• Journal of Life Science
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• v.14 no.2
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• pp.315-319
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• 2004

For antibody development of human serine palmitoyltransferase (SPT, EC 2.3.1.50), SPTLC1 and SPTLC2 genes were subcloned in pRset vector and expressed in E. coli BL21 (DE3)pLys cells. Eucaryotic SPT is a membrane-bound heterodimer enzyme, while all other members are soluble homodimer enzymes. cDNA library were obtained from total RNA from human embryo kidney cell line, HEK293, using RT-PCR and PCR with specific primers was carried out for preparing SPTLC1 and SPTLC2 genes. pRset vector which can express hexahistidine-tag fusion protein was used and the DNA sequences of pRsetB/SPTLC1 and pRsetA/SPTLC2 were confirmed. Recombinant BL21 cells with SPTLC subunits were selected with LB plate containing ampicillin and chroramphenicol. SPTLC1 and SPTLC2 proteins were induced with 1 mM IPTG and seperated on 10% SDS-PAGE gel. Expressed proteins were confirmed by western blotting with His-tag antibody.

• Genomics & Informatics
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• v.6 no.3
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• pp.110-116
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• 2008

Blood pressure refers to the force exerted by circulating blood on the walls of blood vessels, and chronical elevation of blood pressure is known as hypertension. Although hypertension is affected by genetic and environmental factors, the genetic background of hypertension is not fully understood. One of the candidate genetic factors, Prostaglandin-endoperoxide synthase 2 (PTGS2), is a membrane-bound enzyme, catalyzing the conversion of arachidonic acid to prostaglandin, and recently SNPs of PTGS2 gene was associated with hypertension in Japanese population. Therefore the association of PTGS2 polymorphisms was investigated with blood pressure in healthy Korean subjects, 470 unrelated individuals randomly selected from Ansung and Ansan cohorts. The 25 SNPs of PTGS2 gene were identified by the sequencing analysis of 24 Korean samples. Among identified polymorphisms, three SNPs (rs689466, -1329A>G; rs5275, +6365T>C; rs4648308, +8806G> A) were selected for further association analysis, and rs689466 located in promoter region was associated with blood pressure as well as triglyceride level in the blood. By in silico analysis, rs689466 locates in v-Myb transcription factor binding site, and the v-Myb site disappears when the SNP is changed from A to G nucleotide. Individuals with A/G and G/G genotype in rs689466 have higher blood pressure than those with A/A genotype, and the regression p-value is 0.008 for systolic and 0.004 for diastolic blood pressure. In summary, the PTGS2 polymorphism (rs689466) is associated with blood pressure in Asian populations based on this and Japanese studies, shedding light on it as a genetic risk marker of hypertension.

• Biomedical Science Letters
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• v.15 no.1
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• pp.37-45
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• 2009

Insect cell cultures have become important tools in the production of biological substances for use in a variety of research, human and veterinary medicine, and pest control applications. These applications often require the introduction of foreign DNA into the cells and have generally used methods originally developed for use with human and other mammalian cell cultures. While these methods can be successfully employed, they are often less efficient with insect cells and frequently involve complex procedures or require specialized equipment. Even when they do work, they may require substantial modification because of differences in the culture medium or growth patterns of insect cells. In this study, We have optimized transfection conditions of Sf9 cell line using insect expression vector pIZT/V5-His which expresses green fluorescent protein effectively. Human stem cell factor (hSCF) is a glycoprotein that plays a key role in hematopoiesis acting both as a positive and negative regulator, often in synergy with other cytokines. It also plays a key role in mast cell development, gametogenesis, and melanogenesis. It can exist in membrane-bound form and in proteolytically released soluble form. As determined by an enzyme-linked immunosorbent assay performed, hSCF level in supernatant averaged 995ng/ml. The human hSCF was partially purified by immunoaffinity chromatography and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The results show that the hSCF has N-linked carbohydrate and corresponds to the soluble form, at or about 223 amino acids in length. The findings suggest functional importance for soluble hSCF in cells.

• Macromolecular Research
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• v.12 no.1
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• pp.46-52
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• 2004

In a continuation of our previous studies on blood compatibility profiles of anion-substituted poly(vinyl alcohol) (PVA) membranes, in which hydroxyl groups have been replaced with carboxymethyl (C-PVA) and sulfonyl groups (S-PVA), we have studied the activation of complement components and the changes in white cell and platelet count in vitro and compared them with those of unmodified PVA, Cuprophane, and low-density polyethylene. Complement activation of fluid phase components, C3a, Bb, iC3b, and SC5b-9, and of bound phases, C3c, C3d, and SC5b-9, were assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblot, respectively. The changes in the number of white cells and platelets following complement activation were counted using a Coulter counter. C-PVA and S-PVA activated C3 to a lesser extent than did PVA, which we attribute to the diminished level of surface nucleophiles of the samples. In addition, C- and S-PVA exhibit increased inhibition of Bb production, resulting in a decrease in the extent of C5 activation. Consequently, because of the reduced activation of C3 and C5, C- and S-PVA samples cause marked decreases in the SC5b-9 levels in plasma. We also found that the negatively charged sulfonate and carboxylate groups of the samples cause a greater extent of adsorbtion of the positively charged anaphylatoxins, C3a and C5a, because of strong electrostatic attraction, which in turn provides an inhibition of chemotaxis and activation of leukocytes. The ability to inhibit complement production, together with the binding ability of anaphylatoxins of the C- and S-PVA samples, leads to a prominent decrease in lysis of leukocytes as well as activation of platelets.

• Journal of Nutrition and Health
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• v.26 no.5
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• pp.532-546
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• 1993

This study was done to investigate the effects of different dietary oils on hepatic mitochondrial lipid compositon, adenine nucleotide translocase(AdNT) and ATPase activities in carcinogen treated rats. Sixty male Sprague-Dawley rats, weighing 50∼60g, were fed three different types of dietary oil, beef tallow(BT), corn oil(CO) and sardine oil(SO) at 15% by weight for 14 weeks. Three weeks after feeding rats were intraperitoneally injected with a single dose of diethylnitrosamine(200mg/Kg BW). After five weeks rate fed 0.02% acetylaminofluorene contating diet for 6 weeks, and after seven weeks 0.05% phenobarbital containing diet for 7 weeks. At 14th week, rats were sacrificed and hepatic mitochondrial lipid composition, AdNT and ATPase activities were determined. Percent liver weight per body weight was significantly by carcinogen treatment. Analysis of mitochondrial lipid composition showed that body cholesterol and phospholipid contents were not affected by dietary oils but significantly increased by carcinogen treatment. Individual phospholipid composition as well as phosphatidyl ethanolamine/phosphatidyl choline ratio were altered by either dietary oils or carcinogen treatment. Fatty acid composition was changed by dietary oils but not much by carcinogen treatment. AdNT activity was affected by dietary oils in only carcinogen treated groups. ATPase activity was affected by dietary oils in only carcinogen nontreated groups. These data indicate that both dietary oils and caricinogen treatment can change mitochondrial lipid composition and thereby change AdNT and ATPase activities. Particularly effects of carcinogen treatment on cholesterol/phopholipid ratio, phospholipid compositon and ATPase activity were different among dietary oil groups. Therefore it is suggested that different dietary oils can somewhat modulate the changes of mitochnodrial lipid composition and membrane bound enzyme activites during hepatocarcinogenesis.

• Journal of Life Science
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• v.27 no.7
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• pp.849-856
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• 2017

The ${\beta}$-D-fructofuranosidase (EC 3.2.1.26) is an important enzyme from a historical point of view, discovered by French biologist Berthelot in 1860 and was first used to study enzymology. ${\beta}$-D-fructosfuranosidase catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Four biochemical subgroups of ${\beta}$-D-fructofuranosidase have been investigated in plants. There are vacuolar (soluble acid), cytoplasmic (soluble alkaline), membrane-bound (insoluble alkaline), and cell wall-bound (insoluble acid) ${\beta}$-D-fructofuranosidase by purification. Their biochemical characteristics are distinct. It suggested that those enzymes might be different gene products. The contribution of each of these enzymes to sucrose management in the plant is likely to be correlated with their localization. Common localization in developing cells in tissues from a range of developmental stages and plant parts suggests that all of the isoforms may be closely involved in nutrient transport. The ${\beta}$-D-fructofuranosidases were most commonly found associated with maturing tissues in developing fruits, leaves, and roots. The ${\beta}$-D-fructofuranosidase activity varies in the relationship between growth and expansion through cell division, development of storage organs and tissues, and the relationship of plant defense responses. It is necessary to summarize more researches in order to know the definite physiological function.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.412-420
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• 1989

To know how the ribosomes involved in secretory protein synthesis were attached to the cytoplasmic membrane in Bacillus amyloliquefaciens, the cells were treated with puromycin combinated with magnesium at the logarithmic phase, and the variation of cell-bound and extracellular $\alpha$-amylase activity was assayed for determining the $\alpha$-amylase translocation blocking through the cytoplasmic membrane. In the abnormal $\alpha$-amylase producing mutant in which the C-terminal of the $\alpha$-amylase structure was deleted, B. umytotiquefaciens CH10-2, the $\alpha$-amylase was translocated normally through the cytoplasmic membranes, and the translocation blocking by puromycin was revealed to have a similar pattern as that in the wild type. This means that the C-terminal part of the enzyme structure may not have a signal for secretion. The cell death of the logarithmic phase cells in both strains was not affected much under 20$\mu\textrm{g}$/$m\ell$ of puromycin, however, the $\alpha$-amylase translocation was blocked markedly under less than 10$\mu\textrm{g}$/$m\ell$ of the puromycin concentration. The blocking of the enzyme secretion by puromycin may be due to the detachment of the ribosomes from cytoplasmic membranes by disturbing the nascent polypeptide synthesis. Further evidence for confirming this was that the detachment was increased in 50 mM of magnesium ion because the extracellular $\alpha$-amylase activity was decreased more under this condition. If the cells were treated with trypsin combinated with Iysozyme, the extracellular $\alpha$-amylase activity from the cultured medium was reduced markedly, however, the activity from the cells treated with trypsin only was not reduced. This means that the nascent polypeptides protruding from the cytoplasmic membrane were sensitive to the trypsin digestion, whereas the matured ones were not. Therefore, the protruding polypeptides from the cytoplasmic membranes may be truncated by trypsin before forming their final tertiary structures by folding in the cell wall layer.

• Applied Biological Chemistry
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• v.30 no.4
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• pp.357-363
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• 1987

The transition temperature of respiratory activity of mitochondria isolated from panicles of rice plant was measured with 41 varieties bred in Korea, which had been agronomically classified into five groups based on their relative cold-tolerance; S (strong cold-tolerance), S-M (moderate to strong cold-tolerance), M (moderate cold-tolerance), M-W (moderate to weak cold-tolerance) and W (weak cold-tolerance). The temperature was found to be $11.5{\sim}13.5^{\circ}C$ for group S, $13.5{\sim}16^{\circ}C$ for group S-M, $16{\sim}17^{\circ}C$ for group M, $17{\sim}18^{\circ}C$ for group M-W, and ${\geq}18^{\circ}C$ for group W respectively with a few exceptions of minor deviation, demonstrating a close correlation between the transition temperature and the chilling susceptivity of a variety. This observation supports the suggestion that the transition temperature of respiratory activity of panicles be adopted as a reliable index for grading a rice variety as to its overall cold tolerance. Also studied is on the methodology for measurement of the transition temperature in the aspect of analytical biochemistry. In addition to the usual polarographic method, a spectrophotometric method which is based on the enzymatic assay of membrane-bound cytochrome C oxidase was examined. This enzyme was found previousely and confirmed again in the present work to be a key factor governing the respiratory trantion at least in plant mitochondria. The optical method was turned out to be very successful, although it was a little more complicate and time-consuming than the electrochemical method.

• Journal of Life Science
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• v.33 no.8
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• pp.651-662
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• 2023

Peroxisomes, known as microbodies, are a class of morphologically similar subcellular organelles commonly found in most eukaryotic cells. They are 0.2~1.8 ㎛ in diameter and are bound by a single membrane. The matrix is usually finely granular, but occasionally crystalline or fibrillary inclusions are observed. They characteristically contain hydrogen peroxide (H₂O₂) generating oxidases and contain the enzyme catalase, thus confining the metabolism of the poisonous H₂O₂ within these organelles. Therefore, the eukaryotic organelles are greatly dynamic both in morphology and metabolism. Plant peroxisomes, in particular, are associated with numerous metabolic processes, including β-oxidation, the glyoxylate cycle and photorespiration. Furthermore, plant peroxisomes are involved in development, along with responses to stresses such as the synthesis of important phytohormones of auxins, salicylic acid and jasmonic acids. In the past few decades substantial progress has been made in the study of peroxisome biogenesis in eukaryotic organisms, mainly in animals and yeasts. Advancement of sophisticated techniques in molecular biology and widening of the range of genomic applications have led to the identification of most peroxisomal genes and proteins (peroxins, PEXs). Furthermore, recent applications of proteome study have produced fundamental information on biogenesis in plant peroxisomes, together with improving our understanding of peroxisomal protein targeting, regulation, and degradation. Nonetheless, despite this progress in peroxisome development, much remains to be explained about how peroxisomes originate from the endoplasmic reticulum (ER), then assemble and divide. Peroxisomes perform dynamic roles in many phases of plant development, and in this review, we focus on the latest progress in furthering our understanding of plant peroxisome functions, biogenesis, and dynamics.

• Applied Microscopy
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• v.7 no.1
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• pp.21-43
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• 1977

This experiment was undertaken in order to localize the labeled dbcAMP (dibutyryl cyclic AMP) in oocytes whose development has been suppressed by cold dbcAMP for 6 or 19 hours in vitro. Mouse oocytes were obtained from the ovaries of 3-4 week old A strain female mice, by puncturing the Graafian follicles in the modified Krebs-Ringer bicarbonate salt solution under the dissecting microscope. Those oocytes which have intact germinal vesicle were cultured in the basic culture medium supplemented with 0.4% bovine serum albumin (BSA). Cultivation of the oocytes was carried out in a microtube developed by Cho (1974). The cultures were then incubated in a humidified 5% $CO_2$ incubator maintained at $37^{\circ}C$ for 6 or 19 hours (Donahue, 1968). DbcAMP was added to culture medium for a final concentration of 100ug/ml, and $^3H-dbc$ AMP (specific activity 13 Ci/mM) for a final concentration of $40{\mu}Ci/ml$ was also added to the medium. For electron microscopic autoradiography, those oocytes recovered from the culture were washed with phosphate buffer (pH 7.4), and immediately prefixed in a 2.5% glutaraldehyde overnight and postfixed for 2 hours at $4 ^{\circ}C$ in 1% osmium tetroxide in phosphate buffer with pH 7.4 (Palade, 1952). After fixation, the materials were dehydrated in graded alcohol series and embedded in Epon 812 mixture based on the standard procedures (Luft, 1961). The thin sections $600-700{\AA}$ thick were mounted on the grids of 200 meshes. The grids containing sections were coated with a nuclear emulsion Kodak NTB-3 and stored in a cold dark box (at $4^{\circ}C$) for 3 weeks. After exposure, the samples were developed with Kodak D-19 and stained with uranyl acetate and lead citrate. Routine observation was made with Hitachi HU-11E electron microsocope. The results of the observation were as followings: 1. It was found that the labeled dbcAMP penetrated the egg plasma membrane and dispersed at random in the cytoplasm. 2. It was also observed that most of the labeled dbcAMP was attached to microfibrillar lattices portion of the oocyte cytoplasm. There fore, it is presumed that the receptor of the dbcAMP is localized in the microfibrillar lattices of the oocyte. 3. It also seems that some other cell organells such as mitochondria, Golgi complex, cortical granules are not directly related to the action of the dbcAMP. 4. The labeled dbcAMP was neither observed in the membrane nor in the nucleus. Therefore, it seems that there is no relationship between the concentration of dbcAMP and the nuclear membranous permeability. 5. There was no difference in number of dbcAMP particles when oocytes were cultured for 6 hours and 19 hours. 6. However, it was observed that, in same of the oocytes suppressed in germinal vesicle by dbcAMP for 19 hours, cell organells were moved and concentrated to a small portion of the cytoplasm, and that the morphology of the organells greatly changed to an abnormal. form. Therefore, it is supposed that those oocytes were in the process of degeneration. From the above results, it is expected that dbcAMP penetrated the egg membrane and was bound to the receptor which seems to be located in the microfibrillar lattiees portion, and that this dbcAMP-receptor complex inhibited some enzyme system of the oocytes which are essential for the germinal vesicle breakdown.