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Direct Extraction (direct + extraction)
Selected AbstractsSugarcane proteomics: Establishment of a protein extraction method for 2-DE in stalk tissues and initiation of sugarcane proteome reference mapELECTROPHORESIS, Issue 12 2010Ramesh Sundar Amalraj Abstract Sugarcane is an important commercial crop cultivated for its stalks and sugar is a prized commodity essential in human nutrition. Proteomics of sugarcane is in its infancy, especially when dealing with the stalk tissues, where there is no study to date. A systematic proteome analysis of stalk tissue yet remains to be investigated in sugarcane, wherein the stalk tissue is well known for its rigidity, fibrous nature, and the presence of oxidative enzymes, phenolic compounds and extreme levels of carbohydrates, thus making the protein extraction complicated. Here, we evaluated five different protein extraction methods in sugarcane stalk tissues. These methods are as follows: direct extraction using lysis buffer (LB), TCA/acetone precipitation followed by solubilization in LB, LB containing thiourea (LBT), and LBT containing tris, and phenol extraction. Both quantitative and qualitative protein analyses were performed for each method. 2-DE analysis of extracted total proteins revealed distinct differences in protein patterns among the methods, which might be due to their physicochemical limitations. Based on the 2-D gel protein profiles, TCA/acetone precipitation-LBT and phenol extraction methods showed good results. The phenol method showed a shift in pI values of proteins on 2-D gel, which was mostly overcome by the use of 2-D cleanup kit after protein extraction. Among all the methods tested, 2-D cleanup-phenol method was found to be the most suitable for producing high number of good-quality spots and reproducibility. In total, 30 and 12 protein spots commonly present in LB, LBT and phenol methods, and LBT method were selected and subjected to eLD -IT-TOF-MS/MS and nESI-LC-MS/MS analyses, respectively, and a reference map has been established for sugarcane stalk tissue proteome. A total of 36 nonredundant proteins were identified. This is a very first basic study on sugarcane stalk proteome analysis and will promote the unexplored areas of sugarcane proteome research. [source] Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugationENVIRONMENTAL MICROBIOLOGY, Issue 7 2001Sophie Courtois All molecular analyses of soil bacterial diversity are based on the extraction of a representative fraction of cellular DNA. Methods of DNA extraction for this purpose are divided into two categories: those in which cells are lysed within the soil (direct extraction) and those in which cells are first removed from soil (cell extraction) and then lysed. The purpose of this study was to compare a method of direct extraction with a method in which cells were first separated from the soil matrix by Nycodenz gradient centrifugation in order to evaluate the effect of these different approaches on the analysis of the spectrum of diversity in a microbial community. We used a method based on polymerase chain reaction (PCR) amplification of a 16S rRNA gene fragment, followed by hybridization of the amplified fragments to a set of specific probes to assess the phylogenetic diversity of our samples. Control parameters, such as the relationship between amount of DNA template and amount of PCR product and the influence of competing DNA on PCR amplification, were first examined. Comparison between extraction methods showed that less DNA was extracted when cells were first separated from the soil matrix (0.4 µg g,1 dry weight soil versus 38,93 µg g,1 obtained by in situ lysis methods). However, with the exception of the ,-subclass of Proteobacteria, there was no significant difference in the spectrum of diversity resulting from the two extraction strategies. [source] Analysis of hepatic vitamins A1, A2, their fatty acyl esters, and vitamin E for biomonitoring mammals feeding on freshwater fishENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2002Anne Käkelä Abstract In tissues of freshwater fish,feeding mammals, 3,4-didehydroretinol (A2) is a major form of vitamin A. In mink liver, with organochlorine exposure, this analog has been found to decrease more than retinol (A1) and thus has potential as a sensitive freshwater biomarker. The presence of the analogs A1 and A2 as alcohol and different fatty acyl esters, which react to polychlorinated biphenyls differently, necessitates detailed analyses achieved by using direct extraction of tissue homogenate. In direct hexane extraction, compared to total levels of the vitamins obtained in the saponification procedure, a large proportion of the vitamins was released only after repeated and long-time vortex mixing with the extraction solvent. Thus, in tissue extraction, the use of internal standardization alone can lead to a rough underestimation of the levels of these fat-soluble vitamins. For analyses of vitamins A1 and A2 in liver, we applied the argentation high-performance liquid chromatography, which provided good separation of individual A1 and A2 fatty acyl esters. We report retention times for numerous esters of A1 and A2 and, to aid identification, the change in their retention properties after adding AgNO3 to the mobile phase. The argentation did not affect the recoveries of any forms of the retinoids studied but destroyed half the vitamin E. Despite selective acylation of fatty acids into the vitamin A esters, the fatty acids of the esters were the same as those found to be the major fatty acids in the gas,liquid chromatography of total lipids. The goal of this work was to create a methodology that is suitable for biomonitoring alcoholic and esterified vitamins A1 and A2 in tissues of freshwater fish,feeding mammals. [source] A combination of baiting and PCR techniques for the detection of Phytophthora quercina and P. citricola in soil samples from oak standsFOREST PATHOLOGY, Issue 2 2001Nechwatal A description is given of the use of a combination of polymerase chain reaction (PCR) and baiting techniques for the specific detection of Phytophthora quercina and Phytophthora citricola from soil around declining oak trees. The soil was flooded with water and subjected to a specific baiting procedure using Quercus robur leaflets as baits. Single round or nested PCR, respectively, with species-specific primers allowed the detection of P. quercina and P. citricola in infected oak leaflets used as baits and in the water from the same bait tests. PCR detection of both fungi was also possible after soil samples had been thoroughly mixed with water and the floating organic debris had been collected. Phytophthora quercina and P. citricola could be readily detected in almost every case in the water from these tests by PCR but less frequently in the organic debris. The identities of P. quercina and P. citricola were confirmed by restriction digests of the corresponding PCR amplicons. The presence of both fungi was also confirmed in parallel in soil samples tested by baiting with oak leaflets. Nested PCR with the primers used allowed the detection of as few as five zoospores of P. citricola and 300 zoospores of P. quercina in a volume of 100 ,l. The methods presented here allow detection and identification of species of Phytophthora in soil without the need for direct extraction of soil samples, and without specific knowledge of the morphological characteristics of the genus. Détection de Phytophthora quercina et de P. citricola dans le sol de chênaies par une méthode combinant le piégeage et la PCR L'article décrit la détection spécifique de Phytophthora quercina et de P. citricola dans le sol prélevé autour de chênes dépérissants, par une méthode combinant les techniques de piégeage et de PCR. Le sol a été immergé dans l'eau et soumis à la procédure du piégeage avec de très jeunes feuilles de Quercus robur. Une amplification par PCR simple ou par PCR gigogne, respectivement, avec des amorces spécifiques des espèces ont permis de détecter P. quercina et P. citricola dans les feuilles-piège infectées, et dans l'eau de piégeage. La détection des deux champignons par PCR a aussi été possible après que les échantillons de sol aient été soigneusement mélangés à de l'eau et que les débris organiques aient été collectés. Phytophthora quercina et P. citricola ont pu être facilement détectés par PCR dans presque tous les cas dans l'eau de piégeage, mais moins fréquemment dans les débris organiques. L'identité de P. quercina et de P. citricola a été confirmée par les profils de restriction des amplifiats obtenus. La présence des deux champignons a aussi été confirmée en parallèle dans des échantillons de sol par piégeage. L'amplification par PCR gigogne avec les amorces utilisées a permis la détection de seulement 5 zoospores de P. citricola et 300 zoospores de P. quercina, dans un volume de 100 ,l. Les méthodes présentées ici permettent la détection et l'identification des espèces de Phytophthora dans le sol en évitant l'extraction directe d'ADN du sol, et sans connaissances spécifiques sur les caractéristiques morphologiques du genre. Eine Kombination von Köder- und PCR-Techniken zum Nachweis von Phytophthora quercina und P. citricola in Bodenproben von Eichenstandorten Es wird der spezifische Nachweis von Phytophthora quercina und P. citricola in Bodenproben von absterbenden Eichen mit Hilfe einer Kombination von PCR- und Baiting-Methoden beschrieben. Die Bodenproben wurden mit Wasser geflutet und Baiting-Tests unterzogen, bei denen junge Blättchen von Quercus robur als Köder zum Einsatz kamen. Einfache oder nested PCR-Reaktionen mit artspezifischen Primern erlaubten den Nachweis von P. quercina und P. citricola in den infizierten Eichenblättchen aus diesen Tests und im jeweiligen ,Baiting-Wasser'. Der PCR-Nachweis beider Erreger war auch möglich, wenn Bodenproben gründlich mit Wasser gemischt wurden, das aufgeschwemmte organische Material abgesammelt und das Wasser abgenommen wurde. P. quercina und P. citricola wurden dabei in nahezu allen Fällen im Wasser, jedoch weniger regelmäßig im organischen Material nachgewiesen. Die Identität der betreffenden Arten wurde zusätzlich durch Restriktions-Analysen der entsprechenden Amplicons bestätigt. Außerdem wurde die Anwesenheit beider Arten in den untersuchten Bodenproben durch klassische Baiting-Methoden nachgewiesen. Nested PCR mit den verwendeten Primerpaaren erlaubte den Nachweis von nur 5 Zoosporen von P. citricola und 300 Zoosporen von P. quercina in einem Gesamt-Volumen von 100 ,l. Die beschriebenen Methoden ermöglichen Nachweis und Identifizierung von Phytophthora-Arten in Bodenproben, ohne die Notwendigkeit einer direkten Extraktion des Bodens und ohne weitreichende Kenntnis der morphologischen Merkmale der Arten dieser Gattung. [source] Chemical synthesis and biosynthesis of the cyclotide family of circular proteinsIUBMB LIFE, Issue 9 2006Sunithi Gunasekera Abstract Cyclotides are a recently discovered class of proteins that have a characteristic head-to-tail cyclized backbone stabilized by a knotted arrangement of three disulfide bonds. They are exceptionally resistant to chemical, enzymatic and thermal treatments because of their unique structural scaffold. Cyclotides have a range of bio-activities, including uterotonic, anti-HIV, anti-bacterial and cytotoxic activity but their insecticidal properties suggest that their natural physiological role is in plant defense. They are genetically encoded as linear precursors and subsequently processed to produce mature cyclic peptides but the mechanism by which this occurs remains unknown. Currently most cyclotides are obtained via direct extraction from plants in the Rubiaceae and Violaceae families. To facilitate the screening of cyclotides for structure-activity studies and to exploit them in drug design or agricultural applications a convenient route for the synthesis of cyclotides is vital. In this review the current chemical, recombinant and biosynthetic routes to the production of cyclotides are discussed. iubmb Life, 58: 515-524, 2006 [source] Analysis of tricyclic antidepressant drugs in plasma by means of solid-phase microextraction-liquid chromatography-mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 10 2007Claudete Alves Abstract Solid-phase microextraction coupled to liquid chromatography and mass spectrometry (SPME-LC-MS) was used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. SPME was performed by direct extraction on a PDMS/DVB (60 µm) coated fiber, employing a stirring rate of 1200 rpm for 30 min, pH 11.0, and temperature of 30 °C. Drug desorption was carried out by exposing the fiber to the liquid chromatography mobile phase for 20 min, using a labmade SPME-LC interface at 50 °C. The main variables experimentally influencing LC-MS response were evaluated and mathematically modeled. A rational optimization with fewer experiments was achieved using a factorial design approach. The constructed empirical models were adjusted with 96,98% of explained deviation allowing an adequate data set comprehension. The chromatographic separation was realized using an RP-18 column (150 mm × 2.1 mm, 5 µm particles) and ammonium acetate buffer (0.01 mol/l, pH 5.50) : acetonitrile (50 : 50 v/v) as mobile phase. Low detection levels were achieved with electrospray interface (0.1 ng/ml). The developed method showed specificity, linearity, precision, and limit of quantification adequate to assay tricyclic antidepressant drugs in plasma. Copyright © 2007 John Wiley & Sons, Ltd. [source] A novel solid phase for selective separation of flavonoid compoundsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 9 2007Yong-qing Xia Abstract A novel straightforward approach to selective separation for flavonoid compounds was reported. The solid phase material was prepared by copolymerization using allyl-bromide-modified chitosan as macromonomer, and ethylene glycol dimethacrylate as cross-linker. The material was evaluated by chromatographic analysis; it exhibited high selectivity separation for quercetin and its structural analogues using different mobile phases. The material could directly trap a specific class of compounds including quercetin and kaempferol from the hydrolyzate of Ginkgo biloba extract. These results demonstrated the possibility of direct extraction of certain constituents from herb using this material. [source] Estrogenic activity of Nigella damascena extracts, evaluated using a recombinant yeast screenPHYTOTHERAPY RESEARCH, Issue 5 2002E. Agradi Abstract We used the yeast estrogen screen (YES) containing a human estrogen receptor to evaluate the estrogenic activity of extracts obtained from Nigella damascena seeds. Alcohol extracts obtained by direct extraction of seeds showed a low estrogenic activity, while the alcohol extract obtained after extraction with solvents of increasing polarity showed a strong estrogenic activity. This suggests the presence in Nigella of polar components whose activity can be clearly demonstrated after previous elimination of interacting apolar components that may mask the activity of more polar components. The response of both alcohol fractions follow a bell-shaped curve indicating a concentration-dependent relationship. Copyright © 2002 John Wiley & Sons, Ltd. [source] Extraction of functional motion in trypsin crystal structuresACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2005Andrea Schmidt The analysis of anisotropic atomic displacement parameters for the direct extraction of functionally relevant motion from X-ray crystal structures of Fusarium oxysporum trypsin is presented. Several atomic resolution structures complexed with inhibitors or substrates and determined at different pH values and temperatures were investigated. The analysis revealed a breathing-like molecular motion conserved across trypsin structures from two organisms and three different crystal forms. Directional motion was observed suggesting a change of the width of the substrate-binding cleft and a change in the length of the specificity pocket. The differences in direction of motion across the structures are dependent on the mode of substrate or inhibitor binding and the chemical environment around the active-site residues. Together with the occurrence of multiple-residue conformers, they reflect spatial rearrangement throughout the deacylation pathway. [source] Extraction of Proteins from Biological Fluids by Use of an Ionic Liquid/Aqueous Two-Phase SystemCHEMISTRY - A EUROPEAN JOURNAL, Issue 7 2007Zhuo Du Abstract An ionic liquid/aqueous two-phase system based on the hydrophilic ionic liquid 1-butyl-3-methylimidazolium chloride (BmimCl) and K2HPO4 has been employed for direct extraction of proteins from human body fluids for the first time. Proteins present at low levels were quantitatively extracted into the BmimCl-rich upper phase with a distribution ratio of about 10 between the upper and lower phase and an enrichment factor of 5. Addition of an appropriate amount of K2HPO4 to the separated upper phase results in a further phase separation, giving rise to an improved enrichment factor of 20. FTIR and UV spectroscopy demonstrated that no chemical (bonding) interactions between the ionic liquid and the protein functional groups were identifiable, while no alterations of the natural properties of the proteins were observed. The partitioning of proteins in the two-phase system was assumed to have been facilitated by the electrostatic potential difference between the coexisting phases, as well as by salting out effects. The system could be applied successfully for the quantification of proteins in human urine after on-line phase separation in a flow system. The use of an ionic liquid, as a green solvent, offers clear advantages over traditional liquid,liquid extractions, in which the use of toxic organic solvents is unavoidable. [source] | |||||||||||||