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Chromatographic Procedure (chromatographic + procedure)
Selected AbstractsInhibition of pneumococcal choline-binding proteins and cell growth by esters of bicyclic aminesFEBS JOURNAL, Issue 2 2007Beatriz Maestro Streptococcus pneumoniae is one of the major pathogens worldwide. The use of currently available antibiotics to treat pneumococcal diseases is hampered by increasing resistance levels; also, capsular polysaccharide-based vaccination is of limited efficacy. Therefore, it is desirable to find targets for the development of new antimicrobial drugs specifically designed to fight pneumococcal infections. Choline-binding proteins are a family of polypeptides, found in all S. pneumoniae strains, that take part in important physiologic processes of this bacterium. Among them are several murein hydrolases whose enzymatic activity is usually inhibited by an excess of choline. Using a simple chromatographic procedure, we have identified several choline analogs able to strongly interact with the choline-binding module (C-LytA) of the major autolysin of S. pneumoniae. Two of these compounds (atropine and ipratropium) display a higher binding affinity to C-LytA than choline, and also increase the stability of the protein. CD and fluorescence spectroscopy analyses revealed that the conformational changes of C-LytA upon binding of these alkaloids are different to those induced by choline, suggesting a different mode of binding. In vitro inhibition assays of three pneumococcal, choline-dependent cell wall lytic enzymes also demonstrated a greater inhibitory efficiency of those molecules. Moreover, atropine and ipratropium strongly inhibited in vitro pneumococcal growth, altering cell morphology and reducing cell viability, a very different response than that observed upon addition of an excess of choline. These results may open up the possibility of the development of bicyclic amines as new antimicrobials for use against pneumococcal pathologies. [source] Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybeanJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2005T. Zendo Abstract Aims:, Identification of the bacteriocin produced by Enterococcus mundtii QU 2 newly isolated from soybean and fermentative production of the bacteriocin. Methods and Results:, The bacteriocin produced by Ent. mundtii QU 2 inhibited the growth of various indicator strains, including Enterococcus, Lactobacillus, Leuconostoc, Pediococcus and Listeria. The bacteriocin activity was stable at wide pH range and against heat treatment, but completely abolished by proteolytic enzymes. The bacteriocin was purified from the culture supernatant by the three-step chromatographic procedure. Mass spectrometry, amino acid sequencing and DNA sequencing revealed that the bacteriocin was similar to class IIa bacteriocins produced by other Ent. mundtii strains. The bacteriocin production decreased in the absence of glucose, nitrogen sources, or Tween 80 in MRS medium. Additionally, it was strongly suppressed by addition of Ca2+ (CaCO3 or CaCl2). In pH-controlled fermentations, the highest bacteriocin production was achieved at pH 6·0, whereas the highest cell growth was obtained at pH 7·0. Conclusions:,Ent. mundtii QU 2 produced a class IIa bacteriocin. Some growth factors (e.g. Ca2+ and pH) influenced the bacteriocin production. Significance and Impact of the Study:, A new soybean isolate, Ent. mundtii QU 2 was found to be a class IIa bacteriocin producer. Factors influencing the bacteriocin production described herein are valuable for applications of the bacteriocins from Ent. mundtii strains. [source] Identification of [(GS)2AsSe], in rabbit bile by size-exclusion chromatography and simultaneous multielement-specific detection by inductively coupled plasma atomic emission spectroscopyAPPLIED ORGANOMETALLIC CHEMISTRY, Issue 2 2002Jürgen Gailer Abstract An arsenic,selenium metabolite that exhibited the same arsenic and selenium X-ray absorption near-edge spectra as the synthetic seleno-bis(S -glutathionyl) arsinium ion [(GS)2AsSe], was recently detected in rabbit bile within 25,min after intravenous injection of rabbits with sodium selenite and sodium arsenite. X-ray absorption spectroscopy did not (and cannot) conclusively identify the sulfur-donor in the in vivo sample. After similar treatment of rabbits, we analyzed the collected bile samples by size-exclusion chromatography (SEC) using inductively coupled plasma atomic emission spectroscopy (ICP-AES) to monitor arsenic, selenium and sulfur simultaneously. The bulk of arsenic and selenium eluted in a single peak, the intensity of which was greatly increased upon spiking of the bile samples with synthethic [(GS)2AsSe],. Hence, we identify [(GS)2AsSe], as the major metabolite in bile after exposure of rabbits to selenite and arsenite. The reported SEC,ICP-AES method is the first chromatographic procedure to identify this biochemically important metabolite in biological fluids and is thus a true alternative to X-ray absorption spectroscopy, which is not available to many chemists. Copyright © 2001 John Wiley & Sons, Ltd. [source] Photostability studies for micellar liquid chromatographic determination of nifedipine in serum and urine samplesBIOMEDICAL CHROMATOGRAPHY, Issue 2 2006M. T. Gil-Agustí Abstract Nifedipine is a photosensitive compound that is converted into its 4-(2-nitrophenyl) pyridine and 4-(2-nitrosophenyl) pyridine homologue. In order to obtain the most adequate conditions for handling nifedipine solutions in the analytical laboratory, a number of studies on the decomposition of this compound were performed. A simple micellar liquid chromatographic procedure was described to determine nifedipine in different biological matrices such as serum and urine, and to control its decomposition. To perform the analysis, nifedipine was dissolved in 0.1 m SDS at pH 3 and chromatographed using a mobile phase containing 0.125 m SDS,3% pentanol, pH 3 on a C18 column and UV detection at 235 nm. The chromatographic analysis time was 8 min. The response of the drug for both biological matrices was linear in the 1,100 µg/mL range, with r2 > 0.997 at all times. Repeatability, intermediate precision (CV, %) and limits of quantification and detection (ng/mL) were 0.19, 4.3, 104 and 31 in serum and 0.81, 2.1, 136 and 41 in urine. The method developed here does not show interferences or matrix effects produced by endogenous compounds. Micellar media and mobile phases have the advantage of stabilising the compounds, thus preventing photodegradation and allowing the direct injection of biological samples. Copyright © 2005 John Wiley & Sons, Ltd. [source] Challenges and trends in bioseparationsJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 2 2008Juan A Asenjo Abstract In this paper the issues and challenges presented 15 years ago for performing efficient separation processes for recombinant proteins are revised and discussed. Competitive advantage in production was seen as not only dependent on innovations in molecular biology and other areas of basic biological sciences but also on innovation of separations and downstream processes. The trend to develop techniques that exploit fundamental physicochemical principles more efficiently was emphasized, including analysis of the physicochemical properties of proteins and its relation to efficiency in bioseparation. 15 years ago the main thrust was also focused on the development of novel techniques. Clearly the challenges faced today, where highly optimized and efficient production processes exist, are dramatically different. The use of mathematical models for optimizing chromatographic separations and simplifying validation of such operations is extremely advantageous. Their use constitutes an example of how the challenges that bioseparations are facing and will be facing within the next few years can be met. Such models should be extended to a larger number of proteins, chromatographic procedures and experimental conditions. Copyright © 2008 Society of Chemical Industry [source] Expression, purification, crystallization and preliminary X-ray diffraction analysis of the transcriptional repressor SirR from Mycobacterium tuberculosis H37RvACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Baisakhee Saha SirR, a metal-dependent transcriptional repressor from Mycobacterium tuberculosis (Rv2788), was cloned in pQE30 expression vector with an N-terminal His6 tag for heterologous overexpression in Escherichia coli M15 (pREP4) cells and purified to homogeneity using chromatographic procedures. The purified protein was crystallized using the sitting-drop vapour-diffusion technique. The crystals belonged to the tetragonal space group P41212/P43212, with unit-cell parameters a = 105.21, b = 105.21, c = 144.85,Å. The X-ray diffraction data were processed to a maximum resolution of 2.5,Å. The Matthews coefficient suggests the presence of two (VM = 4.01,Å3,Da,1) to four (VM = 2.0,Å3,Da,1) molecules in the asymmetric unit. Calculation of the self-rotation function shows a crystallographic fourfold symmetry axis along the z axis (, = 90°) and also a twofold symmetry axis around the z axis (, = 180°). [source] Preparation of enantiomeric gossypol by crystallization,CHIRALITY, Issue 6 2003Michael K. Dowd Abstract Large enantiomorphic crystals of gossypol-acetone (1:3) were grown from acetone solutions of rac -gossypol-acetic acid (1:1) at 4°C. By controlling the initial gossypol concentration, crystallization time, and solution volume, single crystals were grown that weighed >50 mg, equivalent to >37 mg of enantiomeric gossypol. Even larger crystals were possible, but it was difficult to produce these reliably without contamination of the antipode. Essentially all of the acetone within the crystal form was removed by storing the crystals under vacuum for 3,4 days. By employing these techniques, gram quantities of enantiomeric gossypol were prepared in high chemical and optical purity. Based on measured and reported optical rotations, the optical purity of samples prepared by crystallization was greater than the optical purity of samples prepared by chromatographic separation of gossypol-amine diastereomers. The principal limitation of crystallization as a preparative method is the need to determine the chirality and purity of each product crystal. Nevertheless, the method competes favorably with preparative-scale chromatographic procedures. Chirality 15:486,493, 2003. Published 2003 Wiley-Liss, Inc. [source] | ||||||||||