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Proteomics Approach on Puroindoline Gene of Pre-harvest Sprouting Wheat

  • Kamal, Abu Hena Mostafa;Park, Cheol-Soo;Heo, Hwa-Young;Chung, Keun-Yook;Cho, Yong-Gu;Kim, Hong-Sig;Song, Beom-Heon;Lee, Chul-Won;Woo, Sun-Hee
    • Korean Journal of Breeding Science
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    • v.41 no.3
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    • pp.205-212
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    • 2009
  • Wheat (Triticum aestivum L.) grain texture is an important determinant of milling properties and end product use. Two linked genes, puroindoline a (PINA) and puroindoline b (PINB), control most of the genetic variation in wheat grain texture. Wheat seed proteins were examined to identify PINA and PINB gene using two pre-harvest sprouting wheat cultivars; Jinpum (resistant) and Keumgang (susceptible).Wheat seed proteins were separated by two-dimensional electrophoresis with IEF gels over pH ranges: pH 3-10. A total of 73 spots were digested with trypsin resulting peptide fragmentation were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS). Mass spectra were automatically processed and searched through NCBInr, SWISS-PORT and MSDB database with mono isotopic masses and complete gene sequence were found by UniProt database. Puroindoline a and puroindoline b that is responsible for grain texture related with baking performance and roughness. Two spots were found Pin b (16.7 kDa) and Pin a (16.3 kDa) in Jinpum compare to seven spots were identified Pin a (16.1 kDa, 16.3 kDa) and Pin b (16.7 kDa, 9.5 kDa and 14.4 kDa) in Keumgang. Some selected spots were identified puroindoline like grain softness protein (16.9 kDa, 17 kDa and 18.1 kDa) in Keumgang. Moreover, to gain a better inferring the identification of puroindoline related proteins using proteomics, we accomplished a complete gene sequence of PINA and PINB gene in pre-harvesting sprouting wheat seeds between resistant (Jinpum) and susceptible (Keumgang).

Analgesic Action Mechanism of DA-5018, a New Capsaicin Derivative : Relationship to Opiate :Receptors and Prostanoids (새로운 캅사이신 유도체 DA-5018의 진통활성 기전연구: Opiate 수용체 및 :Prostanoid와의 상관성)

  • 손미원;손문호;배은주;김순희;김원배;양중의
    • Biomolecules & Therapeutics
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    • v.5 no.1
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    • pp.87-93
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    • 1997
  • DA-5018, a new capsaicin derivative, showed potent analgesic effect comparable to that of morphine in various experimental acute pain models. in this study, whether the analgesic mechanism of DA-5018 is related to opiate receptors or prostanoids was investigated. The affinity of DA-5018 for opiate receptor was determined by receptor binding assay. The Ki values of DA-5018 for nonspecific and specific $\mu$, $textsc{k}$, $\delta$-opiate receptor was 299$\pm$8.88, 735$\pm$215, 2930$\pm$ 163, 1550$\pm$813 nM, respectively and DA-5018 exhibited lower affinity than morphine. DA-5018 (10-"~3$\times$10-′M) inhibited electrically-evoked contractions of the guinea ply ileum and rat vas deferens, and these inhibition was not antagonized by naloxone(10 nM), an opiate receptor antagonist. Antagonism of analgesic effect of 7A-5018 by naloxone was examined by tail pinch test. Analgesic action of DA-5018(0.1 ~2 mg/kg, 5.c.) was not antagonized by naloxone(1 mg/rg, i.p.). These results indicate that pharmacological action of DA-5018 is not related with opiate receptor. Cyclooxygenase and 5-lipoxygenase activities in rat peritoneal neutrophil treated with A23187 and arachidonic acid were measured by radioimmunoassay. DA-5018 stimulated the cyclooxygenase activity and the concentration show-ing the two fold increase of activity was 124$\mu$M. DA-5018 slightly inhibited 5-lipoxygenase activity and these results together indicate that analgesic action of 3A-5018 is not mediated through inhibition of cyclooxy genase or lipoxygenase. These results suggest that the analgesic effect of DA-5018 is not due to blocking opiate receptor or to inhibiting the synthesis of prostanoids in the arachidonic acid metabolism pathway.

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신규 항암제 DA-125의 ADME 연구

  • 이명걸;윤은정;심현주
    • Proceedings of the Korean Society of Applied Pharmacology
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    • 1993.04a
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    • pp.133-133
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    • 1993
  • DA-125의 rat blood중 분해반감기는 4.28분으로, DA-125는 정맥 투여후 신속히 M1으로 분해되었으며, DA-125는 투여 10분 후부터 검출되지 않았다. Mouse에 25mg/kg 정맥주사 한 후의 M1의 phamacokinetic parameter는 terminal t$\frac{1}{2}$ 344분, MRT 371분, Vss 25.7 $\ell$/kg, CL$_{total}$ 69.3 ml/min/kg이었으며 투여 2hr후에 M1및 대사체가 폐, 위, 신장등에는 다량 존재하였으나 심장에서는 검출되지 않았다. Rat에 10 mg/kg 정맥주사한 후의 혈장농도는 terminal phase에 도달하지 못하고 혈장농도가 변동을 나타내었다. 담즙으로의 배설은 주로 M2의 형태로 이루어졌으며 M1이 소량 차지하고 M3와 M4는 검출되지 않았다. DA-125는 rat 및 mouse liver homogenate에 의해 대사되어 FT-ADM(M1) ,13-dihy-dro FT-ADM(M2), 7-deoxy-adriamycinone(M3) 및 7-deoxy-13-dihydro adriamyci-none(M4)를 형성하였다. DA-125는 혐기, 호기 및 보효소, 종차에 관계얼이 반응개시 30분후에 약 90%가 분해되었으나 각 대사체의 생성여부 및 속도는 조건에 따라 변화하였다.

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Time-Course of [$^3H$]Spiperone Binding and Dopamine Metabolism in the Rat Striatum after Withdrawal from Haloperidol Ttreatment (Haloperidol 투여후 금단기간에 따른 백서 선조체의 [$^3H$]Spiperone 결합 및 Dopamine 대사물질의 변화)

  • Lee, Jung-Yong;Kong, Bo-Geum;Kim, Yong-Kwan;Jung, Chung;Kim, Sun-Hee;Kim, Young-Hoon
    • Korean Journal of Biological Psychiatry
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    • v.3 no.1
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    • pp.51-56
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    • 1996
  • The effects of 3 week treatment with haloperidol(2mg/kg/day, i.p.) on dopamine(DA) D2 receptor and DA metabolism in rat striata were studied at various time points after withdrawal from the drug treatment. Striatal DA D2 receptors were characterized with the radioligand 0.5nM [$^3H$]Spiperone. Dopamine(DA), homovanillic acid(HVA), 3,4-dihydroxyphenyl acetic acid(DOPAC) in rat striatum were measured with the high performance liquid chromatography. Drug withdrawal for 1 week induced significant increase in the number of D2 receptor in striatum after withdrawal for 1 week(p<0.05), and then this change was restored to control level during the withdrawal time of 2 and 4 weeks. There was no difference in striatal concentrations of DA and its metabolites among the groups. In conclusion, one-week withdrawal from chronic haloperidol treatment induced DA D2 receptor supersensitivity in the striatum, and that was normalized rapidly. Though this adaptive change in DA receptors, it may not affect the metabolism of DA in striatum.

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General Pharmacology of Recombinant Human Granulocyte-colony Stimulating Factor DA-3030 Expressed in E. coli (유전자 재조합 인형 과립구 콜로니 자극인자 DA-3030의 일반약리작용)

  • 배은주;신명수;김순회;강수형;김원배;양중익
    • Biomolecules & Therapeutics
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    • v.2 no.3
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    • pp.281-285
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    • 1994
  • Neutropenia is a major dose-limiting factor in cancer chemotherapy diminishing its usefulness and increase patient's susceptibility to infectious disease. Some recombinant human granulocyte colony stimulating factors(rhG-CSFs) are in use to reduce the risk of this serious side effect. In this study, we examined the pharmacological properties of DA-3030, a rhG-CSF expressed in E. coli. DA-3030 100 and $\mu\textrm{g}$/kg, i. v., had no significant effect on the central nervous, gastrointestinal system in mice and cardiovascular system in rabbits, but it slightly inhibited the spontaneous motility of isolated nonpregnant uterus in rats. It also had no influence on excretion of urinary electrolytes. DA-3030 administered for successive 3 days increased the blood WBC count in zymosan air pouch inflammed rats and in normal rats. These results indicate that DA-3030 has little side effects in animals.

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Effects of Dispersivity of Clay on Thermal Stabilities of PP/Clay Nanocomposites (점토의 분산성이 PP/점토 나노복합재료의 열안정성에 미치는 영향)

  • 박수진;전병렬;송시용;최길영;이종문
    • Polymer(Korea)
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    • v.27 no.5
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    • pp.458-463
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    • 2003
  • The effect of ozone surface treatment of montmorillonite (MMT) was investigated in thermal stabilities of polypropylene (PP) nanocomposites. Sodium montmorillonite (Na$\^$+/-MMT) was organically modified with dodecylammonium chloride. The surface properties of MMT, including the specific surface area (S$\_$BET/), equilibrium spreading pressure ($\pi$$\_$e/), and London dispersive component (${\gamma}$s$\^$L/), were studied by the BET method with $N_2$ adsorption. Also, the thermal stabilities of the nanocomposites were investigated in DSC and TGA. As experimental results, $\pi$$\_$e/ and ${\gamma}$s$\^$L/ of the ozonized dodecylammonium chloride (DA-MK ( $O_3$)) were increased in about 1.7 and 3.5 mJ/ $m^2$, resulting from the increasing of the micropores. From the DSC results, it was found that the melting temperature and crystallization temperature of PP/DA-MK and PP/DA-MK ( $O_3$) were higher that those of pure PP. These results were explained that dodecylammonium chloride of nano-scale led to a nucleation effect for PP crystallization. Also, it was found that E$\_$t/ of the PP/DA-MK ( $O_3$) nanocomposies was increased within about 64 kJ/mol. These results were probably explained by the improvement of dispersivity of DA-MK ( $O_3$) in a PP matrix.

Local Irritation of DA-3285, Recombinant Human Erythropoietin (유전자재조합법으로 생산한 human erythropoietin(DA-3285)의 국소자극성에 관한 연구)

  • 김옥진;김동환;안병옥;김원배;양중익
    • Toxicological Research
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    • v.12 no.1
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    • pp.101-111
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    • 1996
  • The local irritation studies of DA-3285, recombinant human erytropoietin(rHu-EPO), were carried out in rabbits after the following treatment; single application into the conjunctival sac of the eye, single subcutaneous injection, 7-day repeated subcutaneous injection and 8-day repeated infusion into the ear vein. Also, the local irritancy of DA-3285 leaked around vein was studied in mice by single perivascular injection. The results obtained were as follows. In the result of ocular irritation test, DA-3285 could be considered as a non-irritating material. In single and 7-day repeated subcutaneous irritation test, the irritancy of DA-3285 was not so much different from that of saline. The vascular irritancy of DA3285 by 8-day repeated infusion was negligible and similar to that of saline. And the irritancy of DA3285 by perivascular injection was comparable to that of saline. These results indicate that DA-3285 has no irritating activity when injected through subcutaneous or intravenous route for clinical practice as 3.5% solution.

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Antigenicity of DA-3002, a Genuine Recombinant Human growth Hormone, in Guinea Pigs and Mice (천연형 인성장호르몬 DA-3002의 항원성)

  • 강경구;백남기;김원배;양중익
    • Biomolecules & Therapeutics
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    • v.3 no.4
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    • pp.251-255
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    • 1995
  • DA-3002 is a genuine human growth hormone produced by Dong-A Pharm. Co. Ltd. research laboratory using recombinant DNA technic. In this study, antigenic potential of DA-3002 was examined by active systemic anaphyaxis(ASA) in guinea pigs, mouse-rat passive cutaneous anaphylaxis(PCA) and passive hemagglutination(PHA) test as a part of safety research. DA-3002 induced anaphylactic shock in ASA test using guinea pigs Immunized with DA-3002 alone or DA-3002 incoporated into Freund's complete adjutant(FCA) when challenged with 10 times higher dose of anticipated clinical dose of DA-3002. In the mouse-rat PCA and PHA test, DA-3002 also showed positive results. DA-3002, therfore, was considered to produce IgE, IgG, and/or IgM in mice. The results of this study were similar to those of the other human growth hormones and these positive results were thought to be caused due to the fact that both DA-3002 and the other human growth hormones were heterogenous proteins to guinea pigs and mice. Considering the fact that DA-3002 is a genuine human growth hormone of which structure is identical with indigenous human growth hormone, DA-3002 is thought not to cause immunological problems in clinical use.

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Changes in the Organic Compounds and Molecular Weight Distribution in Pig Wastewater by Each Treatment Steps. (양돈폐수의 정화처리 과정별 유기물질 성분과 분자량 분포 변화 연구)

  • 최희철;이덕수;권두중;강희설;유용희;연규영;최영수;곽정훈;최동윤
    • Journal of Animal Environmental Science
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    • v.10 no.1
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    • pp.29-36
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    • 2004
  • These experiments were conducted to analyze the characteristics of organic compounds and molecular weight distribution according to the treatment steps of purifying system for pig wastewater. The results obtained are summarized as follow. 1. Raw pig wastewater contained 31 kinds of organic compounds such as acetic acid, 2-butanone, hexane, chloroform, propionic acid, butanoic acid etc. 2. After ozone treatment, 13 kinds of organic compounds such as ethene, pentane, 2-methylopropanal, hexane, furan and so on were detected in the wastewater. 3. After $TiO_2$ treatment, 12 kinds of organic compounds such as ethene, hexane, chloroform, 1-decene, silane and so on were detected from the effluent. 4. After both ozone and $TiO_2$ treatment, portion of molecular weight smaller than 500 Daltons and molecular weight between 500 to 1,000 Daltons in the wastewater increased.

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Caspase-3-facilitated Stoichiometric Cleavage of a Large Recombinant Polyprotein (카스파제-3 효소를 이용한 폴리-단백질의 정량적 프로세싱 분석)

  • Kim, Moonil
    • Journal of Life Science
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    • v.25 no.4
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    • pp.385-389
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    • 2015
  • In this study, it is reported that a large polyprotein can be stoichiometrically cleaved by the use of caspase-3-dependent proteolysis. Previously, it has been shown that the proteolytic IETD motif was partially processed when treated with caspase-3, while the DEVD motif was completely cleaved. The cleavage efficiency of the DEVD-based substrate was approximately 2.0 times higher than that of the IETD substrate, in response to caspase-3. Based on this, 3 protein genes of interest were genetically linked to each other by adding two proteolytic cleavage sequences, DEVD and IETD, for caspase-3. Particularly, glutathione-S transferase (GST), maltose binding protein (MBP), and red fluorescent protein (RFP) were chosen as model proteins due to the variation in their size. The expressed polyprotein was purified by immobilized metal ion affinity chromatography (IMAC) via a hexa-histidine tag at the C-terminal end, showing 93 kDa of a chimeric GST:MBP:RFP fusion protein. In response to caspase-3, cleavage products, such as MBP:RFP (68 kDa), MBP (42 kDa), RFP (26 kDa), and GST (25 kDa), were separated from a large precursor GST:MBP:RFP (93 kDa) via SDS-PAGE. The results obtained from this study indicate that a multi-protein can be stoichiometrically produced from a large poly-protein by using proteolytic recognition motifs, such as DEVD and IETD tetra-peptides, for caspase-3.