• Title/Summary/Keyword: Isozyme analysis

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Effect of Ethanol on the PKC Isozyme Activities in B103 Neuroblastoma Cells (에탄올이 신경아세포종 B103세포의 Protein Kinase C Isozyme 활성에 미치는 영향)

  • 조효정;정영진;진승하;오우균;김상원;강은정;박진규
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.33 no.2
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    • pp.262-270
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    • 2004
  • It is well known that long-term heavy ethanol intake causes alcoholic dementia, cerebellar degeneracy or Wernicke-Korsakoff syndrome and aggravates the conditions of many other neuro-psychotic disorders. Recently it is indicated that protein kinase C (PKC) plays an important role in the action of ethanol and in the neuro-adaptational mechanisms under chronic ethanol exposure. In order to investigate the effect of ethanol on PKC isoforms levels within the range of not showing any cytotoxicity, B103 neuroblastoma cell line trans-formed from murine central nervous system was employed and western blot analysis was carried out by using PKC isoform-specific antibodies. The changes of PKC-$\alpha$, ${\gamma}$, $\varepsilon$ and ζ level in the range of ethanol concentration 50∼200 mM were examined at the exposure time 1, 2, 8, 18 and 24 hrs in both cytosolic and membrane fraction. A typical ethanol concentration inducing the PKC isozymes was 100 mM, and the transforming time ranges of PKC isozymes could be considered as two different parts to each PKC isoform such as initial (0∼2 hrs) and prolonged (8∼24 hrs) stages. PKC-${\gamma}$ and PKC-$\varepsilon$ were clearly induced during the prolonged stages in cytosol at 18 hrs, and membrane fraction at 8 hrs and 18 hrs, respectively. On the other hand the PKC-$\alpha$ and PKC-ζ isozymes were largely induced in the prolonged stages at 18 hrs and 24 hrs, where the PKC-$\alpha$ isozyme was induced in both cytosol and membrane fractions at 200 mM ethanol concentration while the PKC-ζ isozyme was induced only in the membrane fractions at 100,200 mM. At 200 mM ethanol concentration of 24 hrs incubation in the prolonged stage, the PKC-$\alpha$ was maximally induced by 150% of the control values whereas the PKC-${\gamma}$ was significantly decreased to 47% of the control values. These results suggest that 100∼200 mM ethanol may modulate the signal transduction and neurotransmitter release in the central nervous system through the regulation of PKC isozymes, and the action of these isoforms may act differently each other in the cell.

Lactate Dehydrogenase and Monocarboxylate Transporters 1, 2, and 4 in Tissues of Micropterus salmoides (큰입우럭(Micropterus salmoides) 조직의 젖산탈수소효소 및 Monocarboxylate 수송체(MCT) 1, 2, 4)

  • Yum, Jung-Joo;Yeon, Jun-Hee
    • Journal of Life Science
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    • v.22 no.1
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    • pp.98-109
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    • 2012
  • The properties of lactate dehydrogenase (EC 1.1.1.27, LDH) and expression of monocarboxylate transporters (MCTs) 1, 2, and 4 were studied in tissues from Micropterus salmoides. Native-PAGE revealed that the LDH $A_4$ isozyme was predominantly located in skeletal muscle. The LDH $A_4$, $A_2B_2$, and $B_4$ isozymes were detected in heart, liver, eye, and brain tissues, while eye-specific $C_4$ isozyme was detected in eye tissue. In September, strong LDH $B_4$ isozyme activity was detected in heart tissue. High $A_4$ isozyme activity was noted in all other tissues except heart tissue. However, in November, strong $A_4$ isozyme activity was detected in heart tissue. The LDH/CS (Citrate synthase, EC 4.1.3.7) ratio in skeletal muscle and heart tissues indicated that anaerobic metabolism was high in those tissues. Native-PAGE after immunoprecipitation showed that eye-specific $C_4$ isozyme was more similar to the $A_4$ than the $B_4$ isozyme. The LDH $A_4$ isozyme was purified by affinity chromatography. The molecular weight of subunit A was 37,200. The LDH activity in tissues was consistently 11.05~28.32% due to inhibition by 10 mM pyruvate. The $K_m^{PYR}$ of LDH in eye tissue was very low. The optimum pH for LDH in tissues was pH 7.5~8.0. The LDH $A_4$ isozyme was detected in mitochondria of skeletal muscle, whereas the $B_4$ and $A_2B_2$ isozymes were detected in heart tissue mitochondria. Western blot analysis indicated that MCTs 1, 2, and 4 were located in the plasma membrane and mitochondria of skeletal muscle and heart tissues. The sizes of MCTs 1, 2, and 4 in skeletal muscle were 60, 54~38, and 63 kDa, while those in heart tissue were 57, 54~38, and 55.5 kDa, respectively. In conclusion, M. salmoides appears to use anaerobic metabolism predominantly when adapted to a hypoxic environment. In highly activated skeletal muscle and heart tissue, energy production is controlled by inward and outward flows of pyruvate and lactate through MCTs 1, 2, and 4 in the plasma membrane and mitochondria, with effective adjustment by LDH isozymes.

Identification of Different Species and Dultivars of Brassica by SDS-PAGE, Isozyme and Molecular Marker

  • Mukhlesur Rahman Md.;Hirata Yutaka
    • Journal of Plant Biotechnology
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    • v.7 no.1
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    • pp.27-35
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    • 2005
  • Eighty-five different cultivars of Brassica rapa, B. juncea, B. nap us, B. carinata, B. oleracea and hexaploid Brassica collected from Bangladesh, Japan, China and Denmark were analyzed by SDS-PAGE for seed and leaf protein variations, using esterase, acid phosphatase and peroxidase isozyme analysis. Ten polymorphic bands were identified from seed protein however no identifiable polymorphic band was found in the leaf protein. Polymorphic markers clearly distinguished the different Brassica species as well as yellow sarson (YS) and brown seeded (BS) cultivars of B. rapa. The $F_1$ cross between YS and brown seeded cultivars showed the existance of all poly-morphic bands of the respective parents. The Bangla-deshi and Japanese cultivars of B. rapa differed in the amount of seed protein. In the case of isozyme analysis, esterase showed the highest number of polymorphic bands (13) followed by acid phosphatase (9) and peroxidase (5). These polymorphic markers were very effec-tive for classification of all the species studied in this experiment. In parentage tests using isozymes, the hybridity of intra-and-interspecific crosses of almost all the seedlings could be identified from their respective cross combinations. Esterase polymorphism showed a clear differentiation between YS and BS types of B. rapa. In addition, two esterase polymorphic markers were iden ified to differentiate some cultivars of B. juncea. Segregation patterns in these two esterase bands showed a simple Mendelian monohybrid ratio of 3:1 in $F_2$, 1:1 in test cross and 1:0 in back cross progenies. No polymorphic band was identified to distinguish different cultivars of the same species by acid phosphatase or peroxidase. Polymerase Chain Reaction (PCR) was carried out with seed coat color specific marker of B. juncea. The yellow seeded cultivars produced a strong band at 0.5 kb and weak band 1.2 kb. In the addition of these two specific bands, Japanese yellow-seeded cultivars expressed two more weak bands at 1.0 kb and 1.1 kb. Where the brown seeded cultivars generated a single strong band at 1.1 kb. In segregating population, the yellow seed coat color marker segregated at a ratio 15 (brown) : 1 (yellow), indicating the digenic inheritance pattern of the trait.

Comparative studies on the Hanabusaya asiatica and its allied groups 2. Ultrastructure of epidermis, Palynological characters and Isoyme pat (금강초롱꽃 (Hanabusaya asiatica) 과 근연분류군의 비교연구 2. 표피의 미세구조, 화분학적 형질 및 동위원소 분석)

  • 유기억
    • Korean Journal of Plant Resources
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    • v.8 no.3
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    • pp.303-318
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    • 1995
  • Microscopic structures of epidermis and palynology and isozyme analysis were examined to find out the intergeneric and interspecific relationships and consequently to confirm the position of Hanabusaya aiatica as an endemic genus among 4 genera and 9 species belonging to the Hanabusaya asiatica, Korean endemic, and its allied groups. In the examination of microscopic structures of epidermis, cell patterns of perianth and ultrastructure of seed coat were found to be useful characters for the identification of the 4 genera and 9 species. Palynological characters such as microscopic structure and overall morphology of pollen grains were, however, not enough to distinguish them because of the great variabilities in these traits. Isozyme analysis showed that H. asiatica was very closly related to Campanula punctata and C. takesimana, though there were variations among populations and collected areas in some classified groups, depending on classified groups. Based on these results, the position of H. asiatica as an endemic genus was well confirmed.

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Effects of Organophosphorous Insecticides on Mice Cholinesterase Activity (유기린계 살충제가 흰쥐의 Cholinesterase활성에 미치는 영향)

  • 윤수홍;김신희;박병윤
    • Environmental Analysis Health and Toxicology
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    • v.2 no.1_2
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    • pp.17-24
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    • 1987
  • Changes in cholinesterase (ChE) activity, electrophoretic pattern of ChE and histopathologic state on the mice serum, brain and liver by administration of organophos-phorous insecticides were studied. The mice ChE activities on serum, brain and liver decreased by increasing of concentration and time both administration of malathion and DDVP, whereas on serum and brain the activities of the 7 days after administration decreased, and then presented the gradually slight recovery in course of time. The ChE on serum and liver showed many isozyme bands by polyacrylamide gel electrophoresis but several on brain. And isozyme bands disappeared and diffused by administration of organophosphorous insecticides and development of time. The mice liver with administration group of malathion on histopathologic test showed midzonal necrosis between central vein and portal area, and with administration group of DDVP mainly presented portal necrosis on location of potal area.

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Analysis of the Isozyme Loci of the Beet Armyworm, Spodoptera exigua(H bner) (파밤나방(Spodoptera exigua(H bner))의 동위효소 유전좌위 분석)

  • 김용균;김경성
    • Korean journal of applied entomology
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    • v.37 no.1
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    • pp.19-22
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    • 1998
  • Number of loci, allele frequencies, and subunit structures of 17 kinds of isozymes were analyzed in a laboratory strain of the beet armyworm, Spodoptera exigua (Hubner) to get genetic markers. These isozymes had 30 loci with 21 polymorphic (70.0% polymorphism); effective number of alleles per locus, average heterozygosity (H,), and inbreeding coefficient (F) were 2.52, 32.8%, and 2 1.0%, respectively.

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Genetic Variations of Natural and Hatchery Populations of Korean Ayu (Plecoglossus altivelis) by Isozyme Markers

  • Han, Hyon-Sob;Jin, Deuk-Hee;Lee, Jong-Kwan
    • Journal of Aquaculture
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    • v.16 no.2
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    • pp.69-75
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    • 2003
  • Genetic variability and population structure of 11 natural ayu, Plecoglossus altivelis populations and one hatchery stock were assessed by starch gel electrophoretic analysis with 10 enzyme coding loci. Three loci were polymorphic (lower than 0.95 in major allele frequency) in natural populations,2 loci in hatchery stock. The average number of alleles per locus was 1.38. Observed heterozygosities ranged from 0.0235 to 0.088 (0.055 on the average) in natural population while 0.0925 in hatchery stock. The genetic distance among natural populations measured 0.000047-0.005407 and no significant differentiation was observed among them. On the other hand, a signifcant genetic distance was found between natural populations and the hatchery stock with measuring 0.002032-0.O08605. The results in this study suggest that the hatchery stock has diverged from natural populations, and also that careful to maintain sustainable and effective population size (parents number) should be made.

Somatic hybridization between Nicotiana rustica and N. tabacum through protoplast fusion (Nicotiana tabacum과 N. rustica 체세포 잡종식물의 육성)

  • Choe, Sang-Ju;Lee, S. C.;Hong, B. H.
    • Journal of the Korean Society of Tobacco Science
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    • v.15 no.2
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    • pp.123-129
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    • 1993
  • Mesophyll protoplasts derived from young leaves of Nicotiana rustica and N. tabacum cv Burley 21 were fused with the aid of polyethylene glycol(PEG). Cytological examination of protoplasts after PEG treatment revealed 12.8 % heterokaryocytes. After 7 weeks culture, the hybrid calli showing greenish white with a compact appearance were selected in contrast to parental type calli tinged with white or green color. The somatic hybrid plants were verified by morphological, biochemical and cyclological analysis. A heterosis effect for plant vigor and height was observed but the shape of leaves and flower characteristics were intermediate between N. tabacum and N. rutstica. The isozyme banding patterns for peroxidase of somatic hybrid lines were compared with the parent species. A number of isozyme bands derived from both parental species were found in the hybrids. Somatic hybrid plants have been successfully backcrossed to the parental N. tabacum particularly with somatic hybrid plants as female parents. These hybrid plants yielded small seeds, only few which were germinable.

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Identification of hybride from intra- and interspecific protoplast fusion in trichoderma by electrophoretic patterns of enzymes (효소의 전기영동에 의한 trichoderma속 균의 종내, 종간 잡종의 동정)

  • 민경렴;박희문;하영칠
    • Korean Journal of Microbiology
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    • v.27 no.1
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    • pp.27-34
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    • 1989
  • In order to evaluate the applicability of enzyme electrophoresis for the identification of intra/interspecific hybride obtained by the protoplast fusion in Trichoderma, soluble proteins, intracellular soluble enzymes and extracellular $\beta$-glucosidase were analyzed by polyacrylamide gel electrophorsis. As the results, patterns of soluble protein, and isozyme patterns of peroxidase, malate dehydrogenase, and $\beta$-glucosidase in hydrids were defferent from those in parental and wild type strains. Therefore, it was established that the analysis of protein pattern by electrophoresis could be applied to isolate and identify the hybrids from the protoplast fusion.

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