Home About us Contact
|
Home About us Contact | ||
|
|
||
Purification System (purification + system)
Selected AbstractsDNA Extraction from Olive Oil and PCR Amplification of Microsatellite MarkersJOURNAL OF FOOD SCIENCE, Issue 1 2005Raffaele Testolin And ABSTRACT: DNA was extracted from single-cultivar of cold-pressed (virgin) unfiltered and cotton-filtered olive oils that were stored at 4 °C for up to a year using different DNA extraction kits and protocols. DNA was amplified using original and nested primers designed on 6 microsatellites loci of the UDO series. The most consistent results in terms of successful single sequence repeat amplifications were achieved using the Qiagen QIAamp DNA stool extraction kit, slightly modified and applied to oil sample amounts as small as 200 ,L without any pretreatment. The kit allowed getting polymerase chain reaction (PCR) amplicons visible on gel and scorable peaks at the automatic sequencer for all 6 markers analyzed. Less consistent results were achieved with other kits, such as the Promega Wizard Magnetic DNA Purification System for Food, the LB Link-Biotech ExtMan 50,100 Evolution, the Qiagen Plant Mini kit, and the standard cetyltrimethyl-ammonium bromide-based DNA extraction protocol. The integration in the protocols of further tools, such as the hexane-based phase separation, the addition of water or NaCl solutions to the oil, the precipitation and the use of the pellet, and others, did not result in any substantial use. PCR amplifications that gave low DNA yields were improved by adopting the nested PCR technique, which uses the product of the 1st PCR as a template for a 2nd PCR carried out by means of internal primers. Conclusions are drawn as to the applicability of the method to trace the identity of single-cultivar virgin olive oils. Further work is required to check the sensitivity of the method in determining the varietal composition of blended oils, especially in detecting alleles from cultivars present in only small amounts. [source] Cloning and expression of the gene for an insect haemocyte anti-aggregation protein (VPr3), from the venom of the endoparasitic wasp, Pimpla hypochondriaca,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2009M. Paulina Dani Abstract A venom protein from the endoparasitic wasp, Pimpla hypochondriaca, was recently biochemically isolated. This protein possessed haemocyte anti-aggregation activity in vitro and shares the same N-terminal amino acid sequence as that deduced from a gene termed vpr3. The vpr3 gene was identified by sequence analysis of randomly isolated cDNAs from a P. hypochondriaca venom gland library. Presently, the gene for the full-length sequence of mature VPr3 protein was amplified from the P. hypochondriaca venom gland cDNA library by PCR. The amplicon was directionally cloned into a pET expression vector so that recombinant VPr3 (rVPr3) would have an N-terminal polyhistidine (His) tag. High levels of target protein expression were obtained following addition of IPTG (1,mM) and growth of the bacteria at 37°C for 5,h, or at 24°C for 20,h. Following lysis of bacteria grown at 37°C, the target protein partitioned into the insoluble fraction. However, at 24°C, a small amount of soluble protein was consistently detected. The amount of soluble rVPr3 was subsequently increased when the transformed bacteria were grown in Overnight Express Instant TB medium at 24°C. Soluble rVPr3 was purified utilizing the MagneHis Protein Purification System. Recombinant VPr3 was determined to have adverse effects on the cytoskeleton of Lacanobia oleracea haemocytes and to inhibit the ability of these cells to form aggregates in vitro. © 2009 Wiley Periodicals, Inc. [source] Hyperhomocysteinemia in epileptic patients on new antiepileptic drugsEPILEPSIA, Issue 2 2010Vincenzo Belcastro Summary Purpose:, Older enzyme-inducing antiepileptic drugs (AEDs) may induce supraphysiologic plasma concentrations of total (t) homocysteine (Hcy). The aim of the present study was to investigate the effect of new AEDs on plasma tHcy levels. Methods:, Patients 18,50 years of age, on AEDs monotherapy, with no other known cause of hyper-tHcy were enrolled. Plasma tHcy, folate, vitamin B12, and AEDs levels were determined by standard high-performance liquid chromatography (HPLC) methods. Methylenetetrahydrofolate-reductase (MTHFR) polymorphisms were checked using Puregene genomic DNA purification system (Gentra, Celbio, Italy). A group of healthy volunteers matched for age and sex was taken as control. Results:, Two hundred fifty-nine patients (151 on newer and 108 on older AEDs) and 231 controls were enrolled. Plasma tHcy levels were significantly higher [mean values, standard error (SE) 16.8, 0.4 vs. 9.1, 0.2 ,m; physiologic range 5,13 ,m] and folate lower (6.3, 0.1 vs. 9.3, 0.1 nm; normal > 6.8 nm) in patients compared to controls. Patients treated with oxcarbazepine, topiramate, carbamazepine, and phenobarbital exhibited mean plasma tHcy levels above the physiologic range [mean values (SE) 16 (0.8), 19.1 (0.8), 20.5 (1.0), and 18.5 (1.5) ,m, respectively]. Conversely, normal tHcy concentrations were observed in the lamotrigine and levetiracetam groups [both 11.1 (0.5) ,m]. Discussion:, Oxcarbazepine and topiramate might cause hyper-tHcy, most likely because of the capacity of these agents to induce the hepatic enzymes. Because literature data suggest that hyper-tHcy may contribute to the development of cerebrovascular diseases and brain atrophy, a supplement of folate can be considered in these patients to normalize plasma tHcy. [source] Efficiency of a bagasse substrate in a biological bed system for the degradation of glyphosate, malathion and lambda-cyhalothrin under tropical climate conditionsPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 12 2008Laure de Roffignac Abstract BACKGROUND: After the rinsing of spray equipment, the rinsing water contains polluting products. One way to avoid pollution is to bring the rinsing water over a purification system, a biological bed. The system consists of an impermeable tub filled with a biomix substrate that facilitates biodegradation of pesticides. Usually, straw is one component of the biomix. The objective of this study was to assess the efficiency of an unusual substrate, bagasse, a residue of sugar cane, for the degradation of three pesticides, glyphosate, malathion and lambda-cyhalothrin. RESULTS: Results showed that more than 99% of malathion and glyphosate were degraded in 6 months. In the biological bed, the DT50 value for malathion was 17 days, for glyphosate 33 days and for lambda-cyhalothrin 43 days. The degradation rate of aminomethylphosphonic acid (AMPA) residues from the degradation of glyphosate was slower than that of the other pesticides (DT50 69 days). Finally, the innocuousness of the biomix after 6 months of degradation was confirmed by biological tests. CONCLUSIONS: Although the degradation rates of the three pesticides in the present bagasse-based system were similar to those under temperate conditions, the degradation conditions were improved by comparison with those in soil under the given tropical conditions. Further benefits of this system are pesticide confinement, to avoid their dispersion in the environment by liquids or solids, and a lower overall cost. Finally, possibilities for optimising the bagasse-based system (e.g. management of the water content and nature of the biomix) are discussed. Copyright © 2008 Society of Chemical Industry [source] Analytical method for the quantitative determination of urinary ethylenethiourea by liquid chromatography/electrospray ionization tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 20 2003Cristina Sottani A direct, rapid and selective method for the quantitative determination of the ethylenethiourea (ETU) in human urine has been validated and is reported in the present study. It allows the accurate quantification of ETU in this complex matrix without the use of any internal standard as the sample cleanup is effective enough for the removal of interferences that could lead to ion suppression in the electrospray ionization (ESI) source. This simple and rapid purification system, based on the use of a Fluorosil phase of a BondElut® column followed by a liquid-liquid extraction procedure, achieves mean extracted recoveries, assessed at three different concentrations (2.5, 10.0, and 25.0,,g/L), always more than 85%. High-performance liquid chromatography (HPLC) with positive ion tandem mass spectrometry, operating in selected multiple reaction monitoring (MRM) mode, is used to quantify ETU in human urine. The assay is linear over the range 0,50,,g/L, with a lower limit of quantification (LOQ) of 1.5,,g/L and a coefficient of variation (CV) of 8.9%. The lower limit of detection (LOD) is assessed at 0.5,,g/L. The overall precision and accuracy were determined on three different days. The values for within- and between-day precision are ,,8.3 and 10.1%, respectively, and the accuracy is in the range 97,118%. The relative uncertainties for the LOQ and QC concentrations have been estimated to be 18 and 8%, respectively. The assay was applied to quantify ETU in human urine from growers that regularly handle ethylenebisdithiocarbamate pesticides in large crop plantations. The biological samples were collected at the start and end of the working day, and the ETU urine levels were found to vary between 1.9 and 8.2,,g/L. Copyright © 2003 John Wiley & Sons, Ltd. [source] Study of Protein Splicing and Intein-Mediated Peptide Bond Cleavage under High-Cell-Density ConditionsBIOTECHNOLOGY PROGRESS, Issue 3 2003Shamik Sharma Protein splicing elements (inteins), capable of catalyzing controllable peptide bond cleavage reactions, have been used to separate recombinant proteins from affinity tags during affinity purification. Since the inteins eliminate the use of a protease in the recovery process, the intein-mediated purification system has the potential to significantly reduce recovery costs for the industrial production of recombinant proteins. Thus far, the intein system has only been examined and utilized for expression and purification of recombinant proteins at the laboratory scale for cells cultivated at low cell densities. In this study, protein splicing and in vitro cleavage of intein fusion proteins expressed in high-cell-density fed-batch fermentations of recombinant Escherichia coli were examined. Three model intein fusion constructs were used to examine the stability and splicing/cleavage activities of the fusion proteins produced under high-cell-density conditions. The data indicated that the intein fusion protein containing the wild-type intein catalyzed efficient in vivo protein splicing during high-cell-density cultivation. Also, the intein fusion proteins containing modified inteins catalyzed efficient thiol-induced in vitro cleavage reactions. The results of this study demonstrated the potential feasibility of using the intein-mediated protein purification system for industrial-scale production of recombinant proteins. [source] Towards the high-throughput expression of metalloproteins from the Mycobacterium tuberculosis genomeJOURNAL OF SYNCHROTRON RADIATION, Issue 1 2005John F. Hall The provision of high-quality protein in adequate quantities is a prerequisite for any structural genomics programme. A number of proteins from the Mycobacterium tuberculosis genome have been expressed and the success at each stage of the process assessed. Major difficulties have been encountered in the purification and solubilization of many of these proteins, most likely as a result of mis-folding. Some improvements have been made to the protocol but the overall success rate is still limited; however, the use of a cell-free protein expression system will circumvent some of the difficulties encountered. Alternative purification systems are also required and the properties of a mutant blue copper protein are described, that may offer a combined purification and tagging system. [source] | ||||||||||