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Reliable Protocol (reliable + protocol)
Selected AbstractsRapid and reliable DNA extraction and PCR fingerprinting methods to discriminate multiple biotypes of the nematophagous fungus Pochonia chlamydosporia isolated from plant rhizospheresLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2009R.H. Manzanilla-López Abstract Aims:, To develop a simple, rapid, reliable protocol producing consistent polymerase chain reaction (PCR) fingerprints of Pochonia chlamydosporia var. chlamydosporia biotypes for analysing different fungal isolates during co-infection of plants and nematodes. Methods and Results:, DNA extracted from different P. chlamydosporia biotypes was fingerprinted using enterobacterial repetitive intragenic consensus (ERIC)-PCR. Four extraction methods (rapid alkaline lysis; microLYSIS® -PLUS; DNeasy®; FTA® cards) gave consistent results within each protocol but these varied between protocols. Reproducible fingerprints were obtained only if DNA was extracted from fresh fungal cultures that were free of agar. Some DNA degradation occurred during storage, except with the FTA® cards, used with this fungus for the first time, which provide a method for long-term archiving. Rapid alkaline lysis and ERIC-PCR identified fungal isolates from root and nematode egg surfaces when plants were treated with different combinations of fungal biotypes; the dominant biotype isolated from the rhizosphere was not always the most abundant in eggs. Conclusions:, ERIC-PCR fingerprinting can reliably detect and identify different P. chlamydosporia biotypes. It is important to use fresh mycelium and the same DNA isolation method throughout each study. Significance and Impact of the Study:, This evaluation of methods to assess genetic diversity and identify specific P. chlamydosporia biotypes is relevant to other mycelial fungi. [source] Analysis of pharmaceutical samples of Resina Draconis by HPLC-PADPHYTOCHEMICAL ANALYSIS, Issue 6 2008Wenjun Gong Abstract Introduction: The quality evaluation of traditional Chinese medicine (TCM) represents a particular challenge owing to the complexity of the matrix, which renders separation and identification of the individual components extremely difficult. In recent years, fingerprinting of TCMs has played a dominant role in quality control. Resina Draconis was authorised as a new TCM in 1991, but a satisfactory HPLC fingerprint method for this preparation has not yet been published. Objective: To develop a simple and reliable protocol for the quality control of Resina Draconis using an HPLC-PAD method. Methodology: The TCM was extracted with methanol at room temperature. Chromatography was carried out using a Lichrospher C18 column eluted with a linear gradient of acetonitrile (A) and water containing 0.1% phosphoric acid (B), initially at 30:70 (A:B) and changing to 60:40 in 90 min. UV (PAD) spectra were acquired in the range 210,400 nm. Results: Four chromatograms of samples of Resina Draconis obtained from different pharmaceutical factories showed 20 peaks in common. The average chromatogram was taken as a template from which the correlation coefficients and cosine ratios of the samples were determined. Whereas the contents of individual components in each sample were different, overall the samples were extremely similar one to another, and the products from different pharmaceutical factories were consistent. Conclusion: A reliable and validated HPLC method has been developed for the fingerprint analysis of Resina Draconis that can be applied for the quality control of this TCM. Copyright © 2008 John Wiley & Sons, Ltd. [source] MAXIMAL LACTATE STEADY STATE IN RUNNING MICE: EFFECT OF EXERCISE TRAININGCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 8 2007Julio CB Ferreira SUMMARY 1Maximal lactate steady state (MLSS) corresponds to the highest blood lactate concentration (MLSSc) and workload (MLSSw) that can be maintained over time without continual blood lactate accumulation and is considered an important marker of endurance exercise capacity. The present study was undertaken to determine MLSSw and MLSSc in running mice. In addition, we provide an exercise training protocol for mice based on MLSSw. 2Maximal lactate steady state was determined by blood sampling during multiple sessions of constant-load exercise varying from 9 to 21 m/min in adult male C57BL/6J mice. The constant-load test lasted at least 21 min. The blood lactate concentration was analysed at rest and then at 7 min intervals during exercise. 3The MLSSw was found to be 15.1 ± 0.7 m/min and corresponded to 60 ± 2% of maximal speed achieved during the incremental exercise testing. Intra- and interobserver variability of MLSSc showed reproducible findings. Exercise training was performed at MLSSw over a period of 8 weeks for 1 h/day and 5 days/week. Exercise training led to resting bradycardia (21%) and increased running performance (28%). Of interest, the MLSSw of trained mice was significantly higher than that in sedentary littermates (19.0 ± 0.5 vs 14.2 ± 0.5 m/min; P = 0.05), whereas MLSSc remained unchanged (3.0 mmol/L). 4Altogether, we provide a valid and reliable protocol to improve endurance exercise capacity in mice performed at highest workload with predominant aerobic metabolism based on MLSS assessment. [source] In-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2002S. Kilz Abstract Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide brigded, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF,pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O -glycosylated proteins up to 150 kDa after SDS-PAGE separation. Copyright © 2002 John Wiley & Sons, Ltd. [source] The effect of protein,precipitant interfaces and applied shear on the nucleation and growth of lysozyme crystalsACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2009Nuno M. Reis This paper is concerned with the effect of protein,precipitant interfaces and externally applied shear on the nucleation and growth kinetics of hen egg-white lysozyme crystals. The early stages of microbatch crystallization of lysozyme were explored using both optical and confocal fluorescence microscopy imaging. Initially, an antisolvent (precipitant) was added to a protein drop and the optical development of the protein,precipitant interface was followed with time. In the presence of the water-soluble polymer poly(ethylene glycol) (PEG) a sharp interface was observed to form immediately within the drop, giving an initial clear separation between the lighter protein solution and the heavier precipitant. This interface subsequently became unstable and quickly developed within a few seconds into several unstable `fingers' that represented regions of high concentration-gradient interfaces. Confocal microscopy demonstrated that the subsequent nucleation of protein crystals occurred preferentially in the region of these interfaces. Additional experiments using an optical shearing system demonstrated that oscillatory shear significantly decreased nucleation rates whilst extending the growth period of the lysozyme crystals. The experimental observations relating to both nucleation and growth have relevance in developing efficient and reliable protocols for general crystallization procedures and the controlled crystallization of single large high-quality protein crystals for use in X-ray crystallography. [source] | ||||||||||