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Development of a Simultaneous Analytical Method for Determination of Insecticide Broflanilide and Its Metabolite Residues in Agricultural Products Using LC-MS/MS (LC-MS/MS를 이용한 농산물 중 살충제 Broflanilide 및 대사물질 동시시험법 개발)

  • Park, Ji-Su;Do, Jung-Ah;Lee, Han Sol;Park, Shin-min;Cho, Sung Min;Kim, Ji-Young;Shin, Hye-Sun;Jang, Dong Eun;Jung, Yong-hyun;Lee, Kangbong
    • Journal of Food Hygiene and Safety
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    • v.34 no.2
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    • pp.124-134
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    • 2019
  • An analytical method was developed for the determination of broflanilide and its metabolites in agricultural products. Sample preparation was conducted using the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method and LC-MS/MS (liquid chromatograph-tandem mass spectrometer). The analytes were extracted with acetonitrile and cleaned up using d-SPE (dispersive solid phase extraction) sorbents such as anhydrous magnesium sulfate, primary secondary amine (PSA) and octadecyl ($C_{18}$). The limit of detection (LOD) and quantification (LOQ) were 0.004 and 0.01 mg/kg, respectively. The recovery results for broflanilide, DM-8007 and S(PFP-OH)-8007 ranged between 90.7 to 113.7%, 88.2 to 109.7% and 79.8 to 97.8% at different concentration levels (LOQ, 10LOQ, 50LOQ) with relative standard deviation (RSD) less than 8.8%. The inter-laboratory study recovery results for broflanilide and DM-8007 and S (PFP-OH)-8007 ranged between 86.3 to 109.1%, 87.8 to 109.7% and 78.8 to 102.1%, and RSD values were also below 21%. All values were consistent with the criteria ranges requested in the Codex guidelines (CAC/GL 40-1993, 2003) and the Food and Drug Safety Evaluation guidelines (2016). Therefore, the proposed analytical method was accurate, effective and sensitive for broflanilide determination in agricultural commodities.

Effect of rbST Administrations at Artificial Insemination on Conception and Parturition Rates in Hanwoo (한우 인공수정시 rbST 투여가 수태 및 분만율에 미치는 영향)

  • Han M. H.;Choi S. H.;Choi Y. H.;Kim H. J.;Cho S. R.;Choi C.Y.;Ryu I. S.;Son D. S.;Yeon S. H.;Woo J. S.;Kweon U. G.;Yoon K. Y.;Chang B. S.
    • Journal of Embryo Transfer
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    • v.20 no.2
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    • pp.177-184
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    • 2005
  • This study was conducted to investigate the effects of recombinant bovine somatotropin (rbST) injection on conception and parturition rates in normal or repeat breeding Hanwoo. We treated 462 cows containing 79 repeat-breeding cows of multiparous and allocating 5 treatment groups. Treatment 1 (T1) was injection of 2ml saline (for pseudo treatment), T2 was one injection of rbST 250mg into the tailhead region at the estrus, T3 was twice injection of rbST 250mg both at the time of insemination and again 10 to 14 day later, T4 was once injection of rbST 500mg at insemination and T5 was twice injection of 500mg rbST both at the time of insemination and again 10 to 14day later respectively. In rbST treated groups, timed artificial inseminations (TAI) were performed fellowing estrus synchronization. 100 us GnRH was injected into the scapula region on Day 0, 25mg $PGF_2{\alpha}$ was injected on Day 7 for degeneration of corpus luteum (CL) and 100ug GnRH was injected for inducing the synchronization. The results are as fellows; When normal Hanwoo were inseminated once with rbST administration, the pregnancy rate of T2 $(67.5\pm18.48\%)$ were higher than control $(52.4\pm9.72\%)$, while the pregnancy rate of T4 $(63.3\pm5.77\%)$ were significantly higher (p.<0.05) than control $(39.3\pm12.89\%)$ in repeat breeder Hanwoo. The parturition rates of normal Hanwoo were no differences among the treatments but were significant different in repeat breeder Hanwoo (p<0.05). When the estrous was induced by Ovsynch and inseminated once with rbST administration, the pregnancy rates of T2 was $12.5\%$ higher than control in normal Hanwoo, T4 $(80.0\%)$ was highest among the treatments (p<0.05) in repeat breeder Hanwoo. When normal Hanwoo were inseminated once with rbST administration, the pregnant period was $282.7\~284.8$ days and the body weight was $25.1\~25.9kg$, there were no difference among the treatments. The ratio of sex was almost same without T4 (male vs. female=18:9). In repeat breeder Hanwoo, pregnant period was 280.4~289.3 day and body weight was $23.0\~26.6kg$, it had no difference among the treatments. The sex ratio were similar to normal Hanwoo except T4 (M : F=2 : 8). In conclusion, the pregnancy and parturition rate by once insemination could be improved by the administration of rbST 250mg in normal Hanwoo or 500mg in repeat breeder Hawoo.

The Effects of Isopropyl 2-(1,3-dithioetane-2-ylidene)-2-[N-(4-methyl-thiazol-2-yl)carbamoyl]acetate (YH439) on Potentiated Carbon Tetrachloride Hepatotoxicity (상승적 화학적 간독성에 미치는 YH439의 영향)

  • Kim, Sang-Geon;Cho, Joo-Youn
    • The Korean Journal of Pharmacology
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    • v.32 no.3
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    • pp.407-416
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    • 1996
  • The reactive intermediates formed during the metabolism of therapeutic agents, toxicants and carcinogens by cytochromes P450 are frequently capable of covalently binding to tissue macromolecules and causing tissue damage. It has been shown that YH439, a congener of malotilate, is effective in suppressing hepatic P450 2E1 expression. The present study was designed to further establish the mechanistic basis of YH439 protection against toxicant by assessing its effects against chemical-mediated potentiated hepatotoxicity. Retinoyl palmitate (Vit-A) pretreatment of rats for 7 days substantially enhanced carbon tetrachloride hepatotoxicity, as supported by an ${\sim}5-fold$ increase in serum alanine aminotransferase (ALT) activity, as compared to $CCl_4$ treatment alone. The elevation of ALT activity due to Vit-A was completely blocked by the treatment of $GdCl_3$ a selective inhibitor of Kupffer cell activity. Concomitant pretreatment of rats with both YH439 and Vit-A resulted in a 94% decrease in Vit-A-potentiated $CCl_4$ hepatotoxicity. YH439 was also effective against propyl sulfide-potentiated $CCl_4-induced$ hepatotoxicity. Whereas propyl sulfide (50 mg/kg, 7d) enhanced $CCl_4-induced$ hepatotoxicity by >5-fold, relative to $CCl_4$ treatment alone, concomitant treatment of animals with both propyl sulfide and YH439 at the doses of 100 and 200 mg/kg prevented propyl sulfide-potentiated $CCl_4$ hepatotoxicity by 35% and 90%, respectively. Allyl sulfide, a suppressant of hepatic P450 2E1 expression, completely blocked the propyl sulfide-enhanced hepatotoxicity, indicating that propyl sulfide potentiation of $CCl_4$ hepatotoxicity was highly associated with the expression of P450 2E1 and that YH439 blocked the propyl sulfide-enhanced hepatotoxicity through modulation of P450 2E1 levels. Propyl sulfide- and $CCl_4-induced$ stimulation of lipid peroxidation was also suppressed by YH439 in a dose-related manner, as supported by decreases in malonedialdehyde production. The role of P450 2E1 induction in the potentiation of $CCl_4$ toxicity and the effects of YH439 were further evaluated using pyridine as a P450 2E1 inducer. Pyridine pretreatment substantially enhanced the $CCl_4$ hepatotoicity by 23-fold, relative to $CCl_4$ alone. YH439, however, failed to reduce the pyridine-potentiated toxicity, suggesting that the other form(s) of cytochroms P450 inducible by pyridine, but not suppressible by YH439 treatment, may play a role in potentiating $CCl_4-induced$ hepatotoxicity. YH439 was capable of blocking cadmium chloride-induced liver toxicity in mice. These results demonstrated that YH439 efficiently blocks Vit-A-enhanced hepatotoxiciy through Kupffer cell inactivation and that the suppression of P450 2E1 expression by YH439 is highly associated with blocking of propyl sulfide-mediated hepatotoxicity.

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A Study on Jurisdiction under the International Aviation Terrorism Conventions (국제항공테러협약의 관할권 연구)

  • Kim, Han-Taek
    • The Korean Journal of Air & Space Law and Policy
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    • v.24 no.1
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    • pp.59-89
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    • 2009
  • The objectives of the 1963 Tokyo Convention cover a variety of subjects, with the intention of providing safety in aircraft, protection of life and property on board, and promoting the security of civil aviation. These objectives will be treated as follows: first, the unification of rules on jurisdiction; second, the question of filling the gap in jurisdiction; third, the scheme of maintaining law and order on board aircraft; fourth, the protection of persons acting in accordance with the Convention; fifth, the protection of the interests of disembarked persons; sixth, the question of hijacking of aircraft; and finally some general remarks on the objectives of the Convention. The Tokyo Convention mainly deals with general crimes such as murder, violence, robbery on board aircraft rather than aviation terrorism. The Article 11 of the Convention deals with hijacking in a simple way. As far as aviation terrorism is concerned 1970 Hague Convention and 1971 Montreal Convention cover the hijacking and sabotage respectively. The Problem of national jurisdiction over the offence and the offender was as tangled at the Hague and Montreal Convention, as under the Tokyo Convention. Under the Tokyo Convention the prime base of jurisdiction is the law of the flag (Article 3), but concurrent jurisdiction is also allowed on grounds of: territorial principle, active nationality and passive personality principle, security of the state, breach of flight rules, and exercise of jurisdiction necessary for the performance of obligations under multilateral agreements (Article 4). No Criminal jurisdiction exercised in accordance with national law is excluded [Article 3(2)]. However, Article 4 of the Hague Convention(hereafter Hague Article 4) and Article 5 of the Montreal Convention(hereafter Montreal Article 5), dealing with jurisdiction have moved a step further, inasmuch as the opening part of both paragraphs 1 and 2 of the Hague Article 4 and the Montreal Article 5 impose an obligation on all contracting states to take measures to establish jurisdiction over the offence (i.e., to ensure that their law is such that their courts will have jurisdiction to try offender in all the circumstances covered by Hague Article 4 and Montreal Article 5). The state of registration and the state where the aircraft lands with the hijacker still on board will have the most interest, and would be in the best position to prosecute him; the paragraphs 1(a) and (b) of the Hague Article 4 and paragraphs 1(b) and (c) of the Montreal Article 5 deal with it, respectively. However, paragraph 1(b) of the Hague Article 4 and paragraph 1(c) of the Montreal Article 5 do not specify if the aircraft is still under the control of the hijacker or if the hijacker has been overpowered by the aircraft commander, or if the offence has at all occurred in the airspace of the state of landing. The language of the paragraph would probably cover all these cases. The weaknesses of Hague Article 4 and Montreal Article 5 are however, patent. The Jurisdictions of the state of registration, the state of landing, the state of the lessee and the state where the offender is present, are concurrent. No priorities have been fixed despite a proposal to this effect in the Legal Committee and the Diplomatic Conference, and despite the fact that it was pointed out that the difficulty in accepting the Tokyo Convention has been the question of multiple jurisdiction, for the reason that it would be too difficult to determine the priorities. Disputes over the exercise of jurisdiction can be endemic, more so when Article 8(4) of the Hague Convention and the Montreal Convention give every state mentioned in Hague Article 4(1) and Montreal Article 5(1) the right to seek extradition of the offender. A solution to the problem should not have been given up only because it was difficult. Hague Article 4(3) and Montreal Article 5(3) provide that they do not exclude any criminal jurisdiction exercised in accordance with national law. Thus the provisions of the two Conventions create additional obligations on the state, and do not exclude those already existing under national laws. Although the two Conventions do not require a state to establish jurisdiction over, for example, hijacking or sabotage committed by its own nationals in a foreign aircraft anywhere in the world, they do not preclude any contracting state from doing so. However, it has be noted that any jurisdiction established merely under the national law would not make the offence an extraditable one under Article 8 of the Hague and Montreal Convention. As far as international aviation terrorism is concerned 1988 Montreal Protocol and 1991 Convention on Marking of Plastic Explosives for the Purpose of Detention are added. The former deals with airport terrorism and the latter plastic explosives. Compared to the other International Terrorism Conventions, the International Aviation Terrorism Conventions do not have clauses of the passive personality principle. If the International Aviation Terrorism Conventions need to be revised in the future, those clauses containing the passive personality principle have to be inserted for the suppression of the international aviation terrorism more effectively. Article 3 of the 1973 Convention on the Prevention and Punishment of Crimes Against Internationally Protected Persons, Including Diplomatic Agents, Article 5 of the 1979 International Convention against the Taking of Hostages and Article 6 of the 1988 Convention for the Suppression of Unlawful Acts Against the Safety of Maritime Navigation would be models that the revised International Aviation Terrorism Conventions could follow in the future.

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Hydrochemical and Isotopic Characteristics, and Origin of Noble Gas for Low-temperature Hot Spring Waters in the Honam Area (호남지역 저온형 온천수의 수리지화학적 및 안정동위원소 특성과 영족기체의 기원에 관한 연구)

  • Jeong, Chan-Ho;Hur, Hyun-Sung;Nagao, Keisuke;Kim, Kyu-Han
    • Economic and Environmental Geology
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    • v.40 no.5
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    • pp.635-649
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    • 2007
  • Geochemical composition, stable isotopes $({\delta}^{18}O,\;{\delta}D,\;{\delta}^{34}S)$ and noble gases(He, Ne and Ar) of nine hot spring water and three groundwater for five hot springs(Jukam, Hwasun, Dokog, Jirisan, Beunsan) from the Honam area were analyzed to investigate the hydrogeochemical characteristics and the hydrogeochemical evolution of the hot spring waters, and to interpret the source of sulfur, helium and argon dissolved in the hot spring waters. The hot spring waters show low water temperature ranging from 23.0 to $30.5^{\circ}C$ and alkaline characteristics of pH 7.67 to 9.98. Electrical conductivity of hot spring waters is $153{\sim}746{\mu}S/cm$. Groundwaters in this area were characterized by the acidic to neutral pH range$(5.85{\sim}7.21)$, the wide electrical conductivity range $(44{\sim}165{\mu}S/cm)$. The geochemical compositions of hot spring and groundwaters can be divided into three water types: (1) $Na-HCO_3$ water type, (2) Na-Cl water type and (3) $Ca-HCO_3$ water type. The hot spring water of $Ca-HCO_3$ water type in early stage have been evolved through $Ca(Na)-HCO_3$ water type into $Na-HCO_3$ type in final stage. In particular, Jurim alkaline(pH 9.98) hot spring water plotted at the end point of $Na-HCO_3$ type in the Piper diagram is likely to arrive into the final stage in geochemical evolution process. Hydrogen and oxygen isotopic data of the hot spring water samples indicate that the hot spring waters originated from the local meteoric water showing latitude and altitude effects. The ${\delta}^{34}S$ value for sulfate of the hot spring waters varies widely from 0.5 to $25.9%o$. The sulfur source of most hot spring waters in this area is igneous origin. However, The ${\delta}^{34}S$ also indicates the sulfur of JR1 hot water is originated from marine sulfur which might be derived ken ancient seawater sulfates. The $^3He/^4He\;and\;^4He/^{20}Ne$ ratios of the hot spring waters range from $0.0143{\times}10^{-6}\;to\;0.407{\times}10^{-6}\;and\;6.49{\sim}584{\times}10^{-6}$, respectively. The hot spring waters are plotted on the mixing line between air and crustal components. It means that the He gas in the hot spring waters was mainly originated from crustal sources. However, the JR1 hot spring water show a little mixing ratio of the helium gas of mantle source. The $^{40}Ar/^{36}Ar$ ratios of hot spring water are in the range from $292.3{\times}10^{-6}\;to\;304.1{\times}10^{-6}$, implying the atmospheric argon source.

A Survey of Nutritional Status on Pre-School Children in Korea (학영기전아동(學齡期前兒童)의 영양실태조사(營養實態調査))

  • Ju, Jin-Soon;Oh, Seoung-Ho
    • Journal of Nutrition and Health
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    • v.9 no.2
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    • pp.68-86
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    • 1976
  • The primary purpose of this study is to evaluate the correct nutritional status on pre-school children in Korea. Furthermore, it made an attempt to find and define nutrional problems, and assist in establishment on their nutritional improvement plan. For this, food intake and health condition (physical, clinical, biochemical and parasitological) survey on 109 Pre-school children in both sexes, randomly selected from Yang-Gu area in Gang-Won province and Rea-ju area in Kyong-gy Province, were conducted by means of three-day records, during the two periods of Spring and Fall season in 1975. The results obtained are summerized as follows: 1. The food intake; Average food intake of the subjects per day were $508{\sim}647g$ ($83{\sim}91%$ in vegetable foods and $5.5{\sim}11.7%$ in animal foods) in Yang-gu area, and $587{\sim}698g$ ($88{\sim}89%$ in vegetable foods and $6.3{\sim}7.6%$ in animal foods) in Rea-ju area. 2. The intake of energy and nutrients; a) Calory intake. Average energy intake of subjects per day in Yang-gu area$(1120{\sim}1415kcal)$ were all lower than the Korean Recommended Dietary Allowances (RDA) in either Spring and Fall survey, whereas the subjects in Rea-ju area were lower intake $(1213{\sim}1418kcal)$ than the RDA in the Spring but higher intake$(1516{\sim}1755kcal)$ than the RDA in the Fall, and the average intake were similar level with that of RDA. b) Protein intake. Average protein intake of the subjects per day in Yang-gu area $(33{\sim}43g)$ girl subjects in Rea-ju area $(35{\sim}39g)$ were lower than the RDA in either Spring and Fall survey, whereas the boy subjects in Rea-ju area$(36{\sim}38g)$ were lower in Spring and higher $(49{\sim}57g)$ in the Fall than that of the RDA, but the average $(43{\sim}47g)$ were similar level with the RDA. The protein intake from animal sources in all subjects were much lower $(5.5{\sim}11.7\;of\;total\;protein)$ than the RDA. c) Fat intake. Average fat intake were very lower in all subjects of both area $(14{\sim}24g\;in\;Yang-gu,\;10{\sim}12g\;in\;Rea-ju)$ than that of RDA which is recommended $12{\sim}14%$ of total energy to be supplied from fat. d) Calcium intake. Average calcium intake were very low in all subjects of both area $(264{\sim}355mg\;in\;Yang-gu\;and\;283{\sim}429mg\;in\;Rea-ju)$, especially, these in Spring were about a half level of the RDA, and it was much increased in the Fall due to increased intake of milk, but it was still not enough than the RDA. e) Vitamin A intake. Average intake of V.A ($703{\sim}1465\;IU$ in Ynag-gu and $750{\sim}1521\;IU$ in Rea-ju) were also lower than the RDA, moreover their V-A sources were mainly vegetable, so that the V-A supply might be critical one for the subjected. f) Riboflavin intake. Average riboflavin intake on all subjects in both area except boys in Rea-ju area in Fall, were very lower than the RDA. 3. The physical status; a) Average weight and height of boys aged 4 and 5 in Yang-gu area and girls of aged 5 in Rea-ju area were lower than those of Korean Standard of 1967 report, but those by age of girls in Yang-gu area and boys in Rea-ju area were a little heigher than the Korean Standard. It is, hower, present Korean standard of physical status might be somehow heigher than the 1967, since the socio-economical situation has been much improved during past a decade. So that, if one considered on this sense, the physical status of the subjects on this survey might be somehow lower than those of present Korean standard. b) Average upper arm circumference in both area were no difference each other, and their mean values of age 4, 5 and 6 in boy and girl were 15.6, 16.5, 16.4 and 15.5, 16.5, 16.4cm respectively. c) Average chest girth of boys were similar to those of Korean standard whereas the girls were smaller than the Korea standard. The average head circumference also showed similar tendency with the chest girth. 4. The clinical findings; The most popular clinical signs were angular stomatitis and dental caries, and boys had more heigher incidence then the girls. 5. The biochemical findings; a) Hemoglobin and anemia Average Hb value of boys and girls were 11.4 and 10.9g per 100 ml of blood respectively. The incidence of anemia (Hb value below 11 g/100 ml, by WHO) was increased by age, and girls had more heigher incidence than the boy (34% : 48%). The incidence of anemia in age of 4,5, and 6 in boys and girls were 28%, 41% 34%, and 33%, 50%, 49% respectively. The degree of the anemia was not severe, and the anemia of there subjects may be caused mainly low intake of better quality protein and low iron intake as well. b) Hematocrit. Average Ht value of whole subject were $39.9{\sim}41.6%$. c) Blood plasma protein. Average blood plasma protein contents of whole subjects were $6.6{\sim}7.4gm$ per 100 ml. The incidence of deficient range (<6.0g%, by ICNND) was only one girl of age 4 in yang Gu area. 6. Parasitological findgs; The most popular parasitism were asicris lumbicoides and trichocephalus trichiura, and about 2/3 of the whole subjects were suffering one or more of these parasitism.

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Analytical Studies on The Useful Characters Affecting The Lodging Resistance of Wheat and Barley Varieties (맥류의 도복에 관여하는 유용형질의 분석에 관한 연구)

  • Chang-Hwan Cho
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.11
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    • pp.105-117
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    • 1972
  • This experiment has been made to study the relationship between several characters affecting the field lodging and to establish some useful standards for selection of lodging resistant varieties and to classify the degree of lodging resistance in wheat and barley varieties of different sources. The experiment was carried at the Crop Experiment Station, Suwon in 1968. The results obtained are summarized as followings. a. The lodging index modified with bending moment of culm at breaking seemed to be the most useful character in checking the lodging resistance. Highly significant positive correlation (Barley; r=0.40-0.67, Wheat; r=0.46-0.68) was obtained between the lodging index and actual field lodging. b. Between two essential components expressing bending stiffness of the culm, the bending moment at breaking and secondary moment of inertia, a highly significant positive correlation (Barley; r=0.59, wheat; r=0.46-0.53) was observed. c. The bending stiffness of culm got stronger as the dry weight per unit culm, which express the quantity of accumulated dry matters in culm, increased. The correlation coefficient between those two factors was 0.35 to 0.40 in barley and 0.33 to 0.76 in wheat respectively. d. In both wheat and barley, highly significant negative correlation between lodging index and the other factors such as dry weight per unit culm (Barley; r=-0. 51 to -0.70, Wheat; r=-0.65 to -0.83) and bending moment of culm at breaking (Barley; r=-0.29 to -0.69, Wheat: r=-0.54 to -0.89) were observed. Particulary, weight of culm at breaking, secondary moment of inertia and section modulus showed significant negative correlation with lodging index in wheat. e. Outside diameter of culm expressed more intimate relationship with physical characteristics of culm than inside diameter and also showed highly significant correlation with weight of culm at breaking (Barley; r=0.42-0.56, Wheat; r=0.39-0.44) and with bending moment of culm at breaking (Barley; r=0.40-0.41, Wheat; r=0.38-0.49) and with secondary moment of inertia (Barley; r=0.56-0.57, Wheat; r=0.28-0.98) and with section modulus (Wheat; r=0.22-0.96). Between the thickness of culm and physical characteristics of culm also showed the positive correlation. f. There was positive correlation between the culm length and actual field lodging in several groups of variety among the varieties tested. But the culm length seemed to undesirable as a selection measure for the selection of the lodging resistant variety considering the stiffness of culm. g. In classification of lodging resistance for the varieties tested, many Korean barley varieties expressed excellent lodging resistant than wheat, but most of the wheat and barley varieties from Japan considered quite resistant to lodging. h. In selection of lodging resistant varieties, lodging index lower than 1.67 in barley and 1. 76 in wheat considered highly resistant to actual field lodging.

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Effects of Donor Somatic Cell Conditions on In Vitro Development of Nuclear Transplanted Porcine Embryos (돼지 공여세포의 조건이 핵이식 수정란의 체외발달에 미치는 영향)

  • 홍승표;박준규;이명열;이지삼;정장용
    • Journal of Embryo Transfer
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    • v.16 no.3
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    • pp.213-221
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    • 2001
  • This study was conducted to examine in vitro developmental ability of porcine embryos after somatic cell nuclear transfer. The porcine ear fell was cultured in vitro for confluency in serum-starvation condition(TCM-199 + 0.5% FBS) far 3~6 days of cell confluency. The zona pellucida of IVM oocytes were partially drilled using laser system. Single somatic cell was individually transferred into enucleated oocytes. And the reconstructed embryos were electrically fused(single DC 1.9kv/cm, 30$\mu$ sec) with 0.3M mannitol. After electrofusion, embryos were activated(single AC 5v/mm, 5sec) and cultured in HCSU-23 medium containing 10% FBS at 39$^{\circ}C$, 5% $CO_2$ in air for 6 to 8 days. The fusion rate of donor cells was 45.6, 36.8 and 46.1% in 3~4, 5~6 days of serum starvation and non serum starvation(N-S), and were 52.7. 53.0 and 51.7% in 1~2. 5~6 and 13~14 passages of donor cell culture, respectively. No significant difference was found in the fusion rate of donor cells by the duration of serum starvation treatment or the number of donor cell passages. By the size of donor cells, however, the fusion rate was significantly higher(P<0.05) for reconstructed embryos derived from 25r $\mu$m $\geq$ site of donor cells (65.3%) than that of 25~30$\mu$ m(42.5%) or 30$\mu$ m(45.5%)$\leq$ cells. The cleavage rate was significantly (P<0.05) higher in 3~4 darts of serum starvation treatment(67.1%) than that in N-S (50.7%) or 5~6 days of starvation(57.1%). The activation rate by the size of donor cells in fused oocytes was 56.5, 68.8 and 58.5%, respectively, and was not significant.

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Hsp90 Inhibitor Induces Cell Cycle Arrest and Apoptosis of Early Embryos and Primary Cells in Pigs

  • Son, Myeong-Ju;Park, Jin-Mo;Min, Sung-Hun;Hong, Joo-Hee;Park, Hum-Dai;Koo, Deog-Bon
    • Reproductive and Developmental Biology
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    • v.35 no.1
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    • pp.33-45
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    • 2011
  • Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bel-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MIT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG ($2{\mu}M$) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.

Analysis of the Component and Immunological Efficacy of Chamaecyparis obtusa Leaf Extract (편백나무 잎 추출물의 성분분석과 면역효능에 관한 연구)

  • Kim, Joung Hee;Lee, Syng-Ook;Do, Kook Bae;Ji, Won Dae;Kim, Sun Gun;Back, Young Doo;Kim, Keuk-Jun
    • Korean Journal of Clinical Laboratory Science
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    • v.50 no.1
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    • pp.37-43
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    • 2018
  • Chamaecyparis obtusa (CO) has recently been attracting attention because of its beneficial effects on skin allergies, atopic dermatitis, and skin diseases, such as acne and eczema. In the present study, the extract from CO leaf grown in Jangseong gun, Jeollanam-do, Korea was evaluated for its anti-oxidant, anti-inflammatory, and anti-allergic effects in vitro. The total polyphenol content of the CO leaf extract was $25.89{\pm}0.31mg$ gallic acid equivalents (GAE)/g. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed the presence of six compounds in the CO leaf extract: ${\alpha}-terpinene$ (3.03 mg/g), ${\alpha}-terpineol$ (9.48 mg/g), limonene (5.96 mg/g), borneol (59.78 mg/g), myrcene (4.85 mg/g), and sabinene (11.31 mg/g). The $RC_{50}$ values of the CO leaf extract for $H_2O_2$ and ABTS radical were $5.47{\pm}0.13mg/mL$ and $4.00{\pm}0.01mg/mL$, respectively. In addition, the CO leaf extract showed significant inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells and IgE-induced release of ${\beta}-hexosaminidase$ (degranulation) in mast-cell like RBL-2H3 cells. The cell viability assay showed that the CO leaf extract ($100{\sim}800{\mu}g/mL$) did not affect the viability of human normal skin fibroblast CCD-986sk cells significantly. Overall, these results suggest that the CO leaf extract is a potential functional cosmetic ingredient that can exert anti-oxidant, anti-inflammatory, and anti-allergic effects.