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Selected AbstractsMass spectrometric characterization of the covalent modification of the nitrogenase Fe-protein in Azoarcus sp.FEBS JOURNAL, Issue 13 2009Nitrogenase Fe-protein modification was analyzed in the endophytic ,-proteobacterium Azoarcus sp. BH72. Application of modern MS techniques localized the modification in the peptide sequence and revealed it to be an ADP-ribosylation on Arg102 of one subunit of nitrogenase Fe-protein. A double digest with trypsin and endoproteinase Asp-N was necessary to obtain an analyzable peptide because the modification blocked the trypsin cleavage site at this residue. Furthermore, a peptide extraction protocol without trifluoroacetic acid was crucial to acquire the modified peptide, indicating an acid lability of the ADP-ribosylation. This finding was supported by the presence of a truncated version of the original peptide with Arg102 exchanged by ornithine. Site-directed mutagenesis verified that the ADP-ribosylation occurred on Arg102. With our approach, we were able to localize a labile modification within a large peptide of 31 amino acid residues. The present study provides a method suitable for the identification of so far unknown protein modifications on nitrogenases or other proteins. It represents a new tool for the MS analysis of protein mono-ADP-ribosylations. [source] Methods for inoculum production and inoculation of Cistella japonica, the causal agent of resinous stem canker in Chamaecyparis obtusaFOREST PATHOLOGY, Issue 1 2008T. Yamanobe Summary The ascomycete Cistella japonica was cultured on potato dextrose agar medium (PDA) for inoculation into Chamaecyparis obtusa, enabling the development of an inoculation method suitable for use in a breeding programme aimed at selecting for families of Ch. obtusa resistant to resinous stem canker. Using PDA to generate the inoculum resolved the problems encountered with the previously used mixed medium of rice bran and wheat bran, including unfavourable characteristics, uncertain growth of Ci. japonica mycelia, and a complex culturing operation. The inoculation test induced resin exudation similar to that observed in natural infections, and reproduced clonal differences with regard to damage severity. [source] An SPH shell formulation for plasticity and fracture analysis in explicit dynamicsINTERNATIONAL JOURNAL FOR NUMERICAL METHODS IN ENGINEERING, Issue 7 2008B. Maurel Abstract This paper introduces a new modeling method suitable for the simulation of shell fracture under impact. This method relies on an entirely meshless approach based on the smoothed particle hydrodynamics (SPH) method. The paper also presents the SPH shell formulation being used as well as the different test cases used for its validation. A plasticity model of the global type throughout the thickness is also proposed and validated. Finally, in order to illustrate the capabilities of the method, fracture simulations using a simplified fracture criterion are presented. Copyright © 2008 John Wiley & Sons, Ltd. [source] Predicting the effects of perturbations on ecological communities: what can qualitative models offer?JOURNAL OF ANIMAL ECOLOGY, Issue 5 2005DAVE RAMSEY Summary 1Quantitative predictions of the effects of perturbations on communities of interacting species have often proved to be difficult. However, if precise predictions are not a requirement then qualitative models of community dynamics offer an alternative method for predicting species responses to perturbations. 2We used two qualitative modelling approaches to predict the effects of predator control on the fledging rate of an endangered New Zealand bird, the North Island kokako. The first approach was based on loop analysis and provided predictions on the probable direction of change in species abundances to single species perturbations. The second approach, ,fuzzy interaction webs', used fuzzy logic in the framework of a fuzzy cognitive map to provide predictions on the probable magnitude of change in species abundances to perturbations. 3Using both methods, we predicted the qualitative change in the equilibrium fledging rates of kokako under various regimes of single- and multispecies predator control (ship rats, brushtail possums and stoats). Single species control was insufficient to lift the fledging rate from ,low' to ,moderate'. However, simultaneous control of both ship rats and possums had the greatest influence on the fledging rates compared with any other combination as a result of the additional indirect effect of ship rat control on stoat abundance. 4We propose qualitative modelling of community dynamics as a method suitable for predicting the effects of perturbations in complex ecological communities that can encapsulate diverse sources of knowledge about food web interactions. We believe that these methods are a useful set of heuristic tools that can be used to propose testable hypotheses about ecosystem functioning that can complement existing statistical and quantitative modelling approaches. [source] Combustion of chlorinated hydrocarbons in catalyst-coated sintered metal fleece reactors,JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 2-3 2003K Everaert Abstract Incinerators emit chlorinated hydrocarbons, such as polychlorinated benzenes (PCBz) and phenols (PCPh), polychlorinated biphenyls (PCB) and polychlorinated dibenzodioxins and furans (PCDD/F), as very dilute streams. High temperatures (>1000,°C) are required in traditional oxidizers. From an energy-saving perspective and to avoid de novo synthesis of PCDD/F, exhaust gas clean-up must be performed at low temperatures (250,350,°C). Catalytic combustion can be applied in this temperature range and different reactor layouts are used (eg monoliths, honeycomb). The present investigation uses a novel catalyst-coated sintered metal fleece. Thin metal fibers are sintered (non-woven) to fleece of various thickness, structure and porosity. V,Ti,W catalysts are examined. The paper will briefly review the catalyst coating method suitable to provide a structured fleece reactor with adequate characteristics. Experiments were carried out in the temperature range of 260,340,°C with various hydrocarbons injected in a carrier air stream. The experimental investigations demonstrated: (i) that the conversion of the hydrocarbons (volatile organic compounds, VOC) is independent of the oxygen concentration, corresponding to a zero-order dependence of the reaction rate; (ii) that the conversion of the hydrocarbons is a first-order reaction in the VOC; (iii) that the oxidation of the VOC proceeds to a greater extent with increasing temperature, with chlorine substitution enhancing the reactivity, and (iv) that the reaction rate constant follows an Arrhenius-dependence with activation energies between 37.3 and 58.4,kJ,mol,1. An assessment of the results leads to a model expression with kinetic reaction control. This model can be used in a scale-up strategy. © 2003 Society of Chemical Industry [source] In-vitro release of bupivacaine from injectable lipid formulations investigated by a single drop technique , relation to duration of action in-vivoJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 6 2002Lars Söderberg The aim of this study was to develop an in-vitro release method suitable for injectable slow-release lipid formulations of local anaesthetics (or other drugs). We also aimed that the results of the in-vitro measurements should have a clear relationship to duration of action in-vivo. Six formulations of bupivacaine base in medium-chain triglyceride-glyceryl dilaurate mixtures were developed. A new apparatus was constructed for determination of their in-vitro release profiles. A bulbous glass tube was fixed inside a standard glass bottle, which was then filled with release medium. A stirring magnet was enclosed in the perforated polypropylene cylinder holding the glass tube. The stirring created a continuous, rotating downward flow of medium inside the tube, which kept the lipid phase, introduced by means of a syringe, suspended as a single, free drop. Release profiles were obtained by sampling of the release medium for up to 72 h and analysis by gas-liquid chromatography. The duration of action in-vivo of the respective formulations was tested by the hot-plate method in rats. The release profiles of bupivacaine in-vitro were mono-exponential for four formulations and bi-exponential for the other two. There was a positive correlation between the proportion of glyceryl dilaurate in the formulation and the slow half-life of release of bupivacaine. All formulations showed prolonged duration of action in-vivo, median values within the range 4.5,12 h, as compared with a 2-h effect of bupivacaine hydrochloride solution. A comparison of in-vitro release curves and durations of action in-vivo suggested that to maintain nerve blockade in-vivo the formulations must release bupivacaine at a rate of approximately 350 ,g h,1 under the in-vitro conditions. To conclude, we designed and tested a novel apparatus for measuring release of a local anaesthetic (or other drug) from a fluid or semi-solid formulation in-vitro. Release rates obtained in-vitro by means of this technique may be used to guide the development of formulations with suitable durations of action in-vivo. The apparatus is, however, as yet a prototype. Rigorous evaluation of performance should be carried out on devices built to specific standards according to their intended application. [source] Raman spectroscopic analysis of breast cancer tissues: identifying differences between normal, invasive ductal carcinoma and ductal carcinoma in situ of the breast tissueJOURNAL OF RAMAN SPECTROSCOPY, Issue 10 2007Shazza Rehman Abstract A relatively non-destructive method employing Raman spectroscopy for the analysis of histopathological specimens is described. Raman spectroscopy has allowed qualitative analysis of the same specimen used for histopathological evaluation. Breast cancer tissues have been analysed to demonstrate the feasibility of the chemical changes taking place in the biological tissue, which can be identified precisely, and the results are reproducible. Raman analysis of tissue sections provides distinct spectra that can be used to distinguish between the nuclear grades of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of the breast. Sixty cases of breast carcinoma including DCIS and IDC and seven cases of normal breast tissues were studied employing the Raman spectroscopic technique. This study reports for the first time spectral differences between DCIS grades. It is concluded that Raman spectroscopy can objectively distinguish between DCIS and IDC grades and is non-destructive and reproducible. It should become possible in future to use Raman spectroscopy as an informative and quantitative method suitable for classification of grades and diagnosis of breast carcinoma. Copyright © 2007 John Wiley & Sons, Ltd. [source] Determination of banned Sudan dyes in food samples by molecularly imprinted solid phase extraction-high performance liquid chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 19 2009Claudio Baggiani Abstract A method for molecularly imprinted SPE of banned Sudan azo-dyes from food samples was investigated. The molecularly imprinted polymer was obtained by suspension polymerization using 1-(4-chlorophenyl)azonaphthalen-2-ol as the mimic template. The molecular recognition properties of imprinted beads were evaluated for use as a SPE sorbent, in order to develop a selective extraction protocol for the Sudan class of dyes. The optimized extraction protocol resulted in a reliable molecularly imprinted SPE (MISPE) method suitable for HPLC analysis. It was selective for the main analyte, Sudan I, and the related azo-dyes Sudan II, III, IV, Sudan Red B, and Sudan Red 7B, while the permitted azo-dyes Allura Red AC, Neococcin, and Sunset Yellow FCF were not extracted. The method was tested for Sudan I, II, III, and IV in five different food samples (hot chilli pepper, hot chilli tomato sauce, sausage, tomato sauce, and hard boiled egg yolk) at three concentration levels (15, 100, and 300 ,g/g). It demonstrated itself to be insensitive to the presence of different complex matrices, precise, accurate, and with good recovery rates (85,101%). The LOD and LOQ were satisfactory for most analytical determinations. [source] A simple DNA extraction method suitable for PCR detection of genetically modified maizeJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 14 2007Manuel Porcar Abstract BACKGROUND: The polymerase chain reaction (PCR) is a powerful tool that is being increasingly used for detection of transgenic DNA. PCR requires only a minute quantity of template, but sensitive and accurate testing requires DNA of sufficient purity and free from inhibitors such as plant polysaccharides. Several standard protocols are available for this purpose, but they usually involve several steps, imply destruction of the maize kernel, or are time-consuming. Our aim was to develop a fast and simple extraction method to isolate a raw DNA-containing solution from maize tissues suitable for use as a template in a PCR-based detection assay with specific oligonucleotides directed to the identification of event MON810. RESULTS: The NaOH-based DNA extraction method we report here is time-saving (5 min) and can be used to isolate DNA-containing solutions from a small maize leaf portion (down to 1 mg) or from a single overnight-germinated kernel. PCR performed with selected primers yielded reproducible detection of transgenic DNA. CONCLUSION: The main advantages of the procedure are the quick extraction step, the possibility of non-destructive testing of maize kernels, and the robustness of the PCR-based detection, a consequence of the selection of MON810-matching oligonucleotides yielding intense and highly specific amplicons. Copyright © 2007 Society of Chemical Industry [source] Quantification of lycopene in tomato products: comparing the performances of a newly proposed direct photothermal method and high-performance liquid chromatographyJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 7 2005Dane Bicanic Abstract A new photothermal method suitable for direct, accurate and highly reproducible quantitative measurements of lycopene in tomato products has been introduced. The intrinsic precision of the method is typically better than 0.2%; the repeatability of determination is comparable to that of high-performance liquid chromatography, with 0.86% least overall error. Copyright © 2005 Society of Chemical Industry [source] Evaluation of Insulin Sensitivity in Clinical Practice and in Research SettingsNUTRITION REVIEWS, Issue 12 2003Lais U. Monzillo MD Insulin resistance is the core metabolic abnormality in type 2 diabetes. Its high prevalence and its association with dyslipidemia, hypertension, hyperinsulinemia, and high coronary and cerebrovascular mortality put it in the forefront as the plausible target for aggressive intervention. Measurements of insulin sensitivity provide clinicians and clinical researchers with invaluable instruments to objectively evaluate the efficiency of both current and potentially useful interventional tools. Although several methods had been developed and validated to evaluate insulin sensitivity, none of these methods can be universally used in all patients. Nonetheless, a method suitable for use in clinical or basic research may not necessarily be a practical method for use in clinical practice or for epidemiologic research. We reviewed the currently used methods for assessment of insulin sensitivity. For each method, we summarized its procedure, normal value, cut-off value for defining insulin resistance, advantages and limitations, validity, accuracy for each patient population, and suitability for use in clinical practice and in research settings. The methods reviewed include fasting plasma insulin, homeostatic model assessment, quantitative insulin sensitivity check index, glucose-to-insulin ratio, continuous infusion of glucose with model assessment, indices based on oral glucose tolerance test, insulin tolerance test, and the so called "gold standard" methods, the hyperinsulinemic euglycemic clamp and the frequently sampled-intravenous glucose tolerance test. [source] Electron Backscatter Diffraction Study of Brachiopod Shell Calcite , Microscale Phase and Texture Analysis of a Polycrystalline BiomaterialPARTICLE & PARTICLE SYSTEMS CHARACTERIZATION, Issue 5-6 2008Wolfgang W. Schmahl Abstract Electron backscatter diffraction (EBSD) is an easy to use and highly automated microdiffraction method suitable for the determination of crystallographic phase and crystallite orientation. The high level of hierarchical structural organization in the shells of marine organisms was studied. Calcite brachiopod shell materials were found to belong to three types of microstructure: nano- to microcrystalline layers of acicular crystals, fiber composites with calcite single crystal fibers with [uv0] morphological axes, and material formed by columnar crystals with [001] morphological axes selected by competitive growth. [source] HPLC-PAD-APCI/MS assay of phenylpropanoids in cerealsPHYTOCHEMICAL ANALYSIS, Issue 1 2004Antoine C. Bily Abstract A new, rapid HPLC-PAD-APCI/MS assay has been developed in order to measure accurately the amount of p -coumaric, E - and Z -ferulic acid and the dehydrodimers of ferulic acid in cereal grain. In the positive ionisation mode, MS patterns gave additional information for the identi,cation of the dimers. The time required and the quantities of solvents employed in the developed analytical method are much lower than those involved in previously available assays of these compounds, thus making the method suitable for the screening of cereal genotypes. Application of the method to accessions of maize, wheat and sorghum showed that E -ferulic was the most abundant phenylpropanoid, whilst the major dimer was 8- O -4, dehydrodimer of ferulic acid followed by the 5,5, and then the 8,5, forms. Maize grains, especially of the Mexican landraces, contained the highest levels of these dimers. Copyright © 2004 John Wiley & Sons, Ltd. [source] Calf production from vitrified bovine sexed embryos following in-straw dilutionANIMAL SCIENCE JOURNAL, Issue 4 2010Kiyoshi AKIYAMA ABSTRACT The objective of this study was to develop an in-straw dilution method suitable for direct transfer of vitrified bovine sexed embryos. Embryo sexing was performed by molecular diagnosis. Several sexed and vitrified-warmed embryos were transferred after evaluation of morphologically embryonic survival at warming and in-straw dilution (Evaluation group). The other embryos were immediately directly transferred to recipients without first being expelled from the straws after in-straw dilution (Non-evaluation group). The pregnancy rates of vitrified sexed embryos were 38.7% and 34.8% in the Evaluation group and Non-evaluation group, respectively, which were not significantly different. The viability of lower quality embryos before vitrification tended to be lower (P = 0.087) than that of the higher quality embryos regardless of evaluating embryos after warming and in-straw dilution. The abortion rates were similar, and there was no difference between the two groups (13.9% and 12.5%, respectively). These results demonstrate that vitrified bovine sexed embryos can be vitrified and diluted by the in-straw method and that the vitrified and warmed sexed embryos can develop to term. [source] PCR as a specific, sensitive and simple method suitable for diagnosticsBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 4 2000M. Gonzalo Claros PCR technology is a widespread method that has not reached students laboratory in anything else than a typical amplification reaction. We describe a simple application of PCR in pathogen diagnostics that enables students to identify which ampicillin-resistant organism is present in a cell culture. This experiment has been performed for one year in two "Experimental Biochemistry and Molecular Biology" courses with Biological and Chemical undergraduates. Using specific primers from the Escherichia coli ,-lactamase gene, they have been able to selectively amplify a ,-lactamase DNA fragment in E. coli but not in Staphylococcus aureus and, using different annealing temperatures, test the reaction specificity. By solving the "Study Questions", students understood the specificity and sensitivity of the method, as well as the rationale that should be applied when a molecular weight pattern is used for calculating unknown DNA sizes. © 2000 IUBMB. Published by Elsevier Science Ltd. All rights reserved. [source] Quality assessment for tramadol in pharmaceutical preparations with thin layer chromatography and densitometryBIOMEDICAL CHROMATOGRAPHY, Issue 8 2004Jan Krzek Abstract Research studies have been carried out to develop a chromatographic and densitometric method suitable for identi,cation and determination of tramadol and impurities. In addition, the stability of tramadol in solutions was investigated, including an effect of solution pH, temperature and incubation time. In the ,rst instance the conditions for identi,cation and quantitative determination of tramadol and impurities in pharmaceutical preparations were established. The separation was performed on silica gel-coated chromatographic plates (HPTLC) using two mobile phases: (I) chloroform,methanol,glacial acetic acid (9:2:0.1, v/v/v); (II) chloroform,toluene,ethanol (9:8:1, v/v/v). The UV densitometry was carried out at , = 270 nm. The developed method is of high sensitivity and low detection and determination limits ranging from 0.044 to 0.35 µg. For individual constituents the recovery ranges from 93.23 to 99.66%. The next step was to evaluate the stability of tramadol and determine a method of decomposition under various experimental conditions. It was found that tramadol decomposes in various ways in acidic and basic environments producing (1RS)-[2-(3-methoxyphenyl)cyclohex-2-enyl]- N,N -dimethylmethanamine (imp. B) and (1RS, 2RS)-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl)cyclohexanol (imp. cis -T) or imp. cis -T, respectively. Copyright © 2004 John Wiley & Sons, Ltd. [source] Enlargement of calcium oxalate stones to clinically significant size in an in-vitro stone generatorBJU INTERNATIONAL, Issue 9 2002K. Ananth Objective ,To develop and validate an in vitro method suitable for the quantitative investigation of the growth of calcium oxalate stones through to a clinically significant size. Materials and methods ,Small fragments of calcium oxalate calculi were suspended in a mixed suspension/mixed product removal crystalliser supplied with artificial urine supersaturated with calcium oxalate. The fragments were weighed at regular intervals until they reached ,,500 mg. The results were plotted as weight against time and fitted to equations corresponding to constant increase in diameter, surface area-controlled and constant-deposition growth patterns. The choice of the most appropriate model was based on the squared regression coefficient (r2). Results ,Eight fragments (2,6 mm in diameter) were grown to ,,10 mm in diameter over periods from 137 to 369 h. Seven of the growth curves were best-fitted (r2 , 0.988) by the equation w = kt(3/2) + c, where w is the weight, k is a growth constant, t is the time and c is a constant approximating to the initial weight. This corresponds to a surface area-dependent mechanism. Conclusions ,The growth of these small fragments to a clinically significant size accelerated throughout the experimental period in a way which was consistent with a surface area-dependent mechanism. We have developed a resilient model suitable for studying the kinetics of calcium oxalate stone growth in vitro. [source] Gene trap mutagenesis in mice: New perspectives and tools in cancer researchCANCER SCIENCE, Issue 1 2008Ken-ichi Yamamura The complete human DNA sequence of the human genome was published in 2004 and we entered the postgenomic era. However, many studies showed that gene function is much more complex than we expected, and that mutation of disease genes does not give any clue for molecular mechanisms for disease development. Since the first report on gene knockout mice in 1989, knockout mice have been shown to be a powerful tool for functional genomics and for the dissection of developmental processes in human diseases. In accordance with this successful application of knockout mice, three major mouse knockout programs are now underway worldwide, to mutate all protein-encoding genes in mouse embryonic stem cells using a combination of gene trapping and gene targeting. We developed the exchangeable gene trap method suitable for large scale mutagenesis in mice. In this method we can produce null mutation and post-insertional modification, enabling replacement of the marker gene with a gene of interest and conditional knockout. We herein discuss the effect of this gene-driven type approach for cancer research, especially for finding the genes that are related to cancer, but are paid little attention in hypothesis-driven cancer research. (Cancer Sci 2008; 99: 1,6) [source] | ||||||||||||||||