved oil to fatty acids within the lipid body, which are then further metabolized to acyl-CoA. The acyl-CoA moves from the lipid body to the glyoxysome following β-oxidation for the production of acetyl-CoA. As an initial carbon source supplier, acetyl-CoA is an essential molecule in the glyoxylate cycle within the glyoxysome, which produces the metabolic intermediates of citrate and malate, among others. The glyoxylate cycle is a necessary metabolic pathway for oil seed plant germination because it produces the metabolic intermediates for the tricarboxylic acid (TCA) cycle and for gluconeogenesis, such as the oxaloacetate, which moves to the cytosol for the initiation of gluconeogenesis by phophoenolpyruvate carboxykinase (PEPCK). Following reserved oil mobilization, the production and transport of various metabolic intermediates are involved in the coordinated operation and activation of multiple metabolic pathways to supply directly usable carbohydrate in the form of glucose. Furthermore, corresponding gene expression regulation compatibly transforms the microbody to glyoxysome, which contains the organelle-specific malate synthase (MS) and isocitrate lyase (ICL) enzymes during oil seed germination. Together with glyoxylate cycle, carnitine, which mediates the supplementary route of the acetyl-CoA transport mechanism via the mitochondrial BOU (A BOUT DE SOUFFLE) system, possibly plays a secondary role in lipid metabolism for enhanced plant development.

• Journal of Life Science
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• v.31 no.1
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• pp.66-72
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• 2021

This study developed a species identification method for the salted opossum shrimp of Acetes japonicus, A. chinensis (Korea, China), A. indicus (I, II), and Palaemon gravieri based on PCR-RFLP markers. Genomic DNA was extracted from the salted opossum shrimp. The COI gene was used to amplify 519 base pairs (bp) using specific primers. The amplified products were digested by Acc I and Hinf I, and the DNA fragments were separated by automated electrophoresis for RFLP analysis. When the amplified DNA product (519 bp) was digested with Acc I, A. japonicus, A. chinensis (Korea), and A. indius (II) showed two fragments, whereas a single band of 519 bp was detected in A. chinensis (China) and A. indius (I). Also, in the RFLP patterns digested by Hinf I, A. chinensis (Korea) and A. chinensis (China) showed a single band of 519 bp, while two fragments were observed in A. japonicus and A. indius (I) and four fragments in A. indius (II). The PCR amplicon of P. gravieri was digested by Acc I into 3 bands of 271, 202, and 46 bp and by Hinf I into a single band of 519 bp. Therefore, salted opossum shrimp-specific RFLP markers showing distinct differences between four species and two sub-species by PCR-RFLP analysis. Thus, the PCR-RFLP markers developed in this study are a good method for identifying the six types of salted opossum shrimp.

• Journal of Ginseng Research
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• v.45 no.2
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• pp.295-304
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• 2021

Background: Acute promyelocytic leukemia (APL) is a hematopoietic malignancy driven by promyelocytic leukemia-retinoic acid receptor A (PML-RARA) fusion gene. The therapeutic drugs currently used to treat APL have adverse effects. 20(S)-ginsenoside Rh2 (GRh2) is an anticancer medicine with high effectiveness and low toxicity. However, the underlying anticancer mechanisms of GRh2-induced PML-RARA degradation and apoptosis in human APL cell line (NB4 cells) remain unclear. Methods: Apoptosis-related indicators and PML-RARA expression were determined to investigate the effect of GRh2 on NB4 cells. Z-VAD-FMK, LY294002, and C 87, as inhibitors of caspase, and the phosphatidylinositol 3-kinase (PI3K) and tumor necrosis factor-α (TNF-α) pathways were used to clarify the relationship between GRh2-induced apoptosis and PML-RARA degradation. Results: GRh2 dose- and time-dependently decreased NB4 cell viability. GRh2-induced apoptosis, cell cycle arrest, and caspase3, caspase8, and caspase9 activation in NB4 cells after a 12-hour treatment. GRh2-induced apoptosis in NB4 cells was accompanied by massive production of reactive oxygen species, mitochondrial damage and upregulated Bax/Bcl-2 expression. GRh2 also induced PML/PML-RARA degradation, PML nuclear bodies formation, and activation of the downstream p53 pathway in NB4 cells. Z-VAD-FMK inhibited caspase activation and significantly reversed GRh2-induced apoptosis and PML-RARA degradation. GRh2 also upregulated TNF-α expression and inhibited Akt phosphorylation. LY294002, an inhibitor of the PI3K pathway, enhanced the antitumor effects of GRh2, and C 87, an inhibitor of the TNF-α pathway, reversed NB4 cell viability, and GRh2-mediated apoptosis in a caspase-8-dependent manner. Conclusion: GRh2 induced caspase-dependent PML-RARA degradation and apoptosis in NB4 cells via the Akt/Bax/caspase9 and TNF-α/caspase8 pathways.

• Korean Journal of Clinical Laboratory Science
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• v.53 no.1
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• pp.81-87
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• 2021

The prevalence of type 2 diabetes mellitus (T2DM) is increasing worldwide. T2DM is one of the most common types of diabetes and is caused by increased insulin resistance and reduced insulin secretion. Peroxisome proliferator-activated receptor γ coactivator 1 alpha (PPARGC1A) is a master modulator of mitochondrial biogenesis and of gluconeogenesis in liver. In this study, we analyzed genetic polymorphisms of PPARGC1A gene in a middle-aged Korean population with T2DM. Using the genotype data of 736 T2DM cases and 4544 healthy controls obtained from the Korean Association Resource (KARE), we analyzed genetic correlations between single nucleotide polymorphisms (SNPs) of PPARGC1A and T2DM. Fifteen SNPs of PPARGC1A demonstrated a statistically significant association with T2DM. Of these, rs10212638 exhibited the strongest correlation with T2DM (P-value=0.015, OR=1.29, CI=1.05~1.59), and the minor G allele of PPARGC1A increased the risk of T2DM. This is the first study to report a significant association between genetic polymorphisms in PPARGC1A and T2DM and suggests that SNPs of PPARGC1A display genetic correlations to the etiology of T2DM.

• Korean Journal of Ichthyology
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• v.32 no.4
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• pp.199-209
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• 2020

In order to clarify the taxonomic status of the Korean Macroramphosus species, which were previously confused, we investigated morphological and molecular variations of Macroramphosus (18 individuals) from Korea, and Macroramphosus (35 individuals) from Japan and Taiwan, and compared with those of M. scolopax from type locality (Mediterranean Sea). Although the Korean and Japanese specimens of Macroramphosus were clearly divided into two types in the first dorsal spine length (22.8~32.1% in A-type vs. 15.6~21.4% in B-type), distance between the first dorsal fin and second dorsal fin (6.4~9.7% vs. 8.6~13.3%), and body depth (20.0~28.0% vs. 17.3~22.6%), no genetic differences among all individuals of longspine snipefish between them were found at the specific level [d=0.0~3.3% in control region (CR); 0.0~1.3% in cytochrome b (cytb); 0.0~0.5% in cytochrome c oxidase subunit I (COI)]. Whereas, they were well distinguished in genetics (9.9~11.5% in CR; 3.8~4.6% in cytb; 1.2~3.6% in COI) from those of M. scolopax in Mediterranean Sea. It needs the scientific name of the longspine snipefish (M. scolopax) in Korea be changed as M. japonicus (and/or M. sagifue). However, our results could not find evidence of consistency between morphological and mitochondrial DNA variations which suggests that their differentiation event may occur fairly recently. Further studies using more sensitive markers such as microsatellite are needed to clarify the degree of gene flow between the two types.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.9
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• pp.4517-4525
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• 2016

Background: Sperm DNA damage is underlying aetiology of poor implantation and pregnancy rates but also affects health of offspring and may also result in denovo mutations in germ line and post fertilization. This may result in complex diseases, polygenic disorders and childhood cancers. Childhood cancer like retinoblastoma (RB) is more prevalent in developing countries and the incidence of RB has increased more than three fold in India in the last decade. Recent studies have documented increased incidence of cancers in children born to fathers who consume alcohol in excess and tobacco or who were conceived by assisted conception. The aetiology of childhood cancer and increased disease burden in these children is lin ked to oxidative stress (OS) and oxidative DNA damage( ODD) in sperm of their fathers. Though several antioxidants are in use to combat oxidative stress, the effect of majority of these formulations on DNA is not known. Yoga and meditation cause significant decline in OS and ODD and aid in regulating OS levels such that reactive oxygen speues meditated signal transduction, gene expression and several other physiological functions are not disrupted. Thus, this study aimed to analyze sperm ODD as a possible etiological factor in childhood cancer and role of simple life style interventions like yoga and meditation in significantly decreasing seminal oxidative stress and oxidative DNA damage and thereby decreasing incidence of childhood cancers. Materials and Methods: A total of 131 fathers of children with RB (non-familial sporadic heritable) and 50 controls (fathers of healthy children) were recruited at a tertiary center in India. Sperm parameters as per WHO 2010 guidelines and reactive oxygen species (ROS), DNA fragmentation index (DFI), 8-hydroxy-2'-deoxy guanosine (8-OHdG) and telomere length were estimated at day 0, and after 3 and 6 months of intervention. We also examined the compliance with yoga and meditation practice and smoking status at each follow-up. Results: The seminal mean ROS levels (p<0.05), sperm DFI (p<0.001), 8-OHdG (p<0.01) levels were significantly higher in fathers of children with RB, as compared to controls and the relative mean telomere length in the sperm was shorter. Levels of ROS were significantly reduced in tobacco users (p<0.05) as well as in alcoholics (p<0.05) after intervention. DFI reduced significantly (p<0.05) after 6 months of yoga and meditation practice in all groups. The levels of oxidative DNA damage marker 8-OHdG were reduced significantly after 3 months (p<0.05) and 6 months (p<0.05) of practice. Conclusions: Our results suggest that OS and ODD DNA may contribute to the development of childhood cancer. This may be due to accumulation of oxidized mutagenic base 8OHdG, and elevated MDA levels which results in MDA dimers which are also mutagenic, aberrant methylation pattern, altered gene expression which affect cell proliferation and survival through activation of transcription factors. Increased mt DNA mutations and aberrant repair of mt and nuclear DNA due to highly truncatred DNA repair mechanisms all contribute to sperm genome hypermutability and persistant oxidative DNA damage. Oxidative stress is also associated with genome wide hypomethylation, telomere shortening and mitochondrial dysfunction leading to genome hypermutability and instability. To the best of our knowledge, this is the first study to report decline in OS and ODD and improvement in sperm DNA integrity following adoption of meditation and yoga based life style modification.This may reduce disease burden in next generation and reduce incidence of childhood cancers.

• Tuberculosis and Respiratory Diseases
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• v.41 no.3
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• pp.215-221
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• 1994

Background: It is well known that oxygen free radicals(OFR) play a vital role in the various type of acute lung injury. Among various antioxidant defense mechanisms, the superoxide dismutases(SOD) are thought to be the first line of antioxidant defense by catalyzing the dismutation of two superoxide radicals to yield hydrogen peroxide and oxygen. Eukaryotic cells contain two types of intracellular SOD : cytosolic, dimeric copper/zinc- containing enzyme(CuZnSOD) and mitochondrial, tetrameric manganese-containing enzyme(MnSOD). The purpose of this study is to evaluate the time-dependent gene expression of MnSOD and CuZnSOD in the endotoxin-treated rats, and to compare with the manifestations of LPS-induced acute lung injury in rats. Methods: Total RNA from rat lung was isolated using single step phenol extraction 0, 1, 2, 4, 6, 12, 18, 24 hours after E. coli endotoxin injection(n=3, respectively). RNA was separated by formaldehyde-containing 1.2% agarose gels elctrophoresis, transblotted, baked, prehybridized, and hybridized with $^{32}P$-labeled cDNA probes for rat MnSOD and CuZnSOD, which were kindly donated by Dr. Ho(Duke University, Durham, NC, USA). The probes were labeled by nick translation. Blots were washed and autoradiography were quantitated using laser densitometry. Equivalent amounts of total RNA/gel were assessed by monitoring 28S and 18S rRNA. Results: Endotoxin caused a rise in steady-state MnSOD mRNA levels by 4h with peak mRNA accumulation by 6h. Continued MnSOD mRNA expression was observed at 12h. CuZnSOD mRNA expression was observed from 1h to 24h with peak levels by 18h. Conclusion: These results suggest that SOD palys an important defensive role in the endotoxin-induced acute lung injury in rats.

• KSBB Journal
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• v.23 no.3
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• pp.231-238
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• 2008

The second family member of tissue inhibitors of matrix metalloproteinases, TIMP-2, is a 21kDa protein which inhibits matrix metalloproteinases 2 (MMP-2). Expression of mammalian proteins in E. coli often forms inclusion bodies that are made up of mis-folded or insoluble protein aggregates. The requirement for the formation of 6 disulfide bonds in the process of the TIMP-2 folding is likely to be incompatible with the reducing environment of E. coli. However, this incompatibility can be often overcome by introducing a mutagenesis that could lead to enhancement of the protein solubility. In this reason, we have attempted to express the soluble TIMP-2 in E. coli by applying a modified staggered extension process (StEP), one of the in vitro PCR-based recombinant mutagenesis methods, and error-prone PCR. C-terminally located CAT fusion protein with respect to mutated TIMP-2 proteins enables us to differentiate the soluble TIMP-2 from the insoluble in E. coli by virtue of chloramphenicol resistance. According to this scheme, E. coli harboring properly-folded CAT fused to TIMP-2 protein was selected, and some of the resulting colonies exhibited an enhanced, soluble expression of TIMP-2 compared to the wild type, implying (i) the StEP technique is successfully employed to enhance the proper folding thereby increasing the solubility of TIMP-2, and (ii) the CAT dependent screening may be a simple and effective method to differentiate the soluble protein expression in E. coli.