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Detection of Mitotic Centromere-Associated Kinesin (MCAK) During Cell-Cycle Progression of Human Jurkat T Cells Using Polyclonal Antibody Raised Against Its N- Terminal Region Overexpressed in E. coli

  • Jun, Do-Youn;Rue, Seok-Woo;Kim, Byung-Woo;Kim, Young-Ho
    • Journal of Microbiology and Biotechnology
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    • v.13 no.6
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    • pp.912-918
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    • 2003
  • Mitotic centromere-associated kinesin (MCAK), which is a novel kinesin with a central motor domain, is believed to playa role in mitotic segregation of chromosome during the M phase of the cell cycle. In the present study, it is shown that a rabbit polyclonal antibody has been produced using the N-terminal region (187 aa) of human MCAK expressed in E. coli as the antigen. To express the N-terminal region in E. coli, the MCAK cDNA fragment encoding N-terminal 187 aa was obtained by PCR and was then inserted into the pET 3d expression vector. Molecular mass of the N-terminal region overexpressed in the presence of IPTG was 23.2 kDa on SDS-PAGE, and the protein was insoluble and mainly localized in the inclusion body that could be easily purified from the other cellular proteins. The N-terminal region was purified by electro-elution from the gel after the inclusion body was resolved on the SDS-PAGE. The antiserum obtained after tertiary immunization with the purified protein specifically recognized HsMCAK when subjected to Western blot analysis, and showed a fluctuation of the protein level during the cell cycle of human Jurkat T cells. Synchronization of the cell-cycle progression required for recovery of cells at a specific stage of the cell cycle was performed by either hydroxyurea or nocadazole, and subsequent release from each blocking at 2, 4, and 7 h. Northern and Western analyses revealed that both mRNA and protein of HsMCAK reached a maximum level in the S phase and declined to a basal level in the G1 phase. These results indicate that a polyclonal antibody raised against the N-terminal region (187 aa) of HsMCAK, overexpressed in E. coli, specifically detects HsMCAK (81 kDa), and it can analyze the differential expression of HsMCAK protein during the cell cycle.

Effect of Calving Season on Growth Performance, Feed Intake and Disease Occurrence of Hanwoo Calves (한우 송아지의 분만계절이 성장, 사료섭취량 및 질병 발생에 미치는 영향)

  • Kwon, E.G.;Cho, Y.M.;Park, B.K.;Choi, C.W.;Kim, Y.G.;Paek, B.H.
    • Journal of Animal Science and Technology
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    • v.49 no.1
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    • pp.59-66
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    • 2007
  • Sixty-seven Hanwoo calves were allocated into two groups, 27 spring calves (SC) and 40 fall calves (FC) to investigate the effects of calving season on water and nutrient intakes, growth performance and disease occurrence for the nursing and the post-weaning period. Mean DMI of 3 to 4 mo old FC was lower than that of SC due to low temperature in winter for FC. Water intake was higher for FC (birth to 1 mo old) but lower for SC (2 to 4 mo old) than the others. Mean BW did not differ between SC and FC, but average daily gain for over 2 mo old SC were significantly higher than FC. Diarrhea was mainly observed when the calves were before 60 d old; in particular, it occurred more frequently for less than 30 d old FC compared with SC. The present results indicate that calving season may affect water intake, feed intake and growth performance of Hanwoo calves.

Biological Activities of Chungkugjang Prepared with Black Bean and Changes in Phytoestrogen Content during Fermentation (검정콩 청국장의 생리활성 및 발효중 Phytoestrogen 함량의 변화)

  • Shon, Mi-Yae;Seo, Kwon-Il;Lee, Sang-Won;Choi, Seong-Hee;Sung, Nak-Ju
    • Korean Journal of Food Science and Technology
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    • v.32 no.4
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    • pp.936-941
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    • 2000
  • To assess functional properties of chungkugjang (CK), a traditional Korean soyfood, fermented with black beans at $42^{\circ}C$ for 72 hrs, some biological activities of methanol extract of CK and the changes in phytoesterogen (daidzein, genistein) contents during fermentation were investigated. The methanol extract of CK (MEC) prepared with soybean had no or a little antibacterial activity. MEC prepared with black bean inhibited the growth of all bacteria tested, and MEC of small black bean was higher in antibacterial activity than that of large black bean. Hydrogen-donating activities of MEC of large and small black bean were 76.4 and 75.5%, respectively, which were higher than that of soybean being 67.3%. Nitrite-scavenging activity was found to be above 90% in all MECs tested as compared with control group. MEC showed strong antioxidant activities against both peroxidation of linoleic acid and $H_{2}O_{2}-FeSO_{4}-induced$ peroxidation of rat liver homogenate. The antioxidant activities were high in the order of small black bean, large black bean and soybean. Contents of genistein and daidzein were gradually increased during fermentation of CK. The isoflavones were higher in black bean CK than in soybean CK and higher in large black bean CK than in small black bean CK. In black beans genistein content was about twice as much of daidzein.

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Development of Protocol for the Effective Detection of Feline Calicivirus as Norovirus Surrogate in Oyster and Lettuce (굴과 상추에서 노로바이러스의 대체모델 feline calicivirus의 효율적 검출법 개발)

  • Lee, Soo-Yeon;Jang, Keum-Il;Woo, Gun-Jo;Kwak, Hyo-Sun;Kim, Kwang-Yup
    • Korean Journal of Food Science and Technology
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    • v.39 no.1
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    • pp.71-76
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    • 2007
  • Foodborne illness caused by Noroviruses (NVs) is increasing rapidly in Korea. This study developed an effective detection protocol for NVs found in contaminated oysters and lettuce through an investigation using the major steps of virus particle separation, concentration and RT-PCR. As a surrogate model for NVs, the cultivable feline calicivirus (FCV) that belongs to the same Caliciviridae family was used. Instead of using a time-consuming ultracentrifugation method, efficient methods based on solvent extraction and PEG precipitation procedure were applied. Direct homogenization of a 25g sample of whole oyster and lettuce in 175mL PBS provided the simplicity that would be needed in the actual field of food product examination. The overnight PEG precipitation step at $4^{\circ}C$ was reduced to 3 h by placing the reaction tube in ice and by adjusting the PEG concentrations. The application of the use of chloroform and 0.2 ${\mu}m$ syringe filtration together showed a better detection efficiency than the use of chloroform alone in removing PCR inhibitors for both oyster and lettuce samples. Also, dilution of the extracted RNA solution before PCR provided increased sensitivity. The improved detection protocol developed in this study could be efficiently applied to detect FCV and most likely NVs from oysters and lettuce.

Characterization of Phytase from Bacillus coagulans IDCC 1201 (Bacillus coagulans IDCC 1201이 생산하는 Phytase의 특성)

  • Lee Seung-Hun;Kwon Hyuk-Sang;Koo Kyo-Tan;Kang Byung-Hwa;Kim Tae-Yong
    • Microbiology and Biotechnology Letters
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    • v.34 no.1
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    • pp.28-34
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    • 2006
  • A native extracellular acid phosphatase, phytase (EC 3.1.3.8), from Bacillus coagulans IDCC 1201 (commercially known as Lactobacillus sporogenes) used as probiotics, was characterized. Though some strains of B. coagulans have been evaluated with regard to several health-promoting effects, it has not been reported to produce phytase. Partially purified phytase front the strain IDCC 1201 had a pH optimum of 4.0 and a temperature optimum of $50^{\circ}C$, respectively. The requirement for divalent cations was studied and cobalt ion remarkably increased the enzyme activity. The removal of metal ions from the enzyme by EDTA decreased activity below 50%. The enzyme activity depleted restored when the assay was performed in the presence of $Co^{2+}$. Also, $Co^{2+}$ is the most active stimulator and has unique activation effect at high temperature. The phytase was specific for sodium phytate and p-nitrophenylphosphate, which is different from other known Bacilli phytases. The putative amino acid sequences of the phytase from B. coagulans IDCC 1201 were very similar to that of the phytase from B. subtilis strain 168. Based on these data, we concluded that the phytase from B. coagulans IDCC 1201 is a $Co^{2+}$-dependent acid phosphatase. Therefore, the strain B. coagulans IDCC 1201 is thought to be a valuable addititive for livestocks as well as a beneficial probiotics for human.

Comparison of the Effect of Green Tea By-product and Green Tea Probiotics on the Growth Performance, Meat Quality, and Immune Response of Finishing Pigs

  • Ko, S.Y.;Bae, I.H.;Yee, S.T.;Lee, S.S.;Uuganbayar, D.;Oh, J.I.;Yang, C.J.
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.10
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    • pp.1486-1494
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    • 2008
  • The objective of this experiment was to compare the effects of green tea by-product and green tea probiotics on the growth performance, meat quality and immune response of finishing pigs. A total of 72 crossbred "Landrace$\times$Yorkshire" finishing pigs with an average of 76 kg body weight were assigned to 4 dietary treatments in a completely randomized design. Each treatment had 3 replications with 6 pigs per replication. The four dietary treatments were control, antibiotics (control diet with 0.003% chlortetracycline added), and diets containing 0.5% green tea by-product or 0.5% green tea probiotic supplementation. Weight gain was increased in 0.5% green tea probiotics treatment compared to others, but there was no significant difference (p>0.05). The incorporation of 0.5% green tea probiotics to diets reduced the feed conversion ratio in finishing pigs (p>0.05). The incorporation of 0.5% green tea by-product into the pig diet reduced the crude protein and fat contents of the meat (p>0.05). Pigs fed diets containing 0.5% green tea probiotic supplementation had lowered meat TBA values compared to those fed 0.5% green tea by-product (p<0.05). The proliferation of spleen cells stimulated with Con A (concanavalin: 0.1, 0.3, and $1.0{\mu}g/ml$) significantly increased with 0.5% green tea by-product treatment compared to antibiotic treatment (p<0.05), but was significantly decreased in 0.5% green tea probiotics treatment compared to the antibiotic treatment (p<0.05). When stimulated with $1.0{\mu}g/ml$ Con A, splenocyte production of IL-6 from pigs treated with 0.5% green tea by-product or green tea probiotics was significantly increased compared to the antibiotic treatment group (p<0.05). Splenocyte production of TNF-${\alpha}$ after treatment with $1.0{\mu}g/ml$ Con A was significantly higher following 0.5% green tea probiotics treatment (p<0.05), while TNF-${\alpha}$ production after $10.0{\mu}g/ml$ LPS (lipopolysaccharide) was significantly higher in the 0.5% antibiotic treatment group (p<0.05).

Fimasartan attenuates renal ischemia-reperfusion injury by modulating inflammation-related apoptosis

  • Cho, Jang-Hee;Choi, Soon-Youn;Ryu, Hye-Myung;Oh, Eun-Joo;Yook, Ju-Min;Ahn, Ji-Sun;Jung, Hee-Yeon;Choi, Ji-Young;Park, Sun-Hee;Kim, Chan-Duck;Kim, Yong-Lim
    • The Korean Journal of Physiology and Pharmacology
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    • v.22 no.6
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    • pp.661-670
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    • 2018
  • Fimasartan, a new angiotensin II receptor antagonist, reduces myocyte damage and stabilizes atherosclerotic plaque through its anti-inflammatory effect in animal studies. We investigated the protective effects of pretreatment with fimasartan on ischemia-reperfusion injury (IRI) in a mouse model of ischemic renal damage. C57BL/6 mice were pretreated with or without 5 (IR-F5) or 10 (IR-F10) mg/kg/day fimasartan for 3 days. Renal ischemia was induced by clamping bilateral renal vascular pedicles for 30 min. Histology, pro-inflammatory cytokines, and apoptosis assays were evaluated 24 h after IRI. Compared to the untreated group, blood urea nitrogen and serum creatinine levels were significantly lower in the IR-F10 group. IR-F10 kidneys showed less tubular necrosis and interstitial fibrosis than untreated kidneys. The expression of F4/80, a macrophage infiltration marker, and tumor necrosis factor $(TNF)-{\alpha}$, decreased in the IR-F10 group. High-dose fimasartan treatment attenuated the upregulation of $TNF-{\alpha}$, interleukin $(IL)-1{\beta}$, and IL-6 in ischemic kidneys. Fewer TUNEL positive cells were observed in IR-F10 compared to control mice. Fimasartan caused a significant decrease in caspase-3 activity and the level of Bax, and increased the Bcl-2 level. Fimasartan preserved renal function and tubular architecture from IRI in a mouse ischemic renal injury model. Fimasartan also attenuated upregulation of inflammatory cytokines and decreased apoptosis of renal tubular cells. Our results suggest that fimasartan inhibited the process of tubular injury by preventing apoptosis induced by the inflammatory pathway.

Nutrition Survey in a Korean Mountainous Farming Area (산간지(山間地) 농촌주민(農村住民)의 영양실태조사(營養實態調査))

  • Ham, Jung-Rea;Kim, Hyong-Soo;Lee, Ki-Yull;Kim, Young-Hoo
    • Journal of Nutrition and Health
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    • v.6 no.3
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    • pp.37-45
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    • 1973
  • The main character of the Korean diet has been found to be low in protein both quantity and quality and high in carbohydrate. The purpose of this survey was to study the amount of salt intake related to the dietary pattern in Korea. The nutrition survey was conducted in a mountainous farming area located in Auhchun-ri, Gaebuk-myon, Changsoo-gun, Chunbuk Province, February 14-19 in 1973 (7 days). The precise weighing method was used in evaluating the kinds of foods and nutrients intake for 24 households during a three day period. The physical examinations were performed by a doctor on 120 persons and a detailed biochemical test on both blood and urine was made on 42 persons over 40 years old. The results obtained are summarized as follows: (1) Average nutrients intake of an adult per day: calorie intake was 2,446 Cal and its components-protein(61.1g) was 10 percent, fat(12.9g) was 5 percent and carbohydrate(521g) contributed 85 percent of the total calories. Other nutrients-calcium (443mg), thiamine(1.09mg), riboflavin (0.90mg), niacin (14.4mg) and vitamin C (63.2mg) were lower than the recommended daily allowance but vitamin A(2,083 I.U.), iron(11mg) and phosphorous(998mg) were slightly higher than that. (2) To evaluate the nutritional deficiences, clinical examinations were conducted. Angular stomatitis was present in 16.7 percent of those examiners. No edema was found. The rate of osteoarthritis, hepatomegaly diseases appeared in 20 percent of the total subjects and the symptoms appeared highest among those Iron 50 to 59 years old. (3) The following chemical components of blood serum were analyzed and found to be within the normal range: glucose, blood urea nitrogen, uric acid, total protein, albumin, globulin, bilirubin, total cholesterol, inorganic phosphate, alkaline phosphatase, sodium, potassium, chloride, calcium and lecithine dehydrase. One case of each of the following were found: hyperglycemia, hypocholesterolemia, renal problem, hypoproteinaemia and diabetes mellitus, and two persons were classified as showing hypoglycemia and hyponaturemia. (4) The sodium content in urine was 199.6 mEq/L, potassium content was 24.6 mEq/L. The sugar, pH and specific gravity in the urine was shown to be normal.

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Effect of Molinate, Simetryn and Imazosulfuron U-Granule Application on Bioefficacy and Phytotoxicity in Rice Paddy (Molinate와 Simetryn 및 Imazosulfuron 수면부상성(水面浮上性) 입제(粒劑)의 시용효과(施用效果))

  • Choi, S.Y.;Chung, B.J.;Chae, J.C.
    • Korean Journal of Weed Science
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    • v.18 no.4
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    • pp.341-347
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    • 1998
  • This study was carried out to investigate the effect of U-Granule formulation of molinate(S-ethylhexaphdro-1H-azepine-1-carbothioate) mixtures in green house and paddy field, Five minutes were taken for U-Granule to spread out 7m in irrigated water of direct seeded on flooded paddy surface. The concentration of active ingredient of molinate in molinate U-Granule application was similar to molinate+simetryn U-Granule application. But weeding effect of molinate+simetryn U-Granule on Echinochlor crus-galli was 23% higher Than single application of molinate at 7m from application point. Bioefficacy of molinate+simetryn+imazosulfuron U-Granule on control of Echinochloa crus-galli was higher than that of molinzte+imazosulfuton U-Granule, but those effect on Eleochairs kuroguwai was not significantly different. Only slight rice phytotoxicity was observed at 5m and 2m from application point of U-Granule molinate+simetryn+imazosulfuron and molinate+imazosulfuron, respectively. So it is concluded that there is little phytotoxicity problem in practical application of U-Granule of molinate mixtures.

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Variation of Bolting at Cultivation of Different Regions and Molecular Characterization of FLC homologs in Angelica gigas Nakai (재배 지대에 따른 참당귀의 추대 변이와 FLC 유전자 특성)

  • Kim, Young-Guk;Yeo, Jun-Hwan;An, Tae-Jin;Han, Sin-Hee;Ahn, Young-Sup;Park, Chung-Beom;Jang, Yun-Hee;Kim, Jeong-Kook
    • Korean Journal of Medicinal Crop Science
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    • v.20 no.5
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    • pp.359-364
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    • 2012
  • This study were carried out to find bolting response of cultivation in different regions and to isolate FLC (FLOWERING LOCUS C) homologs in Angelica gigas Nakai. The mean temperature of different regions, ordering in altitude, were as follows: 100 m > 350 m > 530 m > 700 m. The largest amount of rainfall was occurred in the region of 350 m while the longest time of sunshine was occurred in the region of 100 m. The content of soil chemical properties in regions showed pH 6.2 ~ 7.4, T-N 0.17 ~ 26, organic mater $1{\sim}32gkg^{-1}$, $P_2O_5$ ${151{\sim}664_{mgkg}}^{-1}$, exchangeable potassium and calcium and magnesium were 0.78 ~ 1.15, 3.9 ~ 10.0, ${0.7{\sim}3.2_{cmol}}^{+kg-1}$. L5 line of A. gigas was occurred in bolting at all regions, but the bolting ratio was 60.0% in 700 m region with non-mulching treatment. Manchu of A. gigas was not occurred in bolting at all regions. The accumulation bolting ratio of L5 line by non-mulching was higher than that of mulching as 90.4% and 72.8% in 100 m region. The MADS-box transcription factor FLC is one of the well-known examples as a strong floral repressor. We decided to isolate FLC homologs from A. gigas as a starting point of flowering mechanism research of this plant. We have isolated two RT-PCR products which showed very high amino acid sequence homology to Arabidopsis FLC.