• Title/Summary/Keyword: Enzyme assays

검색결과 305건 처리시간 0.021초

Biochemical Study of Recombinant PcrA from Staphylococcus aureus for the Development of Screening Assays

  • Dubaele, Sandy;Martin, Christophe;Bohn, Jacqueline;Chene, Patrick
    • BMB Reports
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    • 제40권1호
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    • pp.7-14
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    • 2007
  • Helicases are ubiquitous enzymes, which utilize the energy liberated during nucleotide triphosphate hydrolysis to separate double-stranded nucleic acids into single strands. These enzymes are very attractive targets for the development of new antibacterial compounds. The PcrA DNA helicase from Staphylococcus aureus is a good candidate for drug discovery. This enzyme is unique in the genome of S. aureus and essential for this bacterium. Furthermore, it has recently been published that it is possible to identify inhibitors of DNA helicases such as PcrA. In this report, we study the properties of recombinant PcrA from S. aureus purified from Escherichia coli to develop ATPase and helicase assays to screen for inhibitors.

Alpha-Amylase Immobilization on Epoxy Containing Thiol-Ene Photocurable Materials

  • Cakmakci, Emrah;Danis, Ozkan;Demir, Serap;Mulazim, Yusuf;Kahraman, Memet Vezir
    • Journal of Microbiology and Biotechnology
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    • 제23권2호
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    • pp.205-210
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    • 2013
  • Thiol-ene polymerization is a versatile tool for several applications. Here we report the preparation of epoxide groups containing thiol-ene photocurable polymeric support and the covalent immobilization of ${\alpha}$-amylase onto these polymeric materials. The morphology of the polymeric support was characterized by scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) coupled with SEM was used to explore the chemical composition. The polymeric support and the immobilization of the enzyme were characterized by FTIR analysis. SEM-EDS and FTIR results showed that the enzyme was successfully covalently attached to the polymeric support. The immobilization efficiency and enzyme activity of ${\alpha}$-amylase were examined at various pH (5.0-8.0) and temperature ($30-80^{\circ}C$) values. The storage stability and reusability of immobilized ${\alpha}$-amylase were investigated. The immobilization yield was $276{\pm}1.6$ mg per gram of polymeric support. Enzyme assays demonstrated that the immobilized enzyme exhibited better thermostability than the free one. The storage stability and reusability were improved by the immobilization on this enzyme support. Free enzyme lost its activity completely within 15 days. On the other hand, the immobilized enzyme retained 86.7% of its activity after 30 days. These results confirm that ${\alpha}$-amylase was successfully immobilized and gained a more stable character compared with the free one.

Enzyme Profiles of Alga-Lytic Bacterial Strain AK-13 Related with Elimination of Cyanobacterium Anabaena cylindrica

  • Kim, Jeong-Dong;Han, Myung-Soo
    • 환경생물
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    • 제22권1호
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    • pp.184-191
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    • 2004
  • To investigate bacteria with algalytic activities against Anabaena cylindrica when water blooming occurs and to study enzyme profiles associated with alga-lytic activity, various bacterial strains were isolated from surface waters and sediments in eutrophic lakes or reservoirs in Korea. Among 178 isolates, only nine isolates exhibited lytic abilities against A cylindrica on the agar plates, and then the isolate AK-13 was selected as the strongest in lysing the cyanobacterium A. cytindrica. The strain AK-13 was characterized and identified as Sinorhizobium sp. based on fatty acid methyl ether profiles and 16S rDNA sequence. According to the results of the enzyme assays, in the strain An-13 of Sinorhizobium sp., alginase, amylase, proteinase (caseinase and gelatinase), carboxymethyl-cellulase (CMCase), laminarinase, and lipase was produced, namely CMCase, laminarinase and protease were highly active. None of glycosidase was produced. Therefore, enzyme systems of Sinorhizobium sp. AK-13 were very complex to degrade cell walls of A. cylindrica. The peptidoglycans of A. cylindrica mat be hydrolyzed and metabolized to a range of easily utilizable monosaccharides or other low molecular weight organic substances by Sinorhizobium sp. AK-13.

해바라기씨 추출물의 천식 완화 효과 (Water-extract of Helianthus annuus Seed Exhibits Potent Anti-asthma Activity In Vitro and In Vivo)

  • 허진철;박자영;우상욱;정신교;정규식;이진만;마진열;이상한
    • 한국식품저장유통학회지
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    • 제13권4호
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    • pp.495-500
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    • 2006
  • 항산화물에 의한 천식 억제 효과를 확인하기 위하여 항산화정도를 알아볼 수 있는 DPPH, FRAP, hydroxyl radical 소거활성 실험을 high throughput-compatible chemical assay로 디자인하여 해바라기씨 추출물에 적용하여 보았다. 해바라기씨 추출물의 용매에 따른 항산화 효과 중 $H_2O_2$에 의한 산화스트레스에 의한 세포사멸을 DW추출물에서 강한 억제능력을 보였다. 이를 이용하여 마우스를 이용한 천식 동물모델에 적용하여 탁월한 천식 억제효과를 $CD4^+$세포, IL-4와 IL-13의 발현정도를 통해 알수 있었다. 해바라기씨 추출물은 천식에 탁월한 효과가 있는 것으로 in vitro 및 in vivo 실험으로 증명하였다.

SH-SY5Y 인간 신경모세포종 세포에서 MPTP 유발 세포 독성에 대한 거저리(Tenebrio molitor) 추출물의 보호효과 (Protective effects of mealworm (Tenebrio molitor) extract on N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced cellular toxicity in SH-SY5Y neuroblastoma cells)

  • 조인호;김유지;김선태
    • 대한임상독성학회지
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    • 제21권2호
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    • pp.81-91
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    • 2023
  • Purpose: Edible insect extracts have been used as an alternative source for medicinal supplements due to their significant antioxidative and anti-inflammatory activity. Recent studies have reported that anti-microbial peptides from insects have neuroprotective effects on dopamine toxins. The purpose of this study was to investigate the protective functions of mealworm (Tenebrio molitor) extract (MWE) on N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced cellular toxicity in SH-SY5Y neuroblastoma cells. Methods: Cellular toxicity induced by the MPTP toxin and the impact of MWE on cell survival were analyzed using MTT assays. DAPI staining was performed to observe apoptotic phenomena caused by MPTP. Changes in caspase-3 activity and protein expression were observed using enzyme activity assays and western blot assays, respectively. Results: MWE exerted significant antioxidant activity, which was measured by both DPPH and ABTS radical assays, with a dose-dependent relationship. Furthermore, MWE resulted in cellular proliferation in SHSY5Y cells in a dose-dependent manner. Furthermore, MWE pretreatment significantly inhibited MPTP-induced cytotoxicity, with a dose-dependent relationship. The morphological characteristics of apoptosis and increased reactive oxygen species induced by MPTP were also significantly reduced by MWE pretreatment. Conclusion: MWE treatment significantly attenuated MPTP-induced changes in the levels of proteins associated with apoptosis, such as caspase-3 and PARP. These findings suggest that MWE exerts neuroprotective effects on human neuroblastoma SH-SY5Y cells subject to MPTP-induced dopaminergic neurodegeneration.

Synthesis of New Diselenide Compounds as Anti-Inflammatory Agents

  • Shen, Liulan;Shin, Kyung-Min;Lee, Kyung-Tae;Jeong, Jin-Hyun
    • Archives of Pharmacal Research
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    • 제27권8호
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    • pp.816-819
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    • 2004
  • Many diselenide compounds are used as antioxidants, enzyme inhibitors and cytokine induc-ers. Three new diselenide compounds, bis-(2-hydroxyphenyl) diselenide, bis-(3-hydroxyphe-nyl) diselenide and bis-(4-hydroxyphenyl) diselenide were designed and synthesized as anti-inflammatory agent. All of them were found to have strong in vitro activity in anti-inflammatory assays.

Assay development and HTS on microfluidic Lab-on-a-chip

  • Yang, Eun-Gyeong
    • 한국응용약물학회:학술대회논문집
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    • 한국응용약물학회 2002년도 창립10주년기념 및 국립독성연구원 의약품동등성평가부서 신설기념 국재학술대회:생물학적 동등성과 의약품 개발 전략을 위한 국제심포지움
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    • pp.73-78
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    • 2002
  • Microfluidic lab-on-a-chip (LOC) systems have enabled a new generation ofassay technologies in chemical and biomedical sciences. Caliper's microfluidic LOC systems contain a network of microscopic channels through which fluids and chemical are moved in order to perform experiments. The main advantages of these continuous-flow devices are integration and automation of multiple steps in complex analytical procedures to improve the reproducibility of the results, and eliminated the manual labor, time and pipetting errors involved in analyses. The present talk is devoted to give a brief introduction of microfluidic basics and to present in applying continuous-flow microchips to drug screening with model enzyme assays.

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Design of Smart Polymer Constructs for Use in Microfluidic Diagnostic Assays

  • Hoffman Allan S.
    • 한국고분자학회:학술대회논문집
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    • 한국고분자학회 2006년도 IUPAC International Symposium on Advanced Polymers for Emerging Technologies
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    • pp.42-43
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    • 2006
  • We have prepared random, block and graft copolymers with single or dual sensitivities to various stimuli. We have conjugated these polymers to proteins at random lysine sites or at specific sites designed into the protein by genetic engineering. We are also grafting the smart polymers to the surfaces of nanobeads. We are applying these smart conjugates and smart nanobeads in microfluidic devices for various applications, including diagnostics, affinity separations and enzyme bioprocesses. In this talk I will update our work with these interesting hybrid systems.

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The Synthesis of New Leuco Squarylium Dyes

  • Park, Soo-Youl;Jun, Kun;Shin, Seung-Rim;Shin, Jong-Il
    • 한국염색가공학회지
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    • 제19권5호
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    • pp.24-29
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    • 2007
  • It is well known that certain classes of dye are reducible to stable colorless leuco compounds, which can be oxidized in air back to the colored species. We now reported that bis(4-dialkylaminophenyl)squaraine dyes are readily reduced in solution by borohydride to give alkali soluble leuco compound, which exists in the 3-hydroxy 2,4-bis(4-dialkylaminophenyl)cyclobuten-one. New alkylamine leuco compounds were synthesized by the reaction of leuco chloro-squaraine with alkylamine derivatives. The leuco compounds are easily isolated and can be air oxidized back to the squaraine dyes. These dyes have many technical application. e.g. in xerography, solar cell, optical recording material, redox indicators, and enzyme assays.