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Purification and Characterization of a Lectin from Arisaema tortuosum Schott Having in-vitro Anticancer Activity against Human Cancer Cell Lines

  • Dhuna, Vikram;Bains, Jagmohan Singh;Kamboj, Sukhdev Singh;Singh, Jatinder;Shanmugavel;Saxena, Ajit Kumar
    • BMB Reports
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    • 제38권5호
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    • pp.526-532
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    • 2005
  • A lectin with in-vitro anticancer activity against established human cancer cell lines has been purified by affinity chromatography on asialofetuin-linked amino activated silica beads from the tubers of Arisaema tortuosum, popularly known as Himalayan Cobra lily, a monocot plant from the family Araceae. The bound Arisaema tortuosum lectin (ATL) was eluted with glycine-HCl buffer, pH 2.5. ATL was effectively inhibited by asialofetuin, a complex desialylated serum glycoprotein as well as by N-acetyl-D-lactosamine, a disaccharide. It gave a single band corresponding to a subunit molecular weight of 13.5 kDa in SDS-PAGE, pH 8.8 both under reducing and non reducing conditions. When subjected to gel-filtration on Biogel P-200, it was found to have a molecular weight of 54 kDa, suggesting a homotetramer structure, in which individual polypeptides are not bound to each other with disulfide bonds. ATL is a glycoprotein with 0.9% carbohydrate content, stable up to $55^{\circ}C$ and at pH 2 to 10. The lectin had no requirement for divalent metal ions i.e. $Ca^{2+}$ and $Mn^{2+}$ for its activity. However, as reported for other monocot lectins, ATL gave multiple bands in isoelectric focusing and Native PAGE, pH 8.3. The lectin was found to inhibit in vitro proliferation of human cancer cell lines HT29, SiHa and OVCAR-5.

포장디자인의 변화가 소비자 구매행동에 미치는 영향 - 자연조미료 포장디자인을 중심으로 - (An Influence on the Change of Consumer Purchase Behavior by Package Design - Focusing on the Natural Flavor Packaging Design -)

  • 김선주;이대영;성정환
    • 한국콘텐츠학회논문지
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    • 제10권2호
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    • pp.25-34
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    • 2010
  • 제품에 대한 소비자의 구매충성도는 구매시점의 새로운 자극에 노출되는 순간 변화할 수 있으며 이 시점에서 타제품과 시각적으로 차별되고 심미적으로 아름답게 제작되어 소비자의 시선을 유도할 수 있는 포장디자인이 필요하다. 본 연구에서는 조미료 포장디자인의 실질적구매자로 판단되는 29세부터 34세까지 평균 31.65세의 기존 조미료의 경험과 새롭게 출시된 조미료 포장디자인에 대한 소비자 인지를 알아보기 위하여 조미료 포장디자인에 대한 디자인분석을 실시하고 설문조사 하였다. 소비자는 기존 조미료 포장디자인에 대하여 비교적 특색이 없다고 하였으며 기존 서체를 재 디자인 로고타입보다 캘리그라피 로고타입에 대하여 '상징성'은 좋은 평가를 보였으나 '가독성'은 낮다고 응답했다. 또한 새롭게 출시된 두 조미료 포장디자인에 대하여 '맛선생'을 '산들애'에 보다 '안전성', '품질보전성', '전시성' 요소를 높게 평가하고 요소들 중 '안전성'을 조미료제품의 구매 욕구를 일으킬 수 있는 요소로 꼽았다.

An Improved Plant Regeneration Protocol using Cotyledonary Explants from Inbred Lines of Chinese Cabbage (Brassica rapa ssp. pekinensis)

  • Yang Zhi Hong;Jin Hua;Plaha Prikshit;Woong Bae Tae;Jiang Guo Bin;Woo Jong Gyu;Yun Han Dae;Lim Yong Pyo;Lee Hyo Yeon
    • Journal of Plant Biotechnology
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    • 제6권4호
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    • pp.235-239
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    • 2004
  • We studied the effect of genotype, explant, age of explant, medium (plant growth regulators and gelling agents), and standardized an efficient regeneration protocol for inbred lines of Chinese cabbage (Brassica rapa ssp. pekinensis). Of the different concentrations of NAA and BA tested, the combination of $5\;\cal{mg/L}\;BA\;and\;0.5\;\cal{mg/L}$ NAA gave the highest frequency of shoot regeneration. The cotyledonary explants had more shoot regeneration frequency ($\ge40\%$) than the hypocotyl ex-plants. Besides, the cotyledonary explants, excised from the 4-day old seedlings, showed higher shoot regene-ration ($56.7\%$). Of the three gelling agents and their concentrations used, 16 g/L agar was found to be the best for shoot regeneration. Shoot regeneration frequency in-creased significantly by supplementing the medium with $4\;\cal{mg/L}\;of\;AgNO_3$ MS medium devoid of NAA showed higher frequency of rooting in the regenerated shoots than the ones supplemented with NAA. Our improved regeneration protocol will be especially useful for the genetic transformation of Chinese cabbage inbred lines to develop transgenic hybrids.

AUA as a Translation Initiation Site In Vitro for the Human Transcription Factor Sp3

  • Hernandez, Eric Moore;Johnson, Anna;Notario, Vicente;Chen, Andrew;Richert, John R.
    • BMB Reports
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    • 제35권3호
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    • pp.273-282
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    • 2002
  • Sp3 is a bifunctional transcription factor that has been reported to stimulate or repress the transcription of numerous genes. Although the size of Sp3 mRNA is 4.0kb, the size of the known Sp3 cDNA sequence is 3.6kb. Thus, Sp3 functional studies have been performed with an artificially introduced start codon, and thus an amino-terminus that differs from the wild-type. Ideally, full-length cDNA expression vectors with the appropriate start codon should be utilized for these studies. Using 5'rapid amplification of cDNA ends, a full-length Sp3 cDNA clone was generated and the sequence verified in nine cell lines. No AUG initiation codon was present. However, stop codons were present in all three frames 5' to the known coding sequence. In vitro translation of this full-length cDNA clone produced the expected three isoforms-one at 100 kDa and two in the mid 60 kDa range. Electrophoretic mobility shift assays showed that the protein products had the ability to bind to the Sp1/3 consensus sequence. In vitro studies, using our Sp3 clone and site directed mutagenesis, identified the translation initiation site for the larger isoform as AUA. AUA has not been previously described as an endogenous initiation codon in eukaryotes.

Usefulness of Oblique Lateral Interbody Fusion at L5-S1 Level Compared to Transforaminal Lumbar Interbody Fusion

  • Mun, Hah Yong;Ko, Myeong Jin;Kim, Young Baeg;Park, Seung Won
    • Journal of Korean Neurosurgical Society
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    • 제63권6호
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    • pp.723-729
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    • 2020
  • Objective : The use of oblique lateral interbody fusion at the L5-S1 level (OLIF51) is increasing, but no study has directly compared OLIF51 and transforaminal lumbar interbody fusion (TLIF) at the L5-S1 level. We evaluated the usefulness of OLIF51 by comparing clinical and radiologic outcomes with those of TLIF at the same L5-S1 level. Methods : We retrospectively reviewed and compared 74 patients who underwent OLIF51 (OLIF51 group) and 74 who underwent TLIF at the L5-S1 level (TLIF51 group). Clinical outcomes were assessed with the visual analogue scale for back pain and leg pain and the Oswestry Disability Index. Mean disc height (MDH), foraminal height (FH), disc angle (DA), fusion rate, and subsidence rate were measured for radiologic outcomes. Results : The OLIF51 group used significantly higher, wider, and larger-angled cages than the TLIF51 group (p<0.001). The postoperative MDH and FH were significantly greater in the OLIF51 group than in the TLIF51 group (p<0.001). The postoperative DA was significantly larger in the OLIF51 group than in the TLIF51 group by more than 10º (p<0.001). The fusion rate was 81.1% and 87.8% at postoperative 6 months in the OLIF51 and TLIF51 groups, respectively, and the TLIF51 group showed a higher fusion rate (p<0.05). The subsidence rate was 16.2% and 25.3% in the OLIF51 and TLIF51 groups, respectively, and the OLIF51 group showed a lower subsidence rate (p<0.05). Conclusion : OLIF51 was more effective for the indirect decompression of foraminal stenosis, providing strong mechanical support with a larger cage, and making a greater lordotic angle with a high-angle cage than with TLIF.

Evaluating Nutritional Quality of Single Stage- and Two Stage-fermented Soybean Meal

  • Chen, C.C.;Shih, Y.C.;Chiou, P.W.S.;Yu, B.
    • Asian-Australasian Journal of Animal Sciences
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    • 제23권5호
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    • pp.598-606
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    • 2010
  • This study investigated the nutritional quality of soybean meal (SBM) fermented by Aspergillus ($FSBM_A$) and/or followed by Lactobacillus fermentation ($FSBM_{A+L}$). Both fermented products significantly improved protein utilization of SBM with higher trichloroacetic acid (TCA) soluble true protein content, in vitro protein digestibility and available lysine content, especially in $FSBM_{A+L}$. Moreover, $FSBM_{A+L}$ produced a huge amount of lactic acid resulting in lower pH as compared to the unfermented SBM or soybean protein concentrate (SPC) (p<0.05). $FSBM_A$ and $FSBM_{A+L}$ raised 4.14% and 9.04% of essential amino acids and 5.38% and 9.37% of non-essential amino acids content, respectively. The ${\alpha}$-galactoside linkage oligosaccharides such as raffinose and stachyose content in $FSBM_A$ and $FSBM_{A+L}$ decreased significantly. The results of soluble protein fractions and distribution showed that the ratio of small protein fractions (<16 kDa) were 42.6% and 63.5% for $FSBM_A$ and $FSBM_{A+L}$, respectively, as compared to 7.2% for SBM, where the ratio of large size fractions (>55 kDa, mainly ${\beta}$-conglycinin) decreased to 9.4%, 5.4% and increased to 38.8%, respectively. There were no significant differences in ileal protein digestibility regardless of treatment groups. SPC inclusion in the diet showed a better protein digestibility than the SBM diet. In summary, soybean meal fermented by Aspergillus, especially through the consequent Lactobacillus fermentation, could increase the nutritional value as compared with unfermented SBM and is compatible with SPC.

할미송이버섯으로부터 혈전용해효소의 정제 및 특성 연구 (II) (Purification and Characterization of Fibrinolytic Enzyme from Tricholoma saponaceum (II))

  • 김준호
    • 대한의생명과학회지
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    • 제6권4호
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    • pp.261-268
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    • 2000
  • 할미송이버섯으로부터 혈전용해효소 (FE-2)를 DEAE-cellulose, Mono-S column으로 분리 정제하였다. 이 효소는 분자량이 18.2 kDa 이었으며, ICP-MS (inductively coupled plasma mass spectrometer) 분석 결과 $Zn^{2+}$을 포함하고 있었다. 15번째까지의 N-terminal amino acid 서열은 A-L-Y-V-G-X-S-P-X-Q-Q-S-L-L-V이고, pH 7.5에서 활성이 가장 큰 염기성 단백질 분해효소로, EDTA와 1,10-phenanthroline에 의해 활성이 감소되는 metalloprotease였다. $Mg^{2+}$, $Zn^{2+}$, Fe$^{2+}$, Co$^{2+}$의 첨가 시 활성이 증가하였으나 Hg$^{2+}$의 경우 활성이 완전히 소멸되었다. 이 효소 (FE-2)는 단백질 분해효소 저해제인 E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane), PMSF (phenylmethylsulfonyl fluoride), pepstatin 과 2-mercaptoethanol의 영향을 받지 않으며, 섬유소원 (fibrinogen)과 반응 시 $A\alpha$ chain과 B$\beta$ chain은 분해시키지만 $\gamma$ chain과는 반응하지 않았다.

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Biochemical Characterization of an Extracellular ${\beta}$-Glucosidase from the Fungus, Penicillium italicum, Isolated from Rotten Citrus Peel

  • Park, Ah-Reum;Hong, Joo-Hee;Kim, Jae-Jin;Yoon, Jeong-Jun
    • Mycobiology
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    • 제40권3호
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    • pp.173-180
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    • 2012
  • A ${\beta}$-glucosidase from Penicillium italicum was purified with a specific activity of 61.8 U/mg, using a chromatography system. The native form of the enzyme was an 88.5-kDa tetramer with a molecular mass of 354 kDa. Optimum activity was observed at pH 4.5 and $60^{\circ}C$, and the half-lives were 1,737, 330, 34, and 1 hr at 50, 55, 60, and $65^{\circ}C$, respectively. Its activity was inhibited by 47% by 5 mM $Ni^{2+}$. The enzyme exhibited hydrolytic activity for p-nitrophenyl-${\beta}$-D-glucopyranoside (pNP-Glu), p-nitrophenyl-${\beta}$-D-cellobioside, p-nitrophenyl-${\beta}$-D-xyloside, and cellobiose, however, no activity was observed for p-nitrophenyl-${\beta}$-D-lactopyranoside, p-nitrophenyl-${\beta}$-D-galactopyranoside, carboxymetyl cellulose, xylan, and cellulose, indicating that the enzyme was a ${\beta}$-glucosidase. The $k_{cat}/K_m\;(s^{-1}mM^{-1})$ values for pNP-Glu and cellobiose were 15,770.4 mM and 6,361.4 mM, respectively. These values were the highest reported for ${\beta}$-glucosidases. Non-competitive inhibition of the enzyme by both glucose ($K_i=8.9mM$) and glucono-${\delta}$-lactone ($K_i=11.3mM$) was observed when pNP-Glu was used as the substrate. This is the first report of non-competitive inhibition of ${\beta}$-glucosidase by glucose and glucono-${\delta}$-lactone.

다발구멍장이버섯으로부터 분리한 혈전용해 세린분해효소의 특성 연구 (Characterization of a Fibrinolytic Serine Protease from an Edible Mushroom, Albatrellus confluens)

  • 김준호
    • 한국균학회지
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    • 제36권2호
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    • pp.183-188
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    • 2008
  • 3단계를 거처 식용 가능한 다발구멍장이버섯으로부터 분리한 혈전용해효소의 비활성은 30.49 U/mg이었으며, MALDI-TOF/MS로부터 분자량이 30086.41 Da이었다. 일차 아미노산 구조는 Glu-Thr-Val-Thr-Glu-Thr-Thr-Ala-Pro-Trp-Gly-Leu-Ser-Arg-Ile으로 밝혀졌으며, pH 8.0에서 pH 10.0의 넓은 범위에서 큰 활성을 나타내는 alkaline protease로, 최적온도는 $50^{\circ}C$이며, $30^{\circ}C$까지는 대체로 열에 안정한 효소였다. 이 효소는 합성 기질 N-Suc-Ala-Ala-Pro-Phe pNA을 강하게 분해하였으며, $Hg^{2+}$ 금속이온의 첨가로 효소 활성이 완전히 사라졌다. 효소저해제인 PMSF의 첨가로 혈전용해활성이 사라지는 결과로부터 serine 분해효소임을 알 수 있었다.

Botrytis cinerea 균주들이 생산하는 Polygalacturonase, Laccase, ${\beta}$-glucosidase의 균주 간 화성 및 병원성과의 상관관계 (Relationships Between Pathogenicty and Activities of Polygalacturonase, Laccase, and ${\beta}$-Glucosidase Produced by Botrytis cinerea)

  • 김종진;김재원;이창원;정영륜
    • 한국식물병리학회지
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    • 제13권4호
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    • pp.225-231
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    • 1997
  • 기주, 분리 연도 및 장소가 다른 Botryis cinerea 20개 균주를 이용하여 오이세포벽을 탄소원으로 첨가한 배지에서 배양한 뒤 병원성과 관련이 있는 것으로 알려진 polygalacturomase, Iaccase, $\beta$-glucosidase의 활성을 측정하고 병원성과의 상관관계를 조사하였다. 조사된 효소들의 활성과 병원성과의 상관관계는 상관계수가 모두 0.5 이하로 직접적인 연관성을 인정할 수 없었다. 그러나 Bovendamm 배지에서의 균사생장 정도의 병원성은 r=0.522(p=0.05)로 정의 상관관계를 보였다. 또한 병원성이 강한 3균주와 약한 3균주를 선발하여 각 기주의 세포벽을 첨가하여 배양 후 poly-galacturonase 활성정도를 비교하였으나 뚜렷한 경향을 찾을 수 없었다. Exo-PG 항체를 이용한 면역혈청 blotting 결과 병원성이 다른 거의 모든 균주가 66kDa 크기의 1 band를 보이는 유사한 효소 pattern을 보였다. 이상의 결과로 볼 때 측정된 이들 효소는 병원성과 관련된 일차적 요인이 아닐 것으로 생각한다.

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