A TLC bioautographic assay for the detection of nitrofurantoin

合集下载

Human reproductive failure

• • • • • • • • • •
Categories of reproductive failure are defined as follows. Infertility is the failure to conceive after frequent unprotected intercourse (Reiss, 1998). Unexplained infertility occurs when no cause of infertility can be identified after full clinical investigation of both partners (Reiss, 1998). Legally, in the UK, miscarriage is defined as the spontaneous loss of a pregnancy with a gestational age of 24weeks or less (Regan,1997; Reiss, 1998). Alternatively, the World Health Organization (WHO) has defined a miscarriage as the loss of a fetus or embryo weighing <500 g, which would normally be at 20±22 complete weeks of gestation (WHO, 1977). Recurrent miscarriage, used synonymously with habitual miscarriage, habitual abortion or recurrent abortion, is defined as the loss of three or more consecutive pregnancies before the 24th week of gestation (Reiss, 1998). Some authors also use the terms recurrent miscarriage (RM) and recurrent spontaneous abortion (RSA) interchangeably. However, RSA has been defined as the loss of two or more, or even three or more, clinically detectable pregnancies with no reference to the week of gestation. Due to the discrepancy between the number of losses and time period of reproductive failure, this review differentiates between studies involving women with RM and RSA. Finally, reproductive success is defined as the ability to conceive and carry a pregnancy to term and reproductive outcome refers to both reproductive failure and success.

薄层色谱生物自显影法筛选新疆莫合烟天然乙酰胆碱酯酶抑制剂的活性成分

薄层色谱生物自显影法筛选新疆莫合烟天然乙酰胆碱酯酶抑制剂的活性成分王健;朱萍萍;倪国柱;温琳豫;支玲;胡曙晨【期刊名称】《中国药业》【年(卷),期】2014(000)020【摘要】Objective To explain nicotine in tobacco can reduce the incidence of degenerative diseases,perform the activity screening of acetyl cholinesterase inhibitors of Xinjiang's unique Mohe-tobacco for illuminating the action mechanism. Methods The thin layer chromatography(TLC)bioautographic method combining TLC with biological activity assay was used to conduct the activity screening of extract solution of Xinjiang Mohe-tobacco. Results Nicotine in the extract solution of Xinjiang Mohe-tobacco appeared the white spots on the purple background by the TLC bioautography,indicating that which possesses the activity,but the white spots were weak, indicating that the nicotine content in the extract solution was unable to reach the significant inhibiting concentration,its activity was lower than that of the reference substance huperzine A;the DPPH development method revealed that the extraction solution of Xinjiang Mohe-tobacco simultaneously possesses the better antioxidant activity. Conclusion Nicotine in Xinjiang Mohe-tobacco can be com-bined with acetyl cholinesterase and inhibits its activity. The TLC bioautographic method is easy to operate with high sensitivity andspecificity in the activity screening for natural acetyl cholinesterase inhibitor.%目的：为阐明烟草中的烟碱降低退行性疾病发病率的作用机理，对新疆特有的莫合烟进行天然乙酰胆碱酯酶抑制剂的活性筛选。

Tet-On 3G Вector Set (with ZsGreen1) 商品说明书

Certificate of Analysis Takara Bio USA, Inc.1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: ********************United States/Canada 800.662.2566 Asia Pacific+1.650.919.7300Europe+33.(0)1.3904.6880Japan+81.(0)77.565.6999Page 1 of 6Tet-On® 3G Vector Set (with ZsGreen1)Table of ContentsDescription (1)pCMV-Tet3G Vector Information (2)pTRE3G-ZsGreen1 Vector and pTRE3G-Luc Control Vector Information (4)Quality Control Data (6)Catalog No. Lot Number631159 (Not sold separately) Specified on product label.DescriptionThe Tet-On 3G Vector Set (with ZsGreen1) is used to create tightly regulated and highly responsive tetracycline (Tet)-inducible mammalian expression systems that are turned on by the addition of doxycycline to the culture medium. The Tet-On 3G Vector Set (with ZsGreen1) allows the simultaneous expression of a gene of interest and a green fluorescent protein marker.Package Contents•20 μl pCMV-Tet3G Vector (500 ng/μl)•20 μl pTRE3G-ZsGreen1 Vector(500 ng/μl)•20 μl pTRE3G-Luc Control Vector(500 ng/μl)•40 μl Linear Hygromycin Marker (50 ng/μl)•40 μl Linear Puromycin Marker (50 ng/μl)Storage Conditions•Store plasmids at –20°C.•Spin briefly to recover contents.•Avoid repeated freeze/thaw cycles.Shelf Life• 1 year from date of receipt under proper storage conditions.Storage Buffer•10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)Shipping Conditions• Dry ice (–70°C)Product DocumentsDocuments for our products are available for download at /manualsThe following documents apply to this product:•Tet-On 3G Expression Systems User Manual (PT5148-1)pCMV-Tet3G Vector InformationFigure 1. pCMV-Tet3G Vector Map.DescriptionThe pCMV-Tet3G Vector expresses Tet-On 3G, a tetracycline-controlled transactivator that exhibits high activity in the presence of the inducer doxycycline (Dox), and exceptionally low activity in its absence. Tet-On 3G results from the fusion of amino acids 1–207 of a mutant Tet repressor (TetR) to 39 amino acids that form three minimal "F"-type transcriptional activation domains from the herpes simplex virus VP16 protein. Tet-On 3G was derived from Tet-On Advanced (Zhou et al. 2006; Urlinger et al. 2000; Gossen and Bujard 1992; Gossen et al. 1995); as a result, it’s fully synthetic, lacks cryptic splice sites, and is codon-optimized for stable expression in mammalian cells. Compared to both of its predecessors, however, this 3rd generation Tet-On transactivator demonstrates increased sensitivity to Dox (Zhou et al. 2006). Constitutive expression of Tet-On 3G is driven by the human cytomegalovirus immediately early promoter (P CMV IE).Location of Features in pCMV-Tet3G•P CMV IE(human cytomegalovirus immediate early promoter): 2–688•Tet-On 3G (transactivator gene): 775–1521•SV40 polyA signal: 1536–1991•pUC origin of replication: 2342–2996•Amp r (ampicillin resistance gene; β-lactamase): 3144–4004 (complementary)•SV40 polyA signal: 4275–4809 (complementary)•Kan r/Neo r (kanamycin/neomycin resistance gene): 5417–6211 (complementary)•P SV40 e (SV40 early promoter): 6532–6891 (complementary)Additional InformationpCMV-Tet3G is used to develop stable Tet-On 3G cell lines, which are hosts for Tet-inducible gene expression systems. To create a Tet-inducible expression system, a vector containing a gene of interest under the control of the Tet-inducible TRE3G promoter(P TRE3G) is transfected into a Tet-On 3G cell line. The addition of Dox to the system causes Tet-On 3G to undergo a conformational change that allows it to bind to P TRE3G, activating transcription of the gene of interest in a highly dose-dependent manner. Additional information on TRE-containing vectors, and protocols describing the construction of Tet-On 3G cell lines can be found in the Tet-On 3G Expression Systems User Manual (PT5148-1).Propagation in E. coli•Suitable host strain: Stellar™ Competent Cells•Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.• E. coli replication origin: pUCpTRE3G-ZsGreen1 Vector and pTRE3G-Luc Control Vector InformationFigure 2. pTRE3G-ZsGreen1 Vector and pTRE3G-Luc Control Vector Maps.Figure 3. pTRE3G-ZsGreen Vector Multiple Cloning Site. The internal start site (ATG) at the IRES2/MCS junction is indicated in bold.DescriptionpTRE3G-ZsGreen1is a Tet-inducible, mammalian expression vector designed to coexpress a gene of interest and the green fluorescent protein ZsGreen1 under the control of the Tet-responsive promoter P TRE3G. This promoter consists of a highly optimized Tet-responsive element (TRE) just upstream of a minimal CMV promoter. P TRE3G exhibits exceptionally low basal activity; it’s induced by the binding of Tet-On 3G but is virtually silent in its absence. The vector is designed to be used as part of our Tet-On 3G Inducible Expression System (Cat. No. 631164).ZsGreen1 is a human codon-optimized variant of the reef coral Zoanthus sp. green fluorescent protein (ZsGreen) that has been engineered for brighter fluorescence (excitation and emission maxima: 493 and 505 nm, respectively; Matz et al. 1999; Haas, Park, and Seed 1996). p TRE3G-ZsGreen allows Dox-inducible coexpression of ZsGreen1 and a gene of interest from a bicistronic mRNA transcript. An encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES2), positioned between ZsGreen1 and the gene of interest, facilitates cap-independent translation of the gene of interest from an internal start site at the IRES2/MCS junction (Jang et al. 1988). This ensures that a high percentage of ZsGreen1-expressing clones also express the gene of interest, allowing ZsGreen1 to be used as an indicator of inducibility and transfection efficiency, as well as a marker for selection by flow cytometry. The vector also contains a pUC origin of replication and an ampicillin resistance gene (Amp r) to allow for propagation and selection in E. coli.The pTRE3G-Luc is a Tet-inducible control vector that expresses firefly luciferase under the control of P TRE3G. When used with standard luciferase detection reagents, this vector can be used as a reporter of induction efficiency (see User Manual for protocol). pTRE3G-Luc is not intended to be used as a cloning vector.Location of Features in pTRE3G-ZsGreen1•P TRE3G (3rd generation Tet-responsive promoter): 7–382•ZsGreen1: 389–1084•IRES2 (encephalomyocarditis virus internal ribosome entry site): 1091–1673•MCS (multiple cloning site): 1686–1721•SV40 polyA signal: 1776–2573•pUC origin of replication: 2838–3481•Amp r (ampicillin resistance gene; β-lactamase): 3629–4489 (complementary)Location of Features in pTRE3G-Luc•P TRE3G (3rd generation Tet-responsive promoter): 7–382•Luciferase: 432–2084•SV40 polyA signal: 2151–2948•pUC origin of replication: 3213–3856•Amp r (ampicillin resistance gene; β-lactamase): 4004–4864 (complementary)Additional InformationpTRE3G-ZsGreen1 is a mammalian expression vector that allows tightly regulated, doxycycline-controlled coexpression of a gene of interest and ZsGreen1. The gene of interest must have both a start and a stop codon. The gene of interest should be cloned in-frame with the start codon at the IRES2/MCS junction (this codon is shown in bold in the MCS sequence in Figure 3, page 3; see the User Manual for details on how to use In-Fusion® to simplify your cloning). Cotransfection of pTRE3G-ZsGreen1 constructs with Linear Hygromycin or Puromycin Markers allows antibiotic selection of stable transfectants. In order to function, the system requires the presence of the Tet-On 3G transactivator protein, supplied by a stable Tet-On 3G cell line created with our Tet-On 3G Inducible Expression System (Cat. No. 631164).Propagation in E. coli•Suitable host strain: Stellar™ Competent Cells•Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.• E. coli replication origin: pUCExcitation and Emission of pTRE3G-ZsGreen1•Excitation: 493 nm•Emission: 505 nmReferences•Gossen, M. et al. Transcriptional activation by tetracyclines in mammalian cells. Science268, 1766–9 (1995).•Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. U. S. A.89, 5547–51 (1992).•Haas, J., Park, E. C. & Seed, B. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr.Biol.6, 315–24 (1996).•Jang, S. K. et al. A segment of the 5’ nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J. Virol.62, 2636–43 (1988).•Matz, M. V et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol.17, 969–73 (1999).•Urlinger, S. et al. Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc. Natl. Acad. Sci. U. S. A.97, 7963–8 (2000).•Zhou, X., Vink, M., Klaver, B., Berkhout, B. & Das, A. T. Optimization of the Tet-On system for regulated gene expression through viral evolution. Gene Ther.13, 1382–90 (2006).Quality Control DataPlasmid Identity & Purity•Digestion with the indicated restriction enzymes produced fragments of the indicated sizes on a 0.8% agarose/EtBr gel:Vector Enzyme(s) Fragment(s)pCMV-Tet3G EcoRI 7.1 kbEcoRI & HindIII 1.2 & 5.9 kbpTRE3G-ZsGreen1 XhoI 4.7 kbEcoRV 1.2 & 3.5 kbpTRE3G-Luc XhoI 5.1 kbEcoRI & BamHI 2.1 & 3.0 kbLinear Hygromycin Marker HindIII & XbaI0.5, 0.6 & 1.1 kbLinear Puromycin Marker HindIII & XbaI0.45, 0.6, & 0.75 kb•Vector identity was confirmed by sequencing.•A260/A280: 1.8–2.0Functional Testing of Linear Markers•HEK 293 cells were transfected with 200 ng of either the Linear Hygromycin Marker or the Linear Puromycin Marker. After 5 hr at 37°C, the transfection solution was removed, and the cells were given fresh medium. 48 hr later, the cells were plated in two 10 cm plates. 48 hr after plating, medium containing either hygromycin orpuromycin (depending on the linear marker used to transfect the cells) was added to the plates. After 2–3 weeks, >20 clones were identified.It is certified that this product meets the above specifications, as reviewed and approved by the Quality Department.CATALOG NO.631159NOTICE TO PURCHASER:Our products are to be used for research purposes only. They may not be used for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. Our products may not betransferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without prior written approval of Takara Bio USA, Inc.Your use of this product is also subject to compliance with the licensing requirements listed below and described on the product´s web page at . It is your responsibility to review, understand and adhere to any restrictions imposed by these statements.STATEMENT 24The RCFPs (including DsRedExpress, DsRedExpress2, and E2-Crimson) are covered by one or more of thefollowing U.S. Patent Nos. 7,166,444; 7,157,565; 7,217,789; 7,338,784; 7,338,783; 7,537,915; 6,969,597; 7,150,979;7,442,522 and 8,012,682.STATEMENT 72Living Colors Fluorescent Protein Products: Not-For-Profit Entities: Orders may be placed in the normal manner by contacting your local representative or Takara Bio USA, Inc. Customer Service. Any and all uses of this product will be subject to the terms and conditions of the Non-Commercial Use License Agreement (the “Non-Commercial License”), a copy of which can be found below. As a condition of sale of this product to you, and prior to using this product, you must agree to the terms and conditions of the Non-Commercial License. Under the Non-Commercial License, Takara Bio USA, Inc. grants Not-For-Profit Entities a non-exclusive, non-transferable, non-sublicensable and limited license to use this product for internal, non-commercial scientific research use only. Such licensespecifically excludes the right to sell or otherwise transfer this product, its components or derivatives thereof to third parties. No modifications to the product may be made without express written permission from Takara Bio USA, Inc.Any other use of this product requires a different license from Takara Bio USA, Inc. For license information, please ***************************************************************************************.For-Profit Entities wishing to use this product are required to obtain a license from Takara Bio USA, Inc. For license information, please contact a licensing representative by phone at 650.919.7320 or by e-mail at ***********************.STATEMENT 42Use of the Tetracycline controllable expression systems (the "Tet Technology") is covered by a series of patents including U.S. Patent # 7541446, # 8383364, # 9181556 , European patents EP # 1200607, # 1954811, #2352833Academic research institutions are granted an automatic license with the purchase of this product to use the Tet Technology only for internal, academic research purposes, which license specifically excludes the right to sell, or otherwise transfer, the Tet Technology or its component parts to third parties. Notwithstanding the above, academicand not-for profit research institutions whose research using the Tet Technology is sponsored by for profitorganizations, which shall receive ownership to any data and results stemming from the sponsored research, shall need a commercial license agreement from TET Systems in order to use the Tet Technology. In accepting this license, all users acknowledge that the Tet Technology is experimental in nature. TET Systems GmbH & Co. KG makes no warranties, express or implied or of any kind, and hereby disclaims any warranties, representations, or guarantees of any kind as to the Tet Technology, patents, or products. All others are invited to request a license from TET Systems GmbH & Co. KG prior to purchasing these reagents or using them for any purpose. Takara Bio USA, Inc. is required by its licensing agreement to submit a report of all purchasers of the Tet-controllable expression system to TET Systems.For license information, please contact:GSF/CEOTET Systems GmbH & Co. KG,Im Neuenheimer Feld 58269120 Heidelberg GermanyTel: +49 6221 5880400Fax: +49 6221 5880404email:*******************or use the electronic licensing request form via /ip-licensing/licensing/for-profit-research TRADEMARKS:© 2015 Takara Bio Inc. All Rights Reserved.All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions.。

蜡样芽孢杆菌GW-01全基因组测序及生物学特性分析

刘珊，蒋杨丹，颜佶沙，等. 蜡样芽孢杆菌GW-01全基因组测序及生物学特性分析[J]. 食品工业科技，2024，45（7）：167−176. doi:10.13386/j.issn1002-0306.2023060031LIU Shan, JIANG Yangdan, YAN Jisha, et al. Whole Genome Sequencing and Biological Characterization of Bacillus cereus GW-01[J]. Science and Technology of Food Industry, 2024, 45(7): 167−176. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023060031· 生物工程 ·蜡样芽孢杆菌GW-01全基因组测序及生物学特性分析刘　珊1,2，蒋杨丹1,2，颜佶沙1,2，谢宇宣1,2，赵思佳1,2，何启田3，赵甲元1,2,*（1.西南土地资源评价与监测教育部重点实验室（四川师范大学），四川成都 610101；2.四川师范大学生命科学学院，四川成都 610101；3.浙江大学生物系统工程与食品工程学院，浙江杭州 310000）摘　要：目的：通过全基因组测序和生物信息学，研究前期筛选可降解高效氯氰菊酯（β-CY ）蜡样芽孢杆菌GW-01的基因组序列信息和生物学特性，为其安全性评估提供参考。

方法：利用HPLC 验证了GW-01降解β-CY 的能力。

GW-01的整个基因组基于二代Illumina NovaSeq 与三代PacBio Sequel 测序平台相结合的测序技术，对菌株GW-01进行全基因组测序，并对测序数据进行基因组组装、基因预测与功能注释、碳水化合物活性酶预测、毒力因子和抗生素抗性分析，此外，还基于gyrA 基因序列对菌株GW-01构建系统发育树。

Genetically modified crops Detection strategies and biosafety issues

ReviewGenetically modi ﬁed crops:Detection strategies and biosafety issuesSuchitra Kamle,Sher Ali ⁎National Institute of Immunology,Aruna Asaf Ali Marg,New Delhi 110067,Indiaa b s t r a c ta r t i c l e i n f o Article history:Accepted 10March 2013Available online 6April 2013Keywords:International regulation LabelingGene expression mRNA transcription Copy number variationGenetically modi ﬁed (GM)crops are increasingly gaining acceptance but concurrently consumers'concerns are also increasing.The introduction of Bacillus thuringiensis (Bt )genes into the plants has raised issues related to its risk assessment and biosafety.The International Regulations and the Codex guidelines regulate the biosafety requirements of the GM crops.In addition,these bodies synergize and harmonize the ethical issues related to the release and use of GM products.The labeling of GM crops and their products are mandatory if the genetically modi ﬁed organism (GMO)content exceeds the levels of a recommended threshold.The new and upcoming GM crops carrying multiple stacked traits likely to be commercialized soon warrant sensitive detection methods both at the DNA and protein levels.Therefore,traceability of the transgene and its protein expression in GM crops is an important issue that needs to be addressed on a priority basis.The advancement in the area of molecular biology has made available several bioanalytical options for the detection of GM crops based on DNA and protein markers.Since the insertion of a gene into the host genome may even cause copy number variation,this may be uncovered using real time PCR.Besides,assessing the exact number of mRNA transcripts of a gene,correlation between the template activity and expressed protein may be established.Here,we present an overview on the production of GM crops,their acceptabilities,detection strategies,biosafety issues and potential impact on society.Further,overall future prospects are also highlighted.©2013Elsevier B.V.All rights reserved.Contents1.Introduction ..............................................................1242.Global status of GM crops........................................................1253.International regulations on GM crops..................................................1254.International consensus on labeling of GM crops .............................................1265.Bt gene and stacked traits........................................................1276.Necessity of GM crop testing ......................................................1287.DNA based detection methods .....................................................1287.1.PCR and real time PCR ......................................................1287.2.Biosensors ...........................................................1298.Protein based detection.........................................................1308.1.ELISA ..............................................................1308.2.Immuno-strip ..........................................................1308.3.Immuno-PCR ..........................................................1309.Future prospects ............................................................13010.Conclusions ..............................................................130Con ﬂict of interest ..............................................................130Acknowledgments ..............................................................131References..................................................................131Gene 522(2013)123–132Abbreviations:ELISA,Enzyme linked immunosorbant assay;GMV,Genetically modi ﬁed varieties;PCR,Polymerase chain reaction.⁎Corresponding author.Tel.:+911126703753;fax:+911126742125.E-mail addresses:suchitrakamle@ (S.Kamle),alisher@nii.ac.in ,sherali5@ (S.Ali).0378-1119/$–see front matter ©2013Elsevier B.V.All rights reserved./10.1016/j.gene.2013.03.107Contents lists available at SciVerse ScienceDirectGenej o ur n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /g e n e1.IntroductionThe evolution of GM crops has come a long way (James,2011)fuelling the processes of their rapid adoption in the context of modern agriculture.Despite this,the global agriculture sector plunged into an enkindled debate over GM crops.Prior to the commercial cultivation of GM crops,consumers'concerns regarding their biosafety have also gained momentum.Arguably,the anti-GM groups (Greenpeace and Gene Campaign)are voicing their reservation fearing the growth of several non-approved varieties and the possibility of cross-contamination of the GM crops (Parlberg,2002;Smythe et al.,2006).To resolve such issues,International Regulatory (IR)bodies are making efforts to deal with the biosafety measures of the GMOs.This includes corporate council chambers and legislative councils besides research laboratories.These bodies,after due deliberation,regulate the release of GM crops accepted the world over (Codex,2003;James,2011;Stewart et al.,2000).Labeling is mandatory to avoid unintended commingling of GM and non-GM crops,thus providing assurance to the consumer (Gruère and Rao,2007).Creating acceptability of GM crops like that of non-GM ones will continue to remain a challenge.With an increased acceptability amongst consumers and society,advanced qualitative and quantitative analytical parameters may be developed for the accurate detection of the GM crops carrying multiple traits and events (Que et al.,2010).Currently,bioanalyticalFig.1.A graphical representation showing current global status of biotech crops.Source:James (2011).Fig.2.Diagrammatic illustration representing the area (in million hectares)covered by biotech crops in major countries of the world.124S.Kamle,S.Ali /Gene 522(2013)123–132tools like PCR,real-time PCR,biosensors,ELISA,immuno-strip and immuno-PCR are routinely used for the detection of DNA/protein.It is envisaged that biosafety and testing procedures both will continue to draw the attention of the policy makers,scientists and consumers alike (Arntzen et al.,2003).2.Global status of GM cropsDuring the past sixteen years,the global area of GM crops has markedly increased by 94-fold covering a total of 160million hectares.About 16.7million farmers across the world planted GM crops.Of the twenty-nine countries known to have advanced biotech-nology,nineteen developing and ten industrial ones (Figs.1–3)planted GM crops (James,2011).Of the seven continents,GM crops were grown in the six continents.The United States of America (USA)is the leading producer of GM crops.Brazil is following this trend and had registered the highest absolute growth of 4.9million hectares.India recorded a phenomenal success of Bt cotton which re ﬂect notable acceptance of GM crops.The European Union (EU)following the approval of the GM crops has reached a record level of 28%of the total production (James,2011).South Africa is the biggest producer of GM crops in the African continent and economically bene ﬁtted from the adoption of GM technology.Mexico had the highest growth rate in the year 2011.GM crop is the fastest adopted crop technology which can contribute to global food security in due course of time (James,2011).3.International regulations on GM cropsThe World Health Organization (WHO)de ﬁned GMOs as those organisms in which the genetic material has been altered in a way that does not occur naturally.Together with the sustainability of GM crops in agriculture for food safety,biodiversity and biosafety issues are equally important.Thus,efforts to regulate biosafety measures are vigorously made both at the international and national levels.Accordingly,GMOs are carefully examined and policies are revised regularly by the regulatory bodies to strengthen the system (Stewart et al.,2000).Worldwide biosafety protocols and amendments on GMOs are strictly implemented.In 1992,the United Nation (UN)conference documented Agenda-21,emphasizing the ecofriendly management of modern biotechnology and the Convention on Biological Diversity (CBD),published the safe guidelines for GMOs (Codex,2003;Haslberger,2003;Ladics,2008).Later,in 1995,the World Trade Organization-Technical Barrier to Trade (WTO-TBT),laid down guidelines for regulations,standards testing,certi ﬁcation process,packaging,marking and mandatory labeling requirements (Codex Alinorm,06/29/34;Report of the APO Study,2002).Similarly,the Cartagena Protocol (2000)on biosafety aims at regulating the safe trans-fer and handling of GMOs protecting the biodiversity (Alexandrova et al.,2005;MacKenzie,2000).The Codex Alimentarius Commission (CAC)an international governmental body of the FAO and the WHO,established in 1962,promulgated the Codex guidelines (2003),for the food safety assessment and evaluation of the immunogenic potency of GMOs.Most of the countries have a speci ﬁc multidisciplinary Inter Institutional advisory group to evaluate scienti ﬁc and technical issues associated with the GMOs (Table 1).To be effective,these regulatory bodies share overall responsibility of GM crops and their products based on empirical data.In 2005,the Bulgarian Parliament adopted the GMO laws and directed the European Commission (Regulation (EC)2001/18)to enforce the GM guidelines (Alexandrova et al.,2005).Later on in 2010,the Bulgaria's parliament released a fresh and stringent law and effectively banned GM crops both for commercial reasons and trial purposes (/cap/bulgaria-approves-law-ban-gmo-cr-news-355729).In 2006,the National Biotechnology Regulatory Authority (NBRA)of India published new legislation known as the Biotechnology Regulatory Authority of India Act regarding GMOs.But under current Indian law,any GM crops before commerciali-zation requires legal approval from the Genetic Engineering Approval Committee (GEAC),the highest body under the Ministry ofEnvironmentFig.3.A graph displaying area (in percent)covered by four major biotech crops worldwide.Table 1List of the regulatory bodies for GMOs.Country Legal regulatory organizations for GMOs ArgentinaNational Advisory Commission on Agricultural Biotechnology (CONABIA)Australia –New Zealand Australia and New Zealand Food Authority (ANZFA)CanadaCanadian Food Inspection Agency (CFIA)European Union 2001/18/EC and 1830/2003IndiaBiotechnology Authority of India (BRAI)Act South Africa South African GMO ActUSAAnimal and Plant Health Protection Inspection Service (APHIS);The Environmental Protection Agency (EPA);Food and Drug Administration (FDA)125S.Kamle,S.Ali /Gene 522(2013)123–132and Forest of India.These regulatory frameworks ensure comprehensive biosafety assessment of GM crops and administer enforcement,compli-ance,accreditation,and national and international policy coordination through its legal units.Every year,a number of new GM crops are approved asynchronous-ly(http://ftp.jrc.es/EURdoc/report_GMOpipeline_online_preprint.pdf). Modern biotechnology can beneﬁt mankind employing GM crops to meet the food requirement thus,ensuring the economic prosperity of the teeming millions in the world.Besides this,there is a pressing requirement of uniﬁed regulations acceptable to all the countries (Gupta,2000).4.International consensus on labeling of GM cropsThe labeling of the GM crops is a contentious issue.The interna-tional authorities are drafting guidelines for proper labeling of GM crops and their products.Exact labeling requires an extensive identity preservation system from granger to the elevator to grain processor to food processing manufacturer andﬁnally to the consumer through the retailer(Maltsbarger and Kalaitzandonakes,2000).Labeling of GM crops is compulsory to inform the consumer.Consumers must know that the GM crop has been declared safe by the authority (Fig.4)(Hansen,2004;McKay White and Veeman,2007;Streiffer and Rubel,2003).Moreover,it helps to enhance surveillance and tracing of GM beling is required when GM crops are substantially different from its conventional counterpart(e.g.a change in composition, nutritional value or allergenic nature).The FDA stance is that the GM and non-GM crops are substantially equivalent.But it is difﬁcult to label each fruit as it would incur additional prices to the products and at the end be shifted to the consumer(Bansal and Ramaswami,2007).GM labeling requirement for food products as a precautionary measure was introduced by the EU(Regulation(EC)258/97)and ap-proved lawfully to provide safety to society.Thus,biosafety measure-ment and regulations are made to create a‘safety net’by testing and labeling GM products.Usually,country speciﬁc labeling policies are made.In many countries,the labeling of grains,feed and foodstuffs is mandatory if the GMO content exceeds a certain threshold level as mentioned earlier. The proposed threshold level is1%but it has been urged to achieve as low as0.01%(Fig.4and Table2).The threshold value is based on the percentage of GMO material in a non-GM background(Hansen,2001). Normally,no GM food labeling would be required if the food contains GM material below the threshold level.Countrywise,the degree of the labeling pattern varies greatly(Bansal and Gruere,2010;Carter and Gruere,2003).The Codex Committee on Food Labeling(CCFL)has drafted advanced recommendationson Fig.4.A pictorial representation of proper labeling of crops:(a)non-GM corn(b)GM corn.Table2A labeling system and threshold level of GM crops/products in major countries.Source:(EC),1829/2003and1830/2003(Regulation(EC)1829/2003).Country Labeling type Threshold level Product/process Country Labeling type Threshold level Product/process China Mandatory0%Process Indonesia Mandatory5%ProductEU Mandatory0.9%Process Taiwan Mandatory5%ProductRussia Mandatory0.9%Product Thailand Mandatory5%ProductAustralia–New Zealand Mandatory1%Product Canada Voluntary5%ProductBrazil Mandatory1%Process Hong-Kong Voluntary5%ProductSaudi Arabia Mandatory1%Product Japan Mandatory5%ProductIsrael Mandatory1%Product Philippine Mandatory5%ProductKorea Mandatory3%Product South Africa Voluntary–ProductChile Mandatory2%Product USA Voluntary–Product Philippines Mandatory5%Product Argentina Voluntary–Product126S.Kamle,S.Ali/Gene522(2013)123–132labeling of the biotech products and is directly linked with the WTO through an agreement.The Codex process for standard development is based on developing an international consensus,to protect the consumer and to facilitate trade by developing the best labeling policies for harmonization(Codex,2003;Haslberger,2003;Ladics,2008).Till date,there is no authentic global approval and legal registration of GM crops and their processed food products.Therefore,GM testing and its legal registration must be made mandatory and operational the world over(Goodman and Tetteh,2011).5.Bt gene and stacked traitsThe modern biotechnological approach allows genes to be intro-duced into a plant genome.These foreign genes may originate from prokaryotes(bacteria)or eukaryotes either from plants or animals. Theﬁrst GMO was Bt,and due to its wide applications was called Bt technology.In itsﬁrst application,Bt genes were transferred into tobacco and tomato(Fischhoff et al.,1987)and following this,many other crops were developed(Jouanin et al.,1988).A GM maize(Bt11) has been developed to express the Cry1Ab insecticidal protein. This Cry1Ab was found to be toxic against some lepidopterons Helicoverpa punctigera,Helicoverpa zea and Pectinophora gossypiella insects(Bruderer and Leitner,2003).Various GM crops harboring Bt genes(cry1Ac,cry1Ab,cry2Aa,cry2Ab,cry2Ac,cry1F,epsps and vip-3A),encoding insecticidal proteins were derived from a ubiquitous soil bacterium Bt.These insecticidal proteins generally have molecular weights between65kDa and88kDa(Hofte and Whiteley,1989)and are known to be lethal against dipteran,coleopteran and lepidopteron insects.Since the commercialization of GM crops,herbicide tolerance(HT) has consistently been the dominant trait and is used in soybean, followed by insect resistance used in Bt maize,Bt cotton,and Bt canola(Fig.3).Such GM crops tolerate more herbicides like glyphosate and ammonium glufosinate and are resistant to different pests.GM crops expressing insecticidal proteins are steadily gaining acceptance and grown throughout the world(James,2011).GMV that have been commercialized are Bt cotton inﬁve different countries,roundup ready(RR)soybean in Argentina,Bt maize in Canada and Argentina and HT maize in Canada.Argentina gave approval to Syngenta to grow four-stack(GA2×Bt11×MIR60×MIR162)Viptera maize(Que et al.,2010).Stacked events are those which in the same plant combine by con-ventional breeding or re-transformation of one or more existing traits (http://ftp.jrc.es/EURdoc/report_GMOpipeline_online_preprint.pdf). Theﬁrst generation GM crop has a single Bt gene(e.g.Bollgard-I: cry1Ac)and now the second and third generations of GM crops were stacked with multiple genes(e.g.Bollgard-II:cry1Ac+cry2Ab) having one copy of each event to achieve long-lasting resistance.GM maize stacked with thirteen double,three triple and one quadruple event and is currently under EU assessment.The stacked GM crops which are likely to be commercialized are—soybean,maize,cotton, rapeseed,rice and potato(Table3).A database for GM crops has been established to provide uniform and updated information the world over (/index.php?action=gm_crop_database).Table3Details of stacked traits in GM crops and their products.Sources:/index.php?action=gm_crop_database;/bcsweb/cropprotection.nsf/id/BioScience;/ country/us/en/Seeds/Pages/Home.aspx;/prod/;/;/.GM crops Trait developer Product GM event Stacked transgenes TargetCanola Bayer crop sciences Invigor,Seed link MS8(DBN230-0028)RF3(DBN212-05)Bar,barnase,barstar Weeds,male fertility Monsanto Genuity‘RR’GT73(RT73)cp4-epsps,gox WeedsCotton Bayer crop sciences Fiber ax Liberty linkBollgard IILLCotton25,MON15985bar,cry1Ac+cry2Ab Lepidopteron,weedsDow Agro sciences Widestrike DAS-21023-5-DAS-24236-5pat,cry1Ac,cry1Fa Lepidopteron,weedsMonsanto‘RR’Bollgard II MON531,MON1445-2cry1Ac,cp4-epsps Lepidopteron,weedsMaize Dow Agro sciences,Pioneer Hi-Bred Herculex Xtra TC1507,DAS-59122-7cry1Fa,cry34Ab1,cry35Ab1Lepidopteron,coleopterans,weedsMonsanto Yieldgard Triple MON810,MON88017cry1Ab,cry3Bb1,cp4-epsps Lepidopteron,coleopterans,weeds Syngenta Agrisure3000GT GA21,Bt-11,MIR604pat,cry1Ab,cry3Aa,mutant maize epsps Lepidopteron,coleopterans,weedsFig.5.A schematic view of detection methods for GM crops.127S.Kamle,S.Ali/Gene522(2013)123–1326.Necessity of GM crop testingGM content based veri ﬁcation requires testing of GM products for the presence of foreign DNA or protein.The enforcement of threshold values has created a pressing demand for the development of reliable GM analysis methods of a rapid and inexpensive character.Reliable screening methods are important both for detection of unauthorized GM crops and labeling control (Morisset et al.,2009).Unauthorized GM crops can challenge the present analytical system on the ground of practical application of detection methods such as regulatory sequences common to all GM crops.Different screening methods based on DNA and proteins are employed for the detection of GM crops and their products (Fig.5).7.DNA based detection methodsPCR is the preferred method for the identi ﬁcation and quanti ﬁcation of Bt gene because of its versatility,sensitivity,speci ﬁcity,and high throughput applications (Morisset et al.,2009).To detect any Bt gene,it is necessary to know the sequence of the genes used in the GM construct.These may include plasmid vector sequences,selectable markers,promoters and terminators.7.1.PCR and real time PCRAs mentioned earlier,commonly used detection methods for GM crops is based on PCR (Stull,2001).To identify GM crops and products,a primer needs to be designed for the ampli ﬁcation of the inserted gene.This basic requirement is ascertained by restriction endonuclease diges-tion of the gene followed by hybridization with a speci ﬁc DNA probe.Alternatively,the PCR product itself may be used for directsequencing.Fig.6.A multiplex PCR assay showing simultaneous ampli ﬁcation of cry2Ab transgene,promoter (P-35S ),terminator (T-nos )and marker gene (Npt-II )in GM cotton (MON15985).Lane M,100bp DNA ladder;1,environmental control;standard single PCR,2–5:2,cry2Ab (326bp);3,P-35S (195bp);4,T-nos (180bp);5,npt-II (215bp);duplex PCR,6–8;6,cry2Ab +P-35S ;7,cry2Ab +T-nos ;8,cry2Ab +npt-II ;triplex PCR,9–11;9,cry2Ab +npt-II +P-35S ;10,cry2Ab +T-nos +P-35S ;11,cry2Ab +npt-II +T-nos ;quadruplex PCR,12,cry2Ab +npt-II +P-35S +T-nos ;13,non-GM cotton.Source:Kamle et al.,(2011a).Fig.7.A schematic plot of real-time quantitative PCR,displaying threshold and CT value.Source:/projects/genome/probe/IMG/PCR_plot.gif.Fig.8.A comparative view of detection of Bt maize (cry1Ab ):(a)PCR based detection;(b)biosensor based detection.Source:Bai et al.(2007).128S.Kamle,S.Ali /Gene 522(2013)123–132In addition,a nested PCR in which two sets of standard primers are used that bind speci ﬁcally to the target sequences may also be employed.A multiplex and transgene construct speci ﬁc PCR assays for cry1Ab ,cry1Ac ,cry2Ab and vip-3A transgenes (Fig.6)have been reported (Kamle et al.,2011a;Randhawa et al.,2010).Real time PCR is used to quantify a targeted DNA molecule.For detection of the products,sequence speci ﬁc oligonucleotides labeled with a ﬂuorescent reporter are used which allow the detection of the ampli ﬁed product as the reaction advances (Fig.7).Real-time PCR has great value in validating and estimating the number of copies of inserted genes into the host genome (Bon ﬁni et al.,2002;Zhang et al.,2003).This has been reported for several GM crops such as maize,cassava,rapeseed,wheat,cotton and brinjal (Aguilera et al.,2008;Ballari et al.,2013;Beltrán et al.,2009;Lee et al.,2006;Li et al.,2004;Wu et al.,2007).Furthermore,a sensitive loop mediatedisothermal amplication method employed for the detection of three GM rice events has been reported (Chen et al.,2012b;Kiddle et al.,2012).Besides these techniques,microarray based detection systems are under development.B t-176transgenic maize (cry1Ab )was quanti ﬁed by ligation detection reaction (LDR)combined with a universal array approach (Bordoni et al.,2004).7.2.BiosensorsA biosensor is an analytical device for the detection of an analyte that combines a biological component with a physicochemical detector component.GM detection has encouraged the development of sensitive sensor technology that promises to generate quick results.Biosensors'prominent attribute is the immobilization oftheFig.9.(a)A schematic view of Cry2Ab sandwich ELISA;(b)a linear graph representing absorbance vs GM protein concentration.Source of panel a:Kamle et al.(2011b).Fig.10.A display of immuno-strip:(a)GM sample having protein of interest (two bands —positive control);(b)non-GM sample having no protein (single band —negative control)of interest.129S.Kamle,S.Ali /Gene 522(2013)123–132probe on an electrode surface like altered cysteamine gold.Currently,different types of biosensors (electrochemical sensors,pizeoelectric biosensors,surface plasmon resonance/optical biosensors)are used to detect transgenes (Fig.8)in GM crops like soybean,maize,cotton,rice,tomato and canola (Bai et al.,2007;Feriotto et al.,2003;Mariotti et al.,2002;Stobiecka et al.,2007;Tichoniuk et al.,2008).Recently,in Surface Enhanced Raman Scattering Spectroscopy (SERS),a barcoded nano-sensor has been developed to detect cry1Ab and cry1Ac transgenes in GM rice (K.Chen et al.,2012;X.Chen et al.,2012).8.Protein based detectionAn immunoassay technique based on antibodies is a standard approach for qualitative and quantitative detection of protein of a known target analyte (Brett et al.,1999).Both monoclonal (highly speci ﬁc)and polyclonal (often more sensitive)antibodies can be used depending on the speci ﬁcity of the detection system.On the basis of typical concentrations of a transgenic material in plant tissues (>10μg per tissue),the limit of detection (LOD)of a protein immuno-assay can predict the presence of recombinant protein in up to 1%of GMOs (Stave,2002).8.1.ELISAELISA has a signi ﬁcant advantage of protein analysis in GM crops and their products.A sandwich ELISA is the preferable immunoassay used for the detection of Bt protein,where an analyte is sandwiched in be-tween the two antibodies;a capture antibody and the detector antibody.In sandwich ELISA protein concentration is directly proportional to the color intensity (the higher the protein concentration,the greater will be the color intensity).ELISA was successfully used for the detection of protein encoded by cp4-epsps gene in a RR soybean (Rogan,1999).Also,monoclonal antibodies are being used for the development of sen-sitive and single epitope speci ﬁc immunoassays for the detection of Bt proteins like Cry1Ac and Cry1Ab (Vázquez-Padrón et al.,2000).For the detection of Cry1Ab,a capillary electrophoresis competitive immu-noassay and a highly sensitive quanti-dot based ﬂuorescence linkedimmunosorbant assay have been developed (Giovannoli et al.,2008;Zhu et al.,2011).Similarly,a monoclonal antibody based sandwich im-munoassay (Fig.9)having a 100ng/g LOD for Cry1Ac and a 1pg/g LOD for Cry2Ab in cotton seed/leaf samples have been reported (Kamle et al.,2011b,2013;Shan et al.,2007).8.2.Immuno-stripUse of a different format like ELISA,using a nitrocellulose-strip rather than microtiter wells,led to the development of lateral ﬂow strip/dipstick/immuno-strip technology.Immobilized double antibodies,speci ﬁc to recognize expressed protein are conjugated to a color reactant (gold nano-particles)and incorporated into a nitrocellulose strip.This nitrocellulose strip when dipped in the protein extract of plant tissue (e.g.GM cotton leaf)harboring a GM protein,leads to an antibody reaction releasing color.This red colored gold conjugated complex ﬂows to the other end of the strip through capillary movement to a porous membrane that has two captured antibody zones.One zone is speci ﬁc for the GM protein and the other one is speci ﬁc for untreated antibodies coupled to the reagent (Fig.10).The immuno-strips can give results as either ‘Yes ’or ‘No ’within 5to 10min.The immuno-strip is an economical,easy and ﬁeld tractable detection method.These immuno-strips are commercially available to detect Cry1Ab,Cry1Ac,Cry2Ab and CP4-EPSPS (Lipton et al.,2000;Fagan et al.,2001).8.3.Immuno-PCRImmuno-PCR potentially offers a sensitive and speci ﬁc method for detecting the antigen,in which a speci ﬁc DNA molecule is used as a marker (Fig.11).It combines the speci ﬁcity of an ELISA with the sensitivity of the assay using PCR (Sano et al.,1992).An immuno-PCR assay has been reported for the detection of Cry proteins expressing GM crops such as Cry1Ac (Allen et al.,2006;Zhang and Guo,2011).9.Future prospectsThe ﬁrst generation of Bt crops (MON810)have been extraordinarily successful with a few examples of pest populations evolving resistance.These crops are already being replaced with a second or third generation of GM crop varieties having two or more traits/events.Even,this is not a matter of complacency and still needs more ef ﬁcacious and potent Bt strains to meet the future requirement (Christou et al.,2006;Crickmore,2006).An engineered Cry1AMod toxin lacking helix α-1has been reported,which does not bind with the receptor-cadherin and therefore kills even insects that are resistant to the parent toxin Cry1Ab (Muñóz-Garay et al.,2009).New Bt strains using a proteomics method can be screened for the presence of the novel toxin Cry60Ba from Bt serovar malayensi .Incidentally,this is also a mosquitocidal toxin (Sun and Park,2010).A recent report showed that the isolated strain LLP29from the phylloplane of Magnolia denudate ,produces a novel toxin (Cyt1Aa6)which is lethal to mosquito larvae (Zhang et al.,2010).This has far reaching implications to control mosquitoes.10.ConclusionsWith the development of newer transgene crops,detection methods are also likely to be improved.The International Regulations and the Codex guidelines acting together with the biosafety issues and labeling of the GMOs seems to be a promising proposition towards the acceptance of GM crops.Con ﬂict of interestThe authors declare they have no con ﬂict ofinterest.Fig.11.A schematic representation of immuno-PCR.Source:.tw/sysdata/81/81/doc/c55122547d93a327/attach/1003.png .130S.Kamle,S.Ali /Gene 522(2013)123–132。

活性物质的提取、分离、表征-综述

EXTRACTION,ISOLATION AND CHARACTERIZATION OF BIOACTIVE COMPOUNDS FROM PLANTS’EXTRACTSS.Sasidharan1,**,Y.Chen1,D.Saravanan2,K.M.Sundram3,L.Yoga Latha11Institute for Research in Molecular Medicine(INFORM),Universiti Sains Malaysia,Minden11800, Malaysia.,2Centre for Drug Research,University Science of Malaysia,11800Minden,Pulau Pinang. Malaysia.,3School of Pharmacy,AIMST University,Jalan Bedong-Semeling,Batu3½,Bukit Air Nasi,Bedong08100,Kedah,Malaysia.*E-mail:srisasidharan@AbstractNatural products from medicinal plants,either as pure compounds or as standardized extracts,provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity.Due to an increasing demand for chemical diversity in screening programs,seeking therapeutic drugs from natural products,interest particularly in edible plants has grown throughout the world.Botanicals and herbal preparations for medicinal usage contain various types of bioactive compounds.The focus of this paper is on the analytical methodologies,which include the extraction,isolation and characterization of active ingredients in botanicals and herbal preparations.The common problems and key challenges in the extraction,isolation and characterization of active ingredients in botanicals and herbal preparations are discussed.As extraction is the most important step in the analysis of constituents present in botanicals and herbal preparations,the strengths and weaknesses of different extraction techniques are discussed.The analysis of bioactive compounds present in the plant extracts involving the applications of common phytochemical screening assays,chromatographic techniques such as HPLC and,TLC as well as non-chromatographic techniques such as immunoassay and Fourier Transform Infra Red(FTIR)are discussed.Key words:Bioactive compound,Plant Extraction,Isolation,Herbal preparations,Natural productsIntroductionNatural products,such as plants extract,either as pure compounds or as standardized extracts,provide unlimited opportunities for new drug discoveries because of the unmatched availability of chemical diversity(Cos et al.,2006).According to the World Health Organization(WHO),more than80%of the world's population relies on traditional medicine for their primary healthcare needs.The use of herbal medicines in Asia represents a long history of human interactions with the environment.Plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases(Duraipandiyan et al.,2006).Due to the development of adverse effects and microbial resistance to the chemically synthesized drugs,men turned to ethnopharmacognosy.They found literally thousands of phytochemicals from plants as safe and broadly effective alternatives with less adverse effect.Many beneficial biological activity such as anticancer, antimicrobial,antioxidant,antidiarrheal,analgesic and wound healing activity were reported.In many cases the people claim the good benefit of certain natural or herbal products.However,clinical trials are necessary to demonstrate the effectiveness of a bioactive compound to verify this traditional claim.Clinical trials directed towards understanding the pharmacokinetics, bioavailability,efficacy,safety and drug interactions of newly developed bioactive compounds and their formulations(extracts) require a careful evaluation.Clinical trials are carefully planned to safeguard the health of the participants as well as answer specific research questions by evaluating for both immediate and long-term side effects and their outcomes are measured before the drug is widely applied to patients.According to the World Health Organization(WHO),nearly20,000medicinal plants exist in91countries including12 mega biodiversity countries.The premier steps to utilize the biologically active compound from plant resources are extraction, pharmacological screening,isolation and characterization of bioactive compound,toxicological evaluation and clinical evaluation.A brief summary of the general approaches in extraction,isolation and characterization of bioactive compound from plants extract can be found in Figure1.This paper provides details in extraction,isolation and characterization of bioactive compound from plants extract with common phytochemical screening assay,chromatographic techniques,such as HPLC,and HPLC/MS and Fourier Transform Mass Spectrometry(FTMS).Figure1:A brief summary of the general approaches in extraction,isolation and characterization of bioactive compound from plants extractExtractionExtraction is the crucial first step in the analysis of medicinal plants,because it is necessary to extract the desired chemical components from the plant materials for further separation and characterization.The basic operation included steps, such as pre-washing,drying of plant materials or freeze drying,grinding to obtain a homogenous sample and often improving the kinetics of analytic extraction and also increasing the contact of sample surface with the solvent system.Proper actions must be taken to assure that potential active constituents are not lost,distorted or destroyed during the preparation of the extract from plant samples.If the plant was selected on the basis of traditional uses(Fabricant and Farnsworth,2001),then it is needed to prepare the extract as described by the traditional healer in order to mimic as closely as possible the traditional‘herbal’drug.The selection of solvent system largely depends on the specific nature of the bioactive compound being targeted.Different solvent systems are available to extract the bioactive compound from natural products.The extraction of hydrophilic compounds uses polar solvents such as methanol,ethanol or ethyl-acetate.For extraction of more lipophilic compounds,dichloromethane or a mixture of dichloromethane/methanol in ratio of1:1are used.In some instances,extraction with hexane is used to remove chlorophyll(Cos et al.,2006).As the target compounds may be non-polar to polar and thermally labile,the suitability of the methods of extraction must be considered.Various methods,such as sonification,heating under reflux,soxhlet extraction and others are commonly used(United States Pharmacopeia and National Formulary,2002;Pharmacopoeia of the People’s Republic of China,2000;The Japanese Pharmacopeia,2001)for the plant samples extraction.In addition,plant extracts are also prepared by maceration or percolation of fresh green plants or dried powdered plant material in water and/or organic solvent systems.A brief summary of the experimental conditions for the various methods of extraction is shown in Table1.The other modern extraction techniques include solid-phase micro-extraction,supercritical-fluid extraction, pressurized-liquid extraction,microwave-assisted extraction,solid-phase extraction,and surfactant-mediated techniques,which possess certain advantages.These are the reduction in organic solvent consumption and in sample degradation,elimination of additional sample clean-up and concentration steps before chromatographic analysis,improvement in extraction efficiency, selectivity,and/kinetics of extraction.The ease of automation for these techniques also favors their usage for the extraction of plants materials(Huie,2002).Identification and characterizationDue to the fact that plant extracts usually occur as a combination of various type of bioactive compounds or phytochemicals with different polarities,their separation still remains a big challenge for the process of identification and characterization of bioactive compounds.It is a common practice in isolation of these bioactive compounds that a number of different separation techniques such as TLC,column chromatography,flash chromatography,Sephadex chromatography and HPLC,should be used to obtain pure compounds.The pure compounds are then used for the determination of structure and biological activity.Beside that,non-chromatographic techniques such as immunoassay,which use monoclonal antibodies (MAbs),phytochemical screening assay,Fourier-transform infrared spectroscopy(FTIR),can also be used to obtain and facilitate the identification of the bioactive compounds.Table 1:A brief summary of the experimental conditions for various methods of extraction for plants materialChromatographic techniquesThin-layer chromatography (TLC)and Bio-autographic methodsTLC is a simple,quick,and inexpensive procedure that gives the researcher a quick answer as to how many components are in a mixture.TLC is also used to support the identity of a compound in a mixture when the R f of a compound is compared with the R f of a known compound.Additional tests involve the spraying of phytochemical screening reagents,which cause color changes according to the phytochemicals existing in a plants extract;or by viewing the plate under the UV light.This has also been used for confirmation of purity and identity of isolated compounds.Bio-autography is a useful technique to determine bioactive compound with antimicrobial activity from plant extract.TLC bioautographic methods combine chromatographic separation and in situ activity determination facilitating the localization and target-directed isolation of active constituents in a mixture.Traditionally,bioautographic technique has used the growth inhibition of microorganisms to detect anti-microbial components of extracts chromatographed on a TLC layer.This methodology has been considered as the most efficacious assay for the detection of anti-microbial compounds (Shahverdi,2007).Bio-autography localizes antimicrobial activity on a chromatogram using three approaches:(i)direct bio-autography,where the micro-organism grows directly on the thin-layer chromatographic (TLC)plate,(ii)contact bio-autography,where the antimicrobial compounds are transferred from the TLC plate to an inoculated agar plate through direct contact and (iii)agar overlay bio-autography,where a seeded agar medium is applied directly onto the TLC plate (Hamburger and Cordell,1987;Rahalison et al.,1991).The inhibition zones produced on TLC plates by one of the above bioautographic technique will be use to visualize the position of the bioactive compound with antimicrobial activity in the TLC fingerprint with reference to R f values (Homans and Fuchs,1970).Preparative TLC plates with a thickness of 1mm were prepared using the same stationary and mobile phases as above,with the objective of isolating the bioactive components that exhibited the antimicrobial activity against the test strain.These areas were scraped from the plates,and the substance eluted from the silica with ethanol or methanol.Eluted samples were further purified using the above preparative chromatography method.Finally,the components were identified by HPLC,LCMS and GCMS.Although it has high sensitivity,its applicability is limited to micro-organisms that easily grow on TLC plates.Other problems are the need for complete removal of residual low volatile solvents,such as n -BuOH,trifluoroacetic acid and ammonia and the transfer of the active compounds from the stationary phase into the agar layer by diffusion (Cos et al.,2006).Because bio-autography allows localizing antimicrobial activities of an extract on the chromatogram,it supports a quick search for new antimicrobial agents through bioassay-guided isolation (Cos et al.,2006).The bioautography agar overlay method is advantageous in that,firstly it uses very little amount of sample when compared to the normal disc diffusion method and hence,it can be used for bioassay-guided isolation of compounds.Secondly,since the crude extract is resolved into its different components,this technique simplifies the process of identification and isolation of the bioactive compounds (Rahalison et al .,1991).Common Solvents used Methanol,ethanol,or mixture ofalcohol and waterMethanol,ethanol,or mixture of alcohol and water Methanol,ethanol,or mixture of alcohol and water Temperature (o C)Depending onsolvent usedCan be heated Room temperature Pressure applied Not applicableNot applicable Not applicable Time required 3–18hr1hr 3-4days Volume of solvent required (ml)150–20050–100Depending on the sample size ReferenceZygmunt and Namiesnik,2003;Huie,2002Zygmunt and Namiesnik,2003;Huie,2002Phrompittayarat et al .,2007;Sasidharan et al.,2008;Cunha et al.,2004;Woisky et al.,1998High performance liquid chromatographyHigh performance liquid chromatography(HPLC)is a versatile,robust,and widely used technique for the isolation of natural products(Cannell,1998).Currently,this technique is gaining popularity among various analytical techniques as the main choice for fingerprinting study for the quality control of herbal plants(Fan et al.,2006).Natural products are frequently isolated following the evaluation of a relatively crude extract in a biological assay in order to fully characterize the active entity.The biologically active entity is often present only as minor component in the extract and the resolving power of HPLC is ideally suited to the rapid processing of such multicomponent samples on both an analytical and preparative scale.Many bench top HPLC instruments now are modular in design and comprise a solvent delivery pump,a sample introduction device such as an auto-sampler or manual injection valve,an analytical column,a guard column,detector and a recorder or a printer.Chemical separations can be accomplished using HPLC by utilizing the fact that certain compounds have different migration rates given a particular column and mobile phase.The extent or degree of separation is mostly determined by the choice of stationary phase and mobile phase.Generally the identification and separation of phytochemicals can be accomplished using isocratic system(using single unchanging mobile phase system).Gradient elution in which the proportion of organic solvent to water is altered with time may be desirable if more than one sample component is being studied and differ from each other significantly in retention under the conditions employed.Purification of the compound of interest using HPLC is the process of separating or extracting the target compound from other(possibly structurally related)compounds or contaminants.Each compound should have a characteristic peak under certain chromatographic conditions.Depending on what needs to be separated and how closely related the samples are,the chromatographer may choose the conditions,such as the proper mobile phase,flow rate,suitable detectors and columns to get an optimum separation.Identification of compounds by HPLC is a crucial part of any HPLC assay.In order to identify any compound by HPLC,a detector must first be selected.Once the detector is selected and is set to optimal detection settings,a separation assay must be developed.The parameters of this assay should be such that a clean peak of the known sample is observed from the chromatograph.The identifying peak should have a reasonable retention time and should be well separated from extraneous peaks at the detection levels which the assay will be performed.UV detectors are popular among all the detectors because they offer high sensitivity(Lia et al.,2004)and also because majority of naturally occurring compounds encountered have some UV absorbance at low wavelengths(190-210nm)(Cannell,1998).The high sensitivity of UV detection is bonus if a compound of interest is only present in small amounts within the sample.Besides UV,other detection methods are also being employed to detect phytochemicals among which is the diode array detector(DAD)coupled with mass spectrometer(MS)(Tsao and Deng, 2004).Liquid chromatography coupled with mass spectrometry(LC/MS)is also a powerful technique for the analysis of complex botanical extracts(Cai et al.,2002;He,2000).It provides abundant information for structural elucidation of the compounds when tandem mass spectrometry(MS n)is applied.Therefore,the combination of HPLC and MS facilitates rapid and accurate identification of chemical compounds in medicinal herbs,especially when a pure standard is unavailable(Ye et al.,2007).The processing of a crude source material to provide a sample suitable for HPLC analysis as well as the choice of solvent for sample reconstitution can have a significant bearing on the overall success of natural product isolation.The source material,e.g.,dried powdered plant,will initially need to be treated in such a way as to ensure that the compound of interest is efficiently liberated into solution.In the case of dried plant material,an organic solvent(e.g.,methanol,chloroform)may be used as the initial extractant and following a period of maceration,solid material is then removed by decanting off the extract by filteration.The filtrate is then concentrated and injected into HPLC for separation.The usage of guard columns is necessary in the analysis of crude extract.Many natural product materials contain significant level of strongly binding components,such as chlorophyll and other endogenous materials that may in the long term compromise the performance of analytical columns. Therefore,the guard columns will significantly protect the lifespan of the analytical columns.Non-chromatographic techniquesImmunoassayImmunoassays,which use monoclonal antibodies against drugs and low molecular weight natural bioactive compounds, are becoming important tools in bioactive compound analyses.They show high specificity and sensitivity for receptor binding analyses,enzyme assays and qualitative as well as quantitative analytical techniques.Enzyme-linked immunosorbent essay (ELISA)based on MAbs are in many cases more sensitive than conventional HPLC methods.Monoclonal antibodies can be produced in specialized cells through a technique known as hybridoma technology(Shoyama et al.,2006).The following steps are involved in the production of monoclonal antibodies via hybridoma technology against plant drugs:(i)A rabbit is immunized through repeated injection of specific plant drugs for the production of specific antibody,facilitated due to proliferation of the desired B cells.(ii)Tumors are produced in a mouse or a rabbit.(iii)From the above two types of animals,spleen cell(these cells are rich in B cells and T cells)are cultured separately.The separately cultured spleen cells produce specific antibodies against the plants drug,and against myeloma cells that produce tumors.(iv)The production of hybridoma by fusion of spleen cells to myeloma cells is induced using polyethylene glycol(PEG).The hybrid cells are grown in selective hypoxanthine aminopterin thymidine(HAT)medium.(v)The desired hybridoma is selected for cloning and antibody production against a plant drug.This process is facilitated by preparing single cell colonies that will grow and can be used for screening of antibody producing hybridomas.(vi)The selected hybridoma cells are cultured for the production of monoclonal antibodies in large quantity against the specific plants drugs.(vii)The monoclonal antibodies are used to determine similar drugs in the plants extract mixture through enzyme-linked immunosorbent essay(ELISA).Phytochemical screening assayPhytochemicals are chemicals derived from plants and the term is often used to describe the large number of secondary metabolic compounds found in plants.Phytochemical screening assay is a simple,quick,and inexpensive procedure that gives the researcher a quick answer to the various types of phytochemicals in a mixture and an important tool in bioactive compound analyses.A brief summary of the experimental procedures for the various phytochemical screening methods for the secondary metabolites is shown in Table2.After obtaining the crude extract or active fraction from plant material,p hytochemical screening can be performed with the appropriate tests as shown in the Table2to get an idea regarding the type of phytochemicals existing in the extract mixture or fraction.Fourier-transform infrared spectroscopy(FTIR)FTIR has proven to be a valuable tool for the characterization and identification of compounds or functional groups (chemical bonds)present in an unknown mixture of plants extract(Eberhardt et al.,2007;Hazra et al.,2007).In addition,FTIR spectra of pure compounds are usually so unique that they are like a molecular"fingerprint".For most common plant compounds, the spectrum of an unknown compound can be identified by comparison to a library of known compounds.Samples for FTIR can be prepared in a number of ways.For liquid samples,the easiest is to place one drop of sample between two plates of sodium chloride.The drop forms a thin film between the plates.Solid samples can be milled with potassium bromide(KBr)to and then compressed into a thin pellet which can be analyzed.Otherwise,solid samples can be dissolved in a solvent such as methylene chloride,and the solution then placed onto a single salt plate.The solvent is then evaporated off,leaving a thin film of the original material on the plate.ConclusionSince bioactive compounds occurring in plant material consist of multi-component mixtures,their separation and determination still creates problems.Practically most of them have to be purified by the combination of several chromatographic techniques and various other purification methods to isolate bioactive compound(s).Table2:A brief summary of phytochemical screening of secondary metabolitesDragendorff’s test Spot a drop of extract on a small piece of precoated TLC plate.Spray the platewith Dragendorff’s reagent.Orange spot(Kumar et al.,2007);Wagner test Add2ml filtrate with1%HCl+steam.Then add1ml of the solution with6drops of Wagner’s reagent.Brownish-red precipitate(Chanda et al.,2006).TLC method1Solvent system:Chloroform:methanol:25%ammonia(8:2:0.5).Spots can be detected after spraying with Dragendorff reagent Orange spot(Tona et al.,1998)1)AlkaloidTLC method2Wet the powdered test samples with a half diluted NH4OH and lixiviated with EtOAc for24hr at room temperature.Separate the organic phase from theacidified filtrate and basify with NH4OH(pH11-12).Then extract it withchloroform(3X),condense by evaporation and use for chromatography.Separate the alkaloid spots using the solvent mixture chloroform and methanol(15:1).Spray the spots with Dragendorff’s reagent.Orange spot(Mallikharjuna et al.,2007).Borntrager's test Heat about50mg of extract with1ml10%ferric chloride solution and1ml of concentrated hydrochloric acid.Cool the extract and filter.Shake the filtratewith equal amount of diethyl ether.Further extract the ether extract with strongammonia.Pink or deep redcoloration of aqueouslayer(Kumar et al.,2007)2)AnthraquinoneBorntrager’s test Add1ml of dilute(10%)ammonia to2ml of chloroform extract.A pink-red color in theammoniacal(lower)layer(Onwukaeme et al.,2007).Kellar–Kiliani test Add2ml filtrate with1ml of glacial acetic acid,1ml ferric chloride and1mlconcentrated sulphuric acid.Green-blue coloration ofsolution(Parekh and Chanda,2007).Kellar-Kiliani test Dissolve50mg of methanolic extract in2ml of chloroform.Add H2SO4to form a layer.Brown ring at interphase(Onwukaeme et al.,2007).3)Cardiac glycosidesTLC method Extract the powdered test samples with70%EtOH on rotary shaker(180 thaws/min)for10hr.Add70%lead acetate to the filtrate and centrifuge at5000rpm/10min.Further centrifuge the supernatant by adding6.3%Na2CO3at10000rpm/10min.Dry the retained supernatant and redissolved in chloroformand use for chromatography.Separate the glycosides using EtOAc-MeOH-H2O(80:10:10)solvent mixture.The color and hR f valuesof these spots can berecorded under ultraviolet(UV254nm)light(Mallikharjuna et al.,2007).Shinoda test To2-3ml of methanolic extract,add a piece of magnesium ribbon and1ml of concentrated hydrochloric acid.Pink red or red colorationof the solution(Kumar et al.,2007).4)FlavonoidTLC method Extract1g powdered test samples with10ml methanol on water bath(60°C/ 5min).Condense the filtrate by evaporation,and add a mixture of water andEtOAc(10:1mL),and mix thoroughly.Retain the EtOAc phase and use for The color and hRf valuesof these spots can berecorded under ultraviolet(Mallikharjuna et al.,2007).chromatography.Separate the flavonoid spots using chloroform and methanol(19:1)solvent mixture.(UV254nm)lightNaOH test Treat the extract with dilute NaOH,followed by addition of dilute HCl.A yellow solution withNaOH,turns colorlesswith dilute HCl(Onwukaeme et al.,2007).5)Phenol Phenol test Spot the extract on a filter paper.Add a drop of phoshomolybdic acid reagentand expose to ammonia vapors.Blue coloration of the spot(Kumar et al.,2007);6)Phlobatannin-2ml extract was boiled with2ml of1%hydrochloric acid HCl.Formation of redprecipitates(Edeoga et al.,2005).7)Pyrrolizidine alkaloid-Prepare1ml of oxidizing agent,consisting of0.01ml hydrogen peroxide(30%w/v)stabilized with tetrasodium pyrophosphate(20mg/ml)and made up to20ml with isoamylacetate,and add to1ml of plant extract.Vortex the sampleand add0.25ml acetic anhydride before heating the sample at60°C for50-70s.Cool the samples to room temperature.Add1ml of Ehrlich reagent and placethe test tubes in water bath(60°C)for5min.Measure the absorbance at562nm.The method of Holstege et al.(1995)should be used to confirm resultsof the screening method Peaks were compared withthe GC–MS library(McGaw et al.,2007;Mattocks,1967;Holstege etal.,1995)8)Reducing sugar Fehling test Add25ml of diluted sulphuric acid(H2SO4)to5ml of water extract in a testtube and boil for15mins.Then cool it and neutralize with10%sodiumhydroxide to pH7and5ml of Fehling solution.Brick red precipitate(Akinyemi et al.,2005)Frothing test/ Foam test Add0.5ml of filtrate with5ml of distilled water and shake well.Persistence of frothing(Parekh and Chanda,2007).9)SaponinTLC method Extract two grams of powdered test samples with10ml70%EtOH by refluxing for10min.Condense the filtrate,enrich with saturated n-BuOH,andmix thoroughly.Retain the butanol,condense and use for chromatography.Separate the saponins using chloroform,glacial acetic acid,methanol andwater(64:34:12:8)solvent mixture.Expose the chromatogram to the iodinevapors.The colour(yellow)andhRf values of these spotswere recorded byexposing chromatogram tothe iodine vapours(Mallikharjuna et al.,2007).Liebermann-Burchardt test To1ml of methanolic extract,add1ml of chloroform,2-3ml of aceticanhydride,1to2drops of concentrated sulphuric acid.Dark green coloration(Kumar et al.,2007).-To1ml of extract,add2ml acetic anhydride and2ml concentrated sulphuric acid H2SO4.Color change to blue orgreen(Edeoga et al.,2005).10)SteroidTLC method Extract two grams of powdered test samples with10ml methanol in water bath (80°C/15min).Use the condensed filtrate for chromatography.The sterols canbe separated using chloroform,glacial acetic acid,methanol and water(64:34:12:8)solvent mixture.The color and hRf values of these spots can berecorded under visible light after spraying the plates with anisaldehyde-sulphuric acid reagent and heating(100°C/6min)The color(Greenish blackto Pinkish black)and hR fvalues of these spots canbe recorded under visiblelight(Mallikharjuna et al.,2007).11)Tannin Braemer’s test10%alcoholic ferric chloride will be added to2-3ml of methanolic extract(1:1)Dark blue or greenish greycoloration of the solution(Kumar et al.,2007);(Parekh and Chanda,2007).Liebermann-Burchardt test To1ml of methanolic extract,add1ml of chloroform,2-3ml of aceticanhydride,1to2drops of concentrated sulphuric acid.Pink or red coloration(Kumar et al.,2007).12)TerpenoidSalkowski test5ml extract was added with2ml of chloroform and3ml of concentrated sulphuric acid H2SO4.Reddish brown color ofinterface(Edeoga et al.,2005).13)Volatile oil-Add2ml extract with0.1ml dilute NaOH and small quantity of dilute HCl.Shake the solution.Formation of whiteprecipitates(Dahiru et al.,2006).。

dual luciferase reporter assay system protocol

Promega CorporaƟon · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-35ቤተ መጻሕፍቲ ባይዱ-9526 · 608-274-4330 · Fax 608-277-2516 TM040 · Revised 6/11
1
1.
Description
Genetic reporter systems are widely used to study eukaryotic gene expression and cellular physiology. Applications include the study of receptor activity, transcription factors, intracellular signaling, mRNA processing and protein folding. Dual reporters are commonly used to improve experimental accuracy. The term “dual reporter” refers to the simultaneous expression and measurement of two individual reporter enzymes within a single system. Typically, the “experimental” reporter is correlated with the eﬀect of speciﬁc experimental conditions, while the activity of the co-transfected “control” reporter provides an internal control that serves as the baseline response. Normalizing the activity of the experimental reporter to the activity of the internal control minimizes experimental variability caused by diﬀerences in cell viability or transfection eﬃciency. Other sources of variability, such as diﬀerences in pipetting volumes, cell lysis eﬃciency and assay eﬃciency, can be eﬀectively eliminated. Thus, dual-reporter assays often allow more reliable interpretation of the experimental data by reducing extraneous inﬂuences. The Dual-Luciferase® Reporter (DLR™) Assay System(a–f) provides an eﬃcient means of performing dual-reporter assays. In the DLR™ Assay, the activities of ﬁreﬂy (Photinus pyralis) and Renilla (Renilla reniformis, also known as sea pansy) luciferases are measured sequentially from a single sample. The ﬁreﬂy luciferase reporter is measured ﬁrst by adding Luciferase Assay Reagent II (LAR II) to generate a stabilized luminescent signal. After quantifying the ﬁreﬂy luminescence, this reaction is quenched, and the Renilla luciferase reaction is simultaneously initiated by adding Stop & Glo® Reagent to the same tube. The Stop & Glo® Reagent also produces a stabilized signal from the Renilla luciferase, which decays slowly over the course of the measurement. In the DLR™ Assay System, both reporters yield linear assays with subattomole sensitivities and no endogenous activity of either reporter in the experimental host cells. Furthermore, the integrated format of the DLR™ Assay provides rapid quantitation of both reporters either in transfected cells or in cell-free transcription/translation reactions. Promega offers the pGL4 series of firefly and Renilla luciferase vectors designed for use with the DLR™ Assay Systems. These vectors may be used to co-transfect mammalian cells with experimental and control reporter genes.

生物技术

生物科学热门话题知识讲座结课论文生物技术在食品检测方面的应用摘要: 综述了DNA探针、PCR、生物芯片、胶体金免疫层析及ELLSA等生物技术的基本原理及其在食品检测方面的应用。

关键词: 生物技术DNA探针PCR 生物芯片胶体金免疫层析ELLSA 食品检测Application of Biological Techn iques for Detection in FoodsXie Xiuzhi(Guangzhou Agricultural S tandard and Supervisory Center, Guangzhou 510315)Abs tra c t: The p rimary p rincip les of several biological techniques, includingDNA p robe, PCR, bio2chip s, immuno2chromatographic assay( ICA) and ELLSA were introduced, and meanwhile, their app lication in the regions of food safety and quality control were mainly discussed1Key wo rd s: Biological techniques DNA p robe PCR Bio2chip s ICA ELLSA Food detection当前,我国农产(食)品质量安全问题受到社会广泛关注,仅靠常规的化学检测已不能满足快速判定的需要。

一些简便、敏感、准确、省力、省成本的快速检测方法越来越多地被运用到食品安全性检测中。

食品安全及质量与人们生活健康息息相关,也是影响食品工业发展及对外贸易的重要因素。

长期以来,广泛应用的物理、化学、仪器等食品检测方法已不能满足现代食品检测的需要。

一些简便、敏感、准确、省力、省成本的快速检测方法越来越多地被运用到食品安全性检测中。

Autophagy in the Pathogenesis of Disease

Leading EdgeReviewCell 132, January 11, 2008 ©2008 Elsevier Inc. 27IntroductionFasting has been an integral part of health and healing practices throughout the recorded history of mankind. This ancient tradition may be partially rooted in a cellular process we are now beginning to understand in modern scientific terms. One of the most evolu-tionarily conserved cellular responses to organismal fasting is the activation of the lysosomal degradation pathway of autophagy, a process in which the cell self-digests its own components. This self-digestion not only provides nutrients to maintain vital cellular functions during fasting but also can rid the cell of superfluous or damaged organelles, misfolded proteins, and invading micro-organisms. Interestingly, self-digestion by autophagy—a process that is potently triggered by fasting—is now emerging as a central biological pathway that functions to promote health and l ongevity.The Autophagic PathwayAutophagy (from the Greek, “auto” oneself, “phagy” to eat) refers to any cellular degradative pathway that involves the delivery of cytoplasmic cargo to the lysosome. At least three forms have been identified—chaperone-mediated autophagy, microau-tophagy, and macroautophagy—that differ with respect to their physiological functions and the mode of cargo delivery to the lyso-some. This Review will focus on macroautophagy (herein referred to as autophagy), the major regulated catabolic mechanism that eukaryotic cells use to degrade long-lived proteins and organelles. This form of autophagy involves the delivery of cytoplasmic cargo sequestered inside double-membrane vesicles to the lysosome (Figure 1). Initial steps include the formation (vesicle nucleation) and expansion (vesicle elongation) of an isolation membrane, which is also called a phagophore. The edges of the phagophore then fuse (vesicle completion) to form the autophagosome, a dou-ble-membraned vesicle that sequesters the cytoplasmic material. This is followed by fusion of the autophagosome with a lysosome to form an autolysosome where the captured material, together with the inner membrane, is degraded (Figure 1).Autophagy occurs at low basal levels in virtually all cells to perform homeostatic functions such as protein and organelle turnover. It is rapidly upregulated when cells need to generate intracellular nutrients and energy, for example, during starva-tion, growth factor withdrawal, or high bioenergetic demands. Autophagy is also upregulated when cells are preparing to undergo structural remodeling such as during developmental transitions or to rid themselves of damaging cytoplasmic com-ponents, for example, during oxidative stress, infection, or pro-tein aggregate accumulation. Nutritional status, hormonal fac-tors, and other cues like temperature, oxygen concentrations, and cell density are important in the control of autophagy. The molecular cascade that regulates and executes autophagy has been the subject of recent, comprehensive reviews (Klion-sky, 2007; Maiuri et al., 2007a; Mizushima and Klionsky, 2007; Rubinsztein et al., 2007).One of the key regulators of autophagy is the target of rapamy-cin, TOR kinase, which is the major inhibitory signal that shuts off autophagy in the presence of growth factors and abundant nutrients. The class I PI3K/Akt signaling molecules link recep-tor tyrosine kinases to TOR activation and thereby repress autophagy in response to insulin-like and other growth factor signals (Lum et al., 2005). Some of the other regulatory mole-cules that control autophagy include 5′-AMP-activated protein kinase (AMPK), which responds to low energy; the eukaryotic initiation factor 2α (eIF2α), which responds to nutrient starvation, double-stranded RNA, and endoplasmic reticulum (ER) stress; BH3-only proteins that contain a Bcl-2 homology-3 (BH3) domain and disrupt Bcl-2/Bcl-X L inhibition of the Beclin 1/class III PI3K complex; the tumor suppressor protein, p53; death-associated protein kinases (DAPk); the ER-membrane-associated protein, Ire-1; the stress-activated kinase, c-Jun-N-terminal kinase; the inositol-trisphosphate (IP 3) receptor (IP 3R); GTPases; Erk1/2; ceramide; and calcium (Criollo et al., 2007; Maiuri et al., 2007a; Meijer and Codogno, 2006; Rubinsztein et al., 2007).Autophagy in the Pathogenesis of DiseaseBeth Levine 1,2,* and Guido Kroemer 3,4,5,*1Department of Internal Medicine 2Department of MicrobiologyUniversity of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA 3Institut Gustave Roussy 4Université Paris Sud, Paris 115INSERM, U848F-94805 Villejuif, France*Correspondence: beth.levine@ (B.L.), kroemer@igr.fr (G.K.)DOI 10.1016/j.cell.2007.12.018Autophagy is a lysosomal degradation pathway that is essential for survival, differentiation, devel-opment, and homeostasis. Autophagy principally serves an adaptive role to protect organisms against diverse pathologies, including infections, cancer, neurodegeneration, aging, and heart disease. However, in certain experimental disease settings, the self-cannibalistic or, paradoxically, even the prosurvival functions of autophagy may be deleterious. This Review summarizes recent advances in understanding the physiological functions of autophagy and its possible roles in the causation and prevention of human diseases.28 Cell 132, January 11, 2008 ©2008 Elsevier Inc.Downstream of TOR kinase, there are more than 20 genes in yeast (known as the ATG genes) that encode proteins (many of which are evolutionarily conserved) that are essential for the execution of autophagy (Mizushima and Klionsky, 2007) (Figure 1). These include a protein serine/threonine kinase complex that responds to upstream signals such as TOR kinase (Atg1, Atg13, Atg17), a lipid kinase signaling complex that mediates vesicle nucleation (Atg6, Atg14, Vps34, and Vps15), two ubiquitin-like conjugation pathways that mediate vesicle expansion (the Atg8 and Atg12 systems), a recycling pathway that mediates the dis-assembly of Atg proteins from mature autophagosomes (Atg2, Atg9, Atg18), and vacuolar permeases that permit the efflux of amino acids from the degradative compartment (Atg22). In mam-mals, proteins that act more generally in lysosomal function are required for proper fusion with autophagosomes—such as the lysosomal transmembrane proteins, LAMP-2 and CLN3—and for the degradation of autophagosomal contents, such as the lysosomal cysteine proteases, cathepsins B, D, and L (Table 1).The identification of signals that regu-late autophagy and genes that execute autophagy has facilitated detection and manipulation of the autophagy pathway. Phosphatidylethanolamine (PE) conju-gation of yeast Atg8 or mammalian LC3 during autophagy results in a nonsoluble form of Atg8 (Atg8-PE) or LC3 (LC3-II) that stably associates with the autopha-gosomal membrane (Figure 1). Conse-quently, autophagy can be detected bio-chemically (by assessing the generation of Atg8-PE or LC3-II) or microscopically (by observing the localization pattern of fluorescently tagged Atg8 or LC3) (Mizushima and Klionsky, 2007). These approaches must be coupled with ancil-lary measures to discriminate between two physiologically distinct scenarios—increased autophagic flux without impair-ment in autophagic turnover (i.e., an increased “on-rate”) versus impaired clearance of autophagosomes (i.e., a “decreased off-rate”), which results in a functional defect in autophagic catabo-lism (Figure 2).Autophagy can be pharmacologically induced by inhibiting negative regulators such as TOR with rapamycin (Rubinsztein et al., 2007); the antiapoptotic proteins Bcl-2 and Bcl-X L that bind to the mammalian ortholog of yeast Atg6, Beclin 1, with ABT-737 (Maiuri et al., 2007b); IP 3R with xestospongin B, an IP 3R antagonist; or lithium, a molecule that lowers IP 3 levels (Criollo et al., 2007). Autophagy can be pharmacologically inhibited by targeting the class III PI3K involved in autopha-gosome formation with 3-methyladenine or by targeting the fusion of autophagosomes with lysosomes, using inhibitors of the lysosomal proton pump such as bafilomycin A1 or lysoso-motropic alkalines such as chloroquine and 3-hydroxychloro-Figure 1. The Cellular, Molecular, and Physiological Aspects of AutophagyThe cellular events during autophagy follow dis-tinct stages: vesicle nucleation (formation of the isolation membrane/phagophore), vesicle elonga-tion and completion (growth and closure), fusion of the double-membraned autophagosome with the lysosome to form an autolysosome, and lysis of the autophagosome inner membrane and break-down of its contents inside the autolysosome. This process occurs at a basal level and is regulated by numerous different signaling pathways (see text for references). Shown here are only the regula-tory pathways that have been targeted pharma-cologically for experimental or clinical purposes. Inhibitors and activators of autophagy are shown in red and green, respectively. At the molecular level, Atg proteins form different complexes that function in distinct stages of autophagy. Shown here are the complexes that have been identified in mammalian cells, with the exception of Atg13 and Atg17 that have only been identified in yeast. The autophagy pathway has numerous proposed physiological functions; shown here are functions revealed by in vivo studies of mice that cannot un-dergo autophagy (see T able 1).quine (Rubinsztein et al., 2007) (Figure 1). It should be noted that all of these pharmacological agents lack specificity for the autophagy pathway. Therefore, although some of these agents such as rapamycin, lithium, and chloroquine are clinically available and may be helpful for treating diseases associated with autophagy deregulation, genetic approaches to inhibiting autophagy—for example, knockout of ATG genes by homolo-gous recombination or knockdown by small-interfering RNA (siRNA)—have yielded more conclusive information about the biologic roles of autophagy in health and disease. Physiological Functions of AutophagyAutophagy Defends against Metabolic Stress Autophagy is activated as an adaptive catabolic process in response to different forms of metabolic stress, including nutrient deprivation, growth factor depletion, and hypoxia. This bulk form of degradation generates free amino and fatty acids that can be recycled in a cell-autonomous fashion or delivered systemically to distant sites within the organism. Presumably, the amino acids generated are used for the de novo synthesis of proteins that are essential for stress adaptation. The molec-ular basis for the recycling function of autophagy has only recently begun to be defined with the identification of yeast Atg22 as a vacuolar permease required for the efflux of amino acids resulting from autophagic degradation (Mizushima and Klionsky, 2007). It is presumed that the recycling function of autophagy is conserved in mammals and other higher organ-isms, although direct data proving this concept are lacking. The amino acids liberated from autophagic degradation can be further processed and, together with the fatty acids, used by the tricarboxylic acid cycle (TCA) to maintain cellular ATP production. The importance of autophagy in fueling theTCACell 132, January 11, 2008 ©2008 Elsevier Inc. 2930 Cell 132, January 11, 2008 ©2008 Elsevier Inc.cycle is supported by studies showing that certain phenotypes of autophagy-deficient cells can be reversed by supplying them with a TCA substrate such as pyruvate (or its membrane-permeable derivative methylpyruvate). For example, meth-ylpyruvate can maintain ATP production and survival in growth factor-deprived autophagy-deficient cells that would otherwise quickly die (Lum et al., 2005). It can also restore ATP produc-tion, the generation of engulfment signals, and effective corpse removal in autophagy-deficient cells during embryonic devel-opment (Qu et al., 2007).This role of autophagy in maintaining macromolecular synthesis and ATP production is likely a critical mechanism underlying its evolutionarily conserved prosurvival function. Gene knockout or knockdown studies in diverse phyla provide strong evidence that autophagy plays an essential function in organismal survival during nutrient stress (Maiuri et al., 2007a). Yeast cells lacking ATG genes display reduced tol-erance to nitrogen or carbon deprivation and are defective in starvation-induced sporulation. Similarly, null mutations in ATG genes in slime molds limit viability and differentiation during nutrient depri-vation. Loss-of-function mutations in ATG genes in plants reduce tolerance to nitrogen or carbon depletion, resulting in enhanced chlorosis, reduced seed set, and accelerated leaf senescence (Bassham et al., 2006). Furthermore, siRNA-mediated knockdown of atg genes in nematodes decreases survival during starvation (Kang et al., 2007). Autophagy also enables mammals to withstand nutrient depletion (Table 1). Mice lacking either atg5−/− or atg7−/− are born at normal Mendelian ratios yet diewithin hours after birth, presumably due to their inability to adapt to the neonatal starvation period.Thus, a critical physiological role of autophagy appears to be the mobilization of intracellular energy resources to meet cellular and organismal demands for metabolic sub-strates. The requirement for this function of autophagy is not limited to settings of nutrient starvation. Because growth factors are often required for nutrient uptake, loss of growth factor signaling can result in reduced intracellular metabo-lite concentrations and activation of autophagy-dependent survival mechanisms (Lum et al., 2005). It is also possible that in certain settings, especially when cells suddenly have high metabolic needs, autophagy may be needed in a cell-autonomous fashion to generate sufficient intracellularmetabolic substrates to maintain cellular energy homeosta-Figure 2. Alterations in Different Stages of Autophagy Have Different ConsequencesAn increased on-rate of autophagy occurs in re-sponse to stress signals, resulting in increased autophagosomal and autolysosomal accumula-tion and successful execution of the adaptive physiological functions of autophagy. In certain disease states or upon treatment with lysosomal inhibitors, there is a reduced off-rate resulting in impaired lysosomal degradation of autophago-somes. This results in increased autophagosomal accumulation and adverse pathophysiological consequences related to unsuccessful comple-tion of the autophagy pathway. A decreased on-rate is observed if signaling activation of au-tophagy is defective or mutations are present in ATG genes. This results in decreased autopha-gosomal accumulation, the accumulation of pro-tein aggregates and damaged organelles, and pathophysiological consequences related to de-ficient protein and organelle turnover. The physi-ological and pathophysiological consequences listed for “increased on-rate,” “reduced off-rate,” and “decreased on-rate” are based on knockout studies of the ATG genes in model organisms.Cell 132, January 11, 2008 ©2008 Elsevier Inc. 31sis. This hypothesis may explain why there are high levels of autophagy in the mouse heart and diaphragm immediately following birth (Kuma et al., 2004).Autophagy Works as a Cellular HousekeeperThe repertoire of routine housekeeping functions performed by autophagy includes the elimination of defective proteins and organelles, the prevention of abnormal protein aggregate accumulation, and the removal of intracellular pathogens. Such functions are likely critical for autophagy-mediated pro-tection against aging, cancer, neurodegenerative diseases, and infection. Although some of these functions overlap with those of the ubiquitin-proteosome system—the other majorcellular proteolytic system—the autophagy pathway is uniquely capable of degrading entire organelles such as mitochondria, peroxisomes, and ER as well as intact intracellular microor-ganisms. Further, the relative role of the autophagy-lysosome system in protein quality control—i.e., in preventing the intra-cellular accumulation of altered and misfolded proteins—may be greater than previously anticipated.Tissue-specific disruption of ATG genes has revealed a critical role for basal autophagy in protein quality control in murine post-mitotic cells (Table 1). Atg7 deletion in hepatocytes, atg5 and atg7 deletion in neurons, and atg5 deletion in cardiomyocytes result in the accumulation of ubiquitin-positive protein aggregates in inclusion bodies that are associated with cellular degeneration. Such abnormalities have not been reported for atg5-deficient dendritic cells or T lymphocytes, perhaps because autophagy is less important for the waste management of rapidly prolifer-ating cells. Moreover, the underlying mechanism for the accu-mulation of ubiquitin-positive aggregates in certain autophagy knockout mouse tissues remains unknown. Cytoplasmic accu-mulation of diffuse ubiquitinated proteins precedes the accu-mulation of aggregates in atg5-deficient neurons (Hara et al., 2006). Thus, aggregate formation may be a secondary result of a general defect in protein turnover rather than a failure of basal autophagy to clear aggregates that are formed constitutively in normal conditions (Mizushima and Klionsky, 2007). According to such a model, in the absence of autophagy, the turnover of cyto-solic proteins is impaired, increasing their propensity to become damaged and misfolded and subsequently ubiquitinated and aggregated (Figure 3). It is not yet clear whether the ubiquit-inated proteins are autophagically sequestered in a random, nonselective fashion, or whether they are selectively targeted to the autophagosome by a mechanism involving p62/SQSTM1, an adaptor protein that binds both ubiquitin and LC3 (Pankiv et al., 2007).Unlike proteasomal degradation, the autophagic breakdown of substrates is not limited by steric considerations and there-fore autophagy can sequester and degrade entire organelles. In yeast, autophagy participates in the selective removal of super-fluous peroxisomes (pexophagy) generated when cells adapt to glucose metabolism (Nair and Klionsky, 2005) and perhaps in the elimination of damaged mitochondria (mitophagy), as atg mutant yeast accumulate dysfunctional mitochondria (Zhang et al., 2007). Selective degradation of peroxisomes or mitochondria was reported in hepatocytes isolated from clofibrate-treated or starved rats, respectively (Kim et al., 2007), but until recently the importance of these forms of selective autophagy in mammalian physiology was unclear. However, under steady-state conditions, atg7-deficient mouse hepatocytes accumulate peroxisomes, deformed mitochondria, and aberrant concentric membranous structures that are contiguous with the ER, and during chemi-cal treatment, atg7-deficient mouse livers display a defect in the removal of excess peroxisomes (Table 1). Furthermore, agents that promote ER stress induce the selective autophagy of ER membranes (reticulophagy) both in yeast and in mammalian cells (Klionsky, 2007). Taken together, these observations indicate that basal and induced autophagy are likely important for the physiological control of number and quality of organelles acrossdiverse phyla and function to eliminate superfluous and damagedFigure 3. Autophagy, Protein Quality Control, and NeurodegenerationNormal proteins are routinely turned over by different protein degradation sys-tems, including the ubiquitin-proteasome system (UPS), chaperone-mediated autophagy (CMA), and macroautophagy (referred to herein as “autophagy”). In autophagy-deficient neurons, there is an accumulation of ubiquitinated pro-tein aggregates that is associated with neurodegeneration. Similar effects of autophagy deficiency are observed in other postmitotic cells (hepatocytes, cardiomyocytes) under basal conditions. Proteins altered by mutations (such as polyglutamine expansion tracts), posttranslational modifications, or stress (such as oxidative stress, UV irradiation, toxins) undergo a conformational change, are recognized by molecular chaperones, and are either refolded and repaired or delivered to protein degradation systems (usually UPS or CMA). If these protein degradation systems are impaired or if the altered proteins form oligomeric complexes that cannot be recognized by the UPS or CMA, au-tophagy may be the primary route for the removal of these abnormal and po-tentially toxic proteins. Impaired autophagy is associated with the formation of protein aggregates and increased neurodegeneration. The mechanisms by which abnormal proteins and impaired autophagy result in neurodegeneration are not known.organelles. Defined or candidate signals for selective organelle recognition by autophagy include the peroxisome membrane tag, Pex14, an outer mitochondrial membrane protein, Uth1p, in yeast (Mizushima and Klionsky, 2007), and the mitochondrial perme-ability transition in mammalian cells (Kim et al., 2007). Autophagy May Be a Guardian of the GenomeRecent studies in ATG gene-deficient immortalized epithelial cells indicate that the autophagic machinery can limit DNA damage and chromosomal instability (Mathew et al., 2007a). Because these studies used cells with simultaneous defects in DNA checkpoints and apoptosis pathways, it is not yet known whether autophagy plays a primary function in preventing genomic instability in nor-mal cells. However, in view of known functions of autophagy in energy homeostasis and in protein and organelle quality control, this seems likely. Such a role of autophagy would mechanistically link effects on the prevention of tumor initiation, tumor progres-sion, aging, and neurodegeneration. The precise mechanisms by which deficient autophagy compromises genomic stability are unclear. Failure to control the damage of checkpoint or repair pro-teins, deregulated turnover of centrosomes, insufficient energy for proper DNA replication and repair, and excessive generation of reactive oxygen species due to inefficient removal of dam-aged mitochondria are possible alterations that may contribute to genomic instability in autophagy-defective cells (Jin and White, 2007; Mathew et al., 2007a).Autophagy in Life and Death Decisions of the Cell Under most circumstances, autophagy constitutes a stress adaptation pathway that promotes cell survival. An appar-ent paradox is that autophagy is also considered a form of nonapoptotic programmed cell death called “type II” or “autophagic” cell death. This type of cell death has been his-torically defined by morphological criteria, but it is now clear that the mere presence of autophagosomes in dying cells is insufficient to distinguish “cell death with autophagy” from “cell death by autophagy.” The knockdown of ATG genes has recently defined whether autophagy functions in the execution of cell death in different settings (see Maiuri et al., 2007a for detailed review).It is not yet understood what factors determine whether autophagy is cytoprotective or cytotoxic and whether cyto-toxicity occurs as the result of self-cannibalism, the specific degradation of cytoprotective factors, or other as of yet unde-fined mechanisms (for an extensive discussion, see Maiuri et al., 2007a). The most intuitive mechanism is self-cannibalism. However, cells subjected to prolonged growth factor depriva-tion or shortage of glucose and oxygen can lose the major-ity of their mass via autophagy and fully recover when placed in optimal culture conditions (Degenhardt et al., 2006; Lum et al., 2005), suggesting that cell death via autophagy may not be simply a matter of crossing a quantitative threshold of self-digestion. Although autophagy can independently influence life and death decisions of the cell (by being cytoprotective or self-destructive), it is also intricately linked to apoptotic death path-ways. Factors that may control the cellular “decision” between the two responses include potentially variable thresholds for each process, molecular links that coordinately regulate apop-tosis and autophagy, and mutual inhibition or activation of each pathway by the other (see Maiuri et al., 2007a for details).There is no evidence currently that the ATG genes promote programmed cell death that occurs physiologically in vivo, for instance during development. In fact, nematodes lacking bec-1, an ortholog of atg6/beclin 1, and mice lacking beclin 1 or atg5 display increased, rather than decreased, numbers of apoptotic cells in embryonic tissues (Qu et al., 2007; Takacs-Vellai et al., 2005; Yue et al., 2003). Given the recently identi-fied role of ATG genes in facilitating the heterophagic removal of apoptotic corpses (Qu et al., 2007) (Figure 1C), it is not yet certain whether the increased numbers of apoptotic cells in autophagy-deficient embryos represent increased cell death events, delayed clearance of dead cells, or a combination of the two. Perhaps clearer evidence for a prosurvival function of autophagy in vivo is provided by tissue-specific ATG gene knockout studies—for example the neuron-specific knockout of atg5or atg7and T cell-specific knockout of atg5—where increased apoptosis is observed in mature animals (Table 1). The intricate interplay between autophagy and life and death decisions of the cell mirrors some of the complexities in deci-phering the roles of autophagy in human diseases and their treatments. For decades, pathologists have noted ultrastruc-tural features of autophagy in a cornucopia of human diseases, including infections, neurodegenerative and myodegenerative diseases, cardiomyopathies, and cancer (de Duve and Wattiaux, 1966; Martinez-Vicente and Cuervo, 2007). These findings were largely either ignored or presumed to reflect a causative role of autophagy in cellular degeneration and disease. The inability to distinguish between defective autophagy (with decreased removal of autophagosomes) and increased autophagic activ-ity (with increased formation of autophagosomes) further con-founded the pathophysiological interpretation of autophagosome accumulation in tissue samples. Now, with the identification of signaling pathways that regulate autophagy, evolutionarily con-served gene products that mediate autophagy, and methods to distinguish between increased on-rates versus decreased off-rates of autophagy, pharmacological, genetic, and biochemical approaches are being used to redefine the role of autophagy in the pathogenesis of human diseases.Autophagy in DiseaseAutophagy and Neurodegenerative DiseasesEarly reports demonstrating that autophagosomes accumulate in the brains of patients with diverse neurodegenerative dis-eases, including Alzheimer’s disease, transmissible spongiform encephalopathies, Parkinson’s disease, and Huntington’s dis-ease (reviewed in Rubinsztein et al., 2007; Williams et al., 2006), led to the initial hypothesis that autophagy contributed to the pathogenesis of these disorders. In mice with cerebellar degen-eration due to mutations in glutamate receptor, autophagy was also postulated to be a mechanism of nonapoptotic cell death (Yue et al., 2002). In contrast, more recent studies provide com-pelling evidence that at least in model organisms autophagy protects against diverse neurodegenerative diseases and that the accumulation of autophagosomes primarily represents the activation of autophagy as a beneficial physiological response or, in the case of Alzheimer’s disease, the consequence of a defect in autophagosomal maturation (Martinez-Vicente and Cuervo, 2007; Rubinsztein et al., 2007; Williams et al., 2006).32Cell 132, January 11, 2008 ©2008 Elsevier Inc.Beyond its role in the clearance of misfolded proteins spon-taneously generated during routine protein turnover (discussed above), autophagy likely plays an important role in the clear-ance of aggregate-prone mutant proteins associated with several different neurodegenerative diseases (Figure 3). These include proteins with polyglutamine (polyQ) expansion tracts such as those seen in Huntington’s disease and spinocerebel-lar ataxia, mutant α-synucleins that cause familial Parkinson’s disease, and different forms of tau including mutations causing frontotemporal dementia (Williams et al., 2006). Because sub-strates need to be unfolded to pass through the narrow pore of the proteasomal barrel, oligomeric and aggregated proteins are poor substrates for proteasomal degradation and better targets for autophagic degradation. The mechanism by which these proteins exert their cellular toxicity is still controversial, but it is generally believed that they are particularly toxic in oligomeric complexes and that higher-order protein aggre-gates may be formed as a last attempt to prevent toxicity in the absence of a properly functioning quality-control system (Martinez-Vicente and Cuervo, 2007). This view is consistent with the model that autophagy functions as a quality-control system that targets oligomeric proteins and with the evidence that autophagy activation reduces, whereas autophagy inhi-bition increases, the formation of protein aggregates and the neurotoxicity of aggregate-prone proteins. Pharmacological activation of autophagy reduces the levels of soluble and aggregated forms of mutant huntingtin protein, proteins mutated in spinocerebellar ataxia, mutant forms of α-synuclein, and mutant tau; it also reduces their cellular toxicity in vitro and their neurotoxicity in either mouse or Drosophila mod-els (Rubinsztein et al., 2007). ATG gene knockdown or knockout increases aggregate formation and toxicity of polyQ expansion proteins in C. elegans(Jia et al., 2007). Autophagy induced by overexpression of histone deacetylase 6 also compensates for impairment in the ubiquitin-proteasome system in a fly model of spinobulbar muscle dystrophy (Pandey et al., 2007). In these models, autophagy-mediated neuroprotection may be due to a quantitative reduction in the amounts of the toxic protein species as well as antiapoptotic effects (Rubinsztein et al., 2007).The development of neurodegenerative disease in patients with proteinopathies implies that the autophagy may reach a saturation point in which its capacity to degrade the mutant aggregate-prone proteins is exceeded, or that concurrentdefects may occur in the autophagy pathway. Acquired defectsCell 132, January 11, 2008 ©2008 Elsevier Inc. 33。

MAK095 Aspartate Assay Kit 产品说明书

mak095pis Rev 09/231Technical BulletinAspartate Assay KitCatalogue number MAK095Product DescriptionAspartate, the carboxylate anion of aspartic acid, is an acidic, non-essential amino acid involved in protein synthesis and multiple other cellular biochemical pathways. Aspartate contributes to nucleotidesynthesis via the synthesis of the precursor inosine monophosphate. In the urea cycle, aspartate is a key metabolite, donating a nitrogen group towards the formation of urea. Aspartate is also critical foroxidative phosphorylation as part of the aspartate-malate shuttle, which transfers reducing equivalents across the mitochondrial membrane.The Aspartate Assay Kit is suitable for aspartate detection in cell and tissue culture supernatants, urine, plasma, serum, and other biological samples. Aspartate concentration is determined by a coupled enzyme assay, which results in a colorimetric(570 nm)/ fluorometric (λEx = 535/λEm = 587 nm)product, proportional to the aspartate present. Typical detection ranges for this kit are 2–10 nmole (colorimetric) and 0.2–1 nmole (fluorometric).ComponentsThe kit is sufficient for 100 assays in 96-well plates. • Aspartate Assay Buffer25 mL Catalogue Number MAK095A • Probe, in DMSO0.2 mLCatalogue Number MAK095B • Serum Clean Up Mix1 vl Catalogue Number MAK095C • Aspartate Enzyme Mix1 vl Catalogue Number MAK095D • Conversion Mix1 vl Catalogue Number MAK095E •Aspartate Standard, 100 mM 0.1 mLCatalogue Number MAK095FReagents and Equipment Required but Not Provided.•96-well flat-bottom plate – It is recommended to use black plates with clear bottoms for fluorescence assays and clear plates forcolorimetric assays. Cell culture or tissue culture treated plates are not recommended.• Fluorescence or spectrophotometric multiwell plate reader•10 kDa Molecular Weight Cut-Off (MWCO) Spin FiltersPrecautions and DisclaimerFor R&D use only. Not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.Preparation InstructionsBriefly centrifuge vials before opening. To maintain reagent integrity, avoid repeated freeze/thaw cycles. Aspartate Assay Buffer – Allow buffer to come to room temperature before use.Probe – Warm to room temperature to melt frozen solution prior to use. Store protected from light and moisture at -20 °C. Upon thawing, the Probe is ready-to-use in the colorimetric assay.For the fluorescence assay, dilute an aliquot of the colorimetric Probe Solution 4-fold with Aspartate Assay Buffer, just prior to use. This will reduce the background of the fluorescence assay.Serum Clean Up Mix, Aspartate Enzyme Mix, and Conversion Mix – Reconstitute each in 220 µL of Aspartate Assay Buffer. Mix well by pipetting, then aliquot each and store at -20 °C. Keep cold while in use and protect from light. Use within 2 months of reconstitution.mak095pis Rev 09/232Storage/StabilityThe kit is shipped on wet ice. Storage at -20 °C, protected from light, is recommended.ProcedureAll Samples and Standards should be run in duplicate.Aspartate Standards for Colorimetric DetectionDilute 10 µL of the 100 mM (100 nmole/µL) Aspartate Standard Solution with 990 µL of water to prepare a 1 mM (1 nmole/µL) Aspartate Standard. PrepareAspartate Standards for colorimetric assay according to Table 1. Mix well.Table 1.Preparation of Aspartate Standards for colorimetricAspartate Standards Fluorometric DetectionPrepare a 1 mM Aspartate Standard solution as for the colorimetric assay. Dilute 100 µL of the1 mM Aspartate Standard with 900 µL of water to make a 0.1 mM (0.1 nmole/µL) Aspartate Standard. Prepare Aspartate Standards for fluorometric assay according to Table 2. Mix well. Table 2.Preparation of Aspartate Standards forfluorometric assay Sample PreparationBoth the colorimetric and fluorometric assays require 50 µL of Sample for each reaction (well).Tissue or cells (1 ⨯ 106) can be homogenized in100 µL of the Aspartate Assay Buffer. Centrifuge the Samples at 13,000 ⨯ g for 10 minutes to removeinsoluble material. Bring Samples to a final volume of 50 µL with Aspartate Assay Buffer.Serum Samples should be pretreated with the Serum Clean Up Mix to remove interfering substances. Add 2 µL of the Serum Clean Up Mix to 100 µL of serum and incubate for 30 minutes at room temperature. Treated Samples should be deproteinized before use in assay with a 10 kDa MWCO spin filter. 1–30 µL of deproteinized serum Samples can be directly diluted to a final volume of 50 µL with the Aspartate Assay Buffer.Note : Due to relatively low levels of aspartate in serum, it is strongly recommended to use thefluorometric assay, which is typically 10-fold more sensitive than the colorimetric assay.For unknown Samples, it is suggested to test several Sample dilutions to ensure the readings are within the linear range of the Standard curve.Pyruvate present in the Sample can generate background. To control for pyruvate background, include a blank Sample for each Sample by omitting the Aspartate Enzyme Mix in the Reaction Mix.mak095pis Rev 09/233Assay Reaction1. Set up the Reaction Mixes according to thescheme in Table 3. 50 µL of the appropriateReaction Mix is required for each reaction (well). Table 3. Reaction Mixture2. Add 50 µl of the appropriate Reaction Mix to eachof the blank, Standard, and test wells. Mix well using a horizontal shaker or by pipetting. 3. Incubate the reaction for 30 minutes at roomtemperature. Protect the plate from light during the incubation. 4. For colorimetric assays, measure the absorbanceat 570 nm (A 570). For fluorometric assays, measure fluorescence intensity (RFU) at λEx = 535/λEm = 587 nm.ResultsCalculations1. The background for the assays is the valueobtained for the 0 (blank) Aspartate Standard. Correct for the background by subtracting the blank value (A 570 or RFU) from all readings.Background values can be significant and must be subtracted from all readings. 2. Use the corrected values obtained from theappropriate Aspartate Standards to plot a Standard curve.Note : A new Standard curve must be set up each time the assay is run.3. Subtract the Sample Blank value (A 570 or RFU)from the Sample readings to obtain the corrected measurement. 4. Using the corrected Sample A 570 or RFUmeasurement, the amount of aspartate present in the Sample may be determined from the Standard curve.Concentration of Aspartate S A /V = CS A = Amount of aspartate in unknown Sample(nmole) from Standard curveV = Sample volume (µl) added into the wells C = Concentration of aspartate in Sample Aspartate molecular weight: 133.11 g/mole.Sample CalculationAmount of aspartate (S A ) = 5.84 nmole Sample volume (V) = 50 µLConcentration of aspartate in Sample: 5.84 nmole/50 µL = 0.1168 nmole/µL0.1168 nmole/µL 133.11 ng/nmole = 15.55 ng/µLTroubleshooting Guidemak095pis Rev 09/23 4The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.Merck and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.© 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. mak095pis Rev 09/235NoticeWe provide information and advice to our customers on application technologies and regulatory matters to the best of our knowledge and ability, but without obligation or liability. Existing laws and regulations are to be observed in all cases by our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve our customers of their own responsibility for checking the suitability of our products for the envisaged purpose.The information in this document is subject to change without notice and should not be construed as a commitment by the manufacturing or selling entity, or an affiliate. We assume no responsibility for any errors that may appear in this document.Technical AssistanceVisit the tech service page at /techservice .Standard WarrantyThe applicable warranty for the products listed in this publication may be found at /terms .Contact InformationFor the location of the office nearest you, go to /offices .。

1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。

Journal of Chromatography B,850(2007)528–530Short communicationA TLC bioautographic assay for the detection of nitrofurantoinresistance reversal compoundAhmad R.Shahverdi a,∗,Farid Abdolpour a,Hamid R.Monsef-Esfahani b,Hasan Farsam ca Department of Pharmaceutical Biotechnology,Faculty of Pharmacy,Tehran University of Medical Sciences,14174Tehran,Iranb Department of Pharmacognosy,Faculty of Pharmacy,Tehran University of Medical Sciences,14174Tehran,Iranc Department of Medicinal Chemistry,Faculty of Pharmacy,Tehran University of Medical Sciences,14174Tehran,IranReceived14April2006;accepted7November2006Available online30November2006AbstractA simple TLC bioautographic method was developed for detection of antibiotic resistance reversal agents.In this study,the retention factor values of the components of some essential oils not previously shown to have any antibacterial activity were evaluated on nitrofurantoin supplemented agar media.The active component of Artemisia annua,Artemisia dracunculus and Eucalyptus globulus essential oils was piperitone which increased the antibacterial activity of nitrofurantoin against Enterobacter cloacae.Piperitone was not detected in the essential oil of Humulus lupulus and we could not observe any clear areas in this bioautographic method.©2006Elsevier B.V.All rights reserved.Keywords:Bioautographic method;Antibiotic resistance;Essential oils;Piperitone1.IntroductionResistance to antimicrobial agents by pathogenic bacteria has emerged in recent years and is a major health problem. Consequently,there has been increasing interest in the use of inhibitors of antibiotic resistance for combination therapy [1,2].These types of compounds have potential in decreas-ing the effective dose of antimicrobial drugs for therapy.In vitro experiments have shown that natural products and some of their components decrease the minimum inhibitory con-centration(MIC)of antibiotics for different microorganisms [3–8].The use of antibiotic resistance reversal agents for the degradation of bacterial resistance is going to be an issue of con-siderable importance,thus a chromatographic method should be developed and tested for detection of this group of compounds in complex plant matrices.Also such assays are particularly important to avoid the time consuming isolation of known sub-stances or inactive ones.TLC bioautographic methods combine chromatographic separation and in situ activity determination facilitating the localization and target-directed isolation of active ∗Corresponding author.Tel.:+982166482706;fax:+982166461178.E-mail address:shahverd@sina.tums.ac.ir(A.R.Shahverdi).constituents in a mixture.Traditionally,bioautographic tech-nique has used growth inhibition of microorganisms to detect anti-microbial components of extracts chromatographed on a TLC layer.This methodology has been considered as the most efﬁcacious assay for the detection of anti-microbial compounds. The objective of this paper was to develop a simple TLC bioau-tographic method for detection of antibiotic resistance reversal agents.In this method,the R f value of components which have not previously shown any antibacterial activity is detected against the resistant strain on antibiotic supplemented agar media.Recently,piperitone,a volatile component of Mentha longi-folia var.chorodictya,has been reported to increase the antibacterial activity of nitrofurans drugs[7].M.longifolia var. chorodictya named commonly in Iran as Poneh Sorkhabadi,and grown in north part of Iran and Afghanistan.In this investi-gation,the synergistic activity of the essential oils from four common plants wormwood,tarragon,hops and Tasmanian blue gum was tested using agar dilution method and compared with the essential oil of M.longifolia against Enterobacter cloacae. Using this bioautographic method the active compounds of the samples which increased the antibacterial effect of nitrofuran-toin was localized on a chromatogram and identiﬁed by GC–MS analysis.1570-0232/$–see front matter©2006Elsevier B.V.All rights reserved. doi:10.1016/j.jchromb.2006.11.011A.R.Shahverdi et al./J.Chromatogr.B850(2007)528–5305292.Experimental2.1.MaterialsNatural product essential oils,including Artemisia annua, Artemisia dracunculus,Eucalyptus globulus,Humulus lupu-lus and M.longifolia were obtained from Prof.Farsam of the Department of Medicinal Chemistry at our Faculty and Dr. M.Bagheri from Azarbaijan Medicinal Plant Center,Ardabil, Iran.dl-piperitone(3-methyl-6-(1-methylethyl)-2-cyclohexen-1-one)was provided by Charabot Grasse(France).2.2.MIC determinationThe MICs of nitrofurantoin in the presence and absence of essential oils(1␮l/ml)were studied by conventional agar dilution methods using E.cloacae.Different concentrations of nitrofurantoin were included in Muller-Hinton agar plates. The MIC was deﬁned as the lowest concentration of antibiotic where bacterial growth was not detected.The test strain used in this study was obtained from Shariati University Hospital, Tehran(Iran).The identity of this strain was conﬁrmed using conventional methods.Polyethylene glycol(PEG)4000at a con-centration of1%(w/v)was used to facilitate oil diffusion in all of the media used in this study.2.3.Detection methodA TLC bioautographic method was used to detect active com-ponents.Piperitone and M.longifolia essential oil were used as positive controls in all experiments.After application of the sub-inhibitory content of essential oils(10␮l)and piperi-tone(3␮l)on a silica gel60F254plate(Merck,Darmstadt, Germany),thin layer chromatography(TLC)was developed using toluene-ethyl acetate(93:7)as the eluent system[9]and dried for complete removal of solvents.The plates were either Table1Enhancement of bactericidal activity of nitrofurantoin for Enterobacter cloacae by various essential oils a,bSelected essential oils Synergistic MICs(␮g/ml)MICreduction Mentha longifolia2511×Artemisia annua70 3.9×Artemisia dracunculus50 5.5×Eucalyptus globules30 6.1×Humulus lupulus125 2.2×a Tested at a sub-inhibitory concentrations of1␮l/ml.b Nitrofurantoin MIC for Enterobacter cloacae was275␮g/ml.visualized using sulfuric vanillin[9]or biologically(bioauto-graphy)to evaluate the activity of the different essential oils. An inoculum of E.cloacae in the0.1%triphenyl-tetrazolium chloride containing Muller-Hinton agar media(with or without 30␮g/ml nitrofurantoin)was distributed over TLC plates,and the plates were incubated at35◦C for24h.Triphenyl-tetra-zolium chloride was supplied by Aldrich Chemical Co.,UK. Inhibition zones were observed as clear areas against a red colored background.Preparative TLC plates with a thickness of1mm were prepared using the same stationary and mobile phases as above,with the objective of isolating the components of essential oils that enhanced the antibacterial activity of nitro-furantoin against the test strain.These areas were scraped from the plates,and the substance eluted from the silica with ethanol. Eluted samples were further puriﬁed using the above preparative chromatography method.Finally,the components were identi-ﬁed by gas chromatography–mass spectrometry(GC/MS)on a ThermoFinnigan ThermoQuest instrument(Applied Science Uk)using a DB-1capillary column30m×250␮m×0.1␮m). Helium was used as carrier gas at a constantﬂow rate of 1.5ml/min.Electron impact mass spectra were obtained at70eV, the instrument scanning from40–300amu.Scan rate was2.5 per second.The oven temperature was initially50◦C(isother-Fig.1.Detection of active compounds of some essential oils involved in augmentation of nitrofurantoin activity by bioautography and thin layer chromatography methods.TLC plates,composed of Merk Silica gel60F254,received10␮l of the following essential oils:Mentha longifolia(1),Artemisia annua(2),Artemisia dracunculus(3),Eucalyptus globulus(4),Humulus lupulus(5).Piperitone(3␮l)was applied as reference control(line6).The panels demonstrate the result in the presence(with drug)and absence(without drug)of nitrofurantoin.530 A.R.Shahverdi et al./J.Chromatogr.B850(2007)528–530Fig.2.The structure of piperitone(CAS Number89-81-6). mal,5min)and then was increased to250◦C at2.5◦C/min. The injector temperature was250◦C.Identiﬁcation of the com-ponents was made by comparison of their retention times and mass spectra with standards[7,10,11].3.Results and discussionsThe effects of essential oils on the bactericidal activity of nitrofurantoin were studied using the agar dilution method (Table1).The results of these experiments showed that all sam-ples reduced the MICs of nitrofurantoin against the test strain (2–11times).Essential oils of M.longifolia and E.globules were most effective in increasing the antibacterial activity of nitrofu-rantoin against resistant Enterobacteria.The active components of essential oils involved in this process were detected using a bioautographic method.No selected essential oils showed any intrinsic antibacterial activity in absence of the drug(Fig.1). By contrast,analysis of these volatile oils in nitrofurantoin con-taining agar media(30␮g/ml)allowed detection of components (R f=0.35)which were effective in enhancing nitrofurantoin activity(Fig.1).These active components had R f=0.35on TLC and UVλmax=232.5.They had the same R f andλmax values as the piperitone standard(Fig.2).Piperitone was not detected in the essential oil of H.lupulus oil(data was not shown)and we could not observe any clear zone of inhibition against test strain in the presence of drug.The identiﬁcation of the fractions as piperitone was further conﬁrmed by GC/MS analysis.The mass spectroscopic data of the active fractions are as follow: MS(EI)m/z(%)152(M+,C10H16O,43),153(4),69(15),137 (53),95(60),82(94),110(100).To the best of our knowledge,based on a literature search,no studies have been conducted on the detection of antibiotic rever-sal agents using bioautographic technique.This bioautographic method can be conducted for screening of these agents in other plant or microbial extracts.References[1]T.E.Renau,S.J.Hecker,V.J.Lee,Ann.Rep.Med.Chem.33(1998)121.[2]G.D.Wright,Chem.Biol.7(2000)R127.[3]S.Shin,Yakhak Hoeji46(2002)203.[4]S.M.Jaber,S.E.Farouk,M.Ilias,Phytother.Res.18(2004)934.[5]R.Giordani,P.Regli,J.Kaloustian,C.Mikail,L.Abou,H.Portugal,Phytother Res.8(2004)990.[6]M.Sato,H.Tanaka,T.Oh-Uchi,T.Fukai,H.Etoh,R.Yamaguchi,Phy-tother.Res.18(2004)906.[7]A.R.Shahverdi,F.Raﬁi,F.Tavassoli,M.Bagheri,F.Attar,A.Ghahraman,Phytother.Res.18(2004)911.[8]Y.Sakagami,M.Iinuma,K.G.Piyasena,H.R.Dharmaratne,Phytomedicine12(2005)203.[9]H.Wagner,S.Bladt(Eds.),Plant Drug Analysis:A Thin layer Chro-matography Atlas,second ed.,Springer-Verlag,Berlin Heidellberg,1996, p.166.[10]Eight Peak Index of Mass Spectra,third ed.,Mass Spectrometry DataCentre,Royal Society of Chemistry,The University of Nottingham,Not-tingham,UK,1983.[11]R.P.Adams(Ed.),Identiﬁcation of Essential Oil Components by GC–MS,Allured Publishing Corporation,Carol Stream,Illinois,1995.。

A TLC bioautographic assay for the detection of nitrofurantoin

Human reproductive failure

薄层色谱生物自显影法筛选新疆莫合烟天然乙酰胆碱酯酶抑制剂的活性成分

Tet-On 3G Вector Set (with ZsGreen1) 商品说明书

蜡样芽孢杆菌GW-01全基因组测序及生物学特性分析

Genetically modified crops Detection strategies and biosafety issues

活性物质的提取、 分离、表征-综述

dual luciferase reporter assay system protocol

生物技术

Autophagy in the Pathogenesis of Disease

MAK095 Aspartate Assay Kit 产品说明书

活性物质的提取、分离、表征-综述