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Reagents Used (reagent + used)
Selected Abstracts31P Solid state NMR study of structure and chemical stability of dichlorotriphenylphosphoraneMAGNETIC RESONANCE IN CHEMISTRY, Issue 6 2009Nina C. Gonnella Abstract Solid state 31P NMR spectroscopy was used to examine, monitor and quantify the compound integrity of the chemical reagent dichlorotriphenylphosphorane. Comparison was also made with solution 31P NMR spectra which showed that this highly reactive species could be observed in dry benzene prior to conversion to the hydrolyzed product. This is the first reported solid state NMR study of the stability and reactivity of dichlorotriphenylphosphorane and the first account of its observation and comparison in the solution state. In the solid state, the ionic and covalent forms for dichlorotriphenylphosphorane were observed along with hydrolyzed products, however, the degree of hydrolysis was dependent upon the rotor packing conditions. Calculation of the relative percent composition of dichlorotriphenylphosphorane with hydrolyzed product was made for samples prepared in air versus under nitrogen atmosphere. This information was critical in adjusting the amount of reagent used in chemical development syntheses and scale up laboratories. All hydrolyzed products were identified, based upon chemical comparisons with spectra of pure materials. Copyright © 2009 John Wiley & Sons, Ltd. [source] Characterization and comparison of commercially available mite extracts for in vivo diagnosisALLERGY, Issue 2 2010B. Brunetto To cite this article: Brunetto B, Tinghino R, Braschi MC, Antonicelli L, Pini C, Iacovacci P. Characterization and comparison of commercially available mite extracts for in vivo diagnosis. Allergy 2010; 65: 184,190. Abstract Background:, Assessment of sensitization by allergen-specific IgE testing and skin prick testing (SPT) are primary tools in routine clinical diagnosis of allergies. To perform a correct diagnosis, it is critical that the allergen reagent used contains an adequate amount of all relevant components. This study aimed at evaluating commercially available mite extracts for in vivo diagnosis from eight manufacturers. Methods:, Eight extracts from Dermatophagoides pteronyssinus and eight from Dermatophagoides farinae were analysed for total protein content by Bradford and for major allergen content by ELISA. SDS-PAGE, immunoblotting and SPT were also carried out. Results:, The protein amount ranged from 27.7 ,g/ml extract to 361.1 ,g/ml (D. pteronyssinus) and from 20.3 to 353.0 ,g/ml (D. farinae). In regards major allergen concentration, Der p 1 ranged from 9.6 to 36.2 ,g/ml, Der f 1 26.5,196.1 ,g/ml, mite group 2 0.7,31.7 ,g/ml in D. pteronyssinus and 1.3,10.4 ,g/ml in D. farinae. SDS-PAGE experiments showed that some components are poorly represented or absent in extracts from most manufacturers. Similar results were obtained by IgE-immunoblotting and SPT with 10 mite allergic patients confirmed a broad spectrum of reactivity of the extracts in the same subject. Conclusions:, Immunochemical analysis showed a heterogeneous amount of component/s among mite extracts from different manufacturers. These data were confirmed by in vivo testing, suggesting that, for some of the patient tested, the absence of relevant allergens could strongly affect the diagnosis. [source] Stable transgene expression in human embryonic stem cells after simple chemical transfectionMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2009Jun Liu In this study we used plasmid-based vectors to investigate the transcriptional activities of three commonly used promoters in transient and stable transfection of MEL-1, a human embryonic stem (ES) cell line, using ExGen500, Fugene HD, and Lipofectamine. We demonstrated that cytomegalovirus (CMV), phosphoglycerate kinase (PGK) and human elongation factor-1, (EF1,) promoters all resulted in robust activity of a reporter gene in MEL-1 ES cell transient transfections regardless of the transfection reagent. Stable transfection outcomes varied, depending on the promoter and the transfection reagent used in the study. The phenomenon of transgene silencing was observed, most notably with the CMV vector, with which no positive stably transfected clones were obtained. Of the methods used in the study, Fugene HD resulted in the highest stable transfection rate, estimated by antibiotic selection, with plasmids containing genes under the control of the EF1, or PGK promoters. Stably transfected cells maintained typical hES cell morphology, with immunostaining exhibiting expression of the hES cell markers: Oct4, SSEA4, Tra-1-60, and Tra-1-81. Further, embryoid bodies formed by suspension culture retained reporter gene expression. Following injection into immunodeficient mice, the transfected cell lines showed robust formation of teratomas with cell types representative of the three germ layers. Mol. Reprod. Dev. 76: 580,586, 2009. © 2008 Wiley-Liss, Inc. [source] The factors that influence the elution order for the resolution of amino acids on vancomycin phase using the polar-organic mobile phases after their pre-column derivatization with electrophilic reagentsBIOMEDICAL CHROMATOGRAPHY, Issue 6 2005S. Chen Abstract A variety of amino acids were enantioresolved on a vancomycin bonded chiral phase using the polar-organic mobile phases after their pre-column derivatization with electrophilic reagents in alkaline medium. The resolution was highly dependent on the analyte's structure and was enhanced as the aromatic side-chain group on the skeleton of analyte for ,,, interaction with the chiral selector became available. The steric hindrance resulting from the bulky side-chain group on the analyte also affected the resolution. Elution reversal, not found on the teicoplanin phase under the same chromatographic conditions, was possible through altering the type of reagent used in the derivatizing reaction (e.g. 2,4-difluorophenylisothiocyanate to 2,4-difluorophenylisocyanate). It is believed that the steric hindrance, as a result of the bulky sulfur atom in reagents such as methylisothiocyanate and others examined in this study, was responsible for the reversed elution order. The bulkiness of the substituent on the aromatic ring of derivatizing reagents (i.e. 2,3- and 3,5-dichlorophenylisothiocyanate) was observed to affect the resolution and alter the elution order as well. Copyright © 2005 John Wiley & Sons, Ltd. [source] Reaction of Cytidine Nucleotides with Cyanoacetylene: Support for the Intermediacy of Nucleoside-2,,3,-cyclic Phosphates in the Prebiotic Synthesis of RNACHEMBIOCHEM, Issue 6 2006Michael A. Crowe Abstract A robust and prebiotically plausible synthesis of RNA is a key requirement of the "RNA World" hypothesis, but, to date, no such synthesis has been demonstrated. Monomer synthesis strategies involving attachment of preformed nucleobases to sugars have failed, and, even if activated 5,-nucleotides could be made, the hydrolysis of these intermediates in water makes their efficient oligomerisation appear unlikely. We recently reported a synthesis of cytidine-2,,3,-cyclic phosphate 1 (C>p) in which the nucleobase was assembled in stages on a sugar-phosphate template. However, 2,,3,-cyclic nucleotides (N>p's) also undergo hydrolysis, in this case giving a mixture of the 2,- and 3,-monophosphates. This hydrolysis has previously been seen as making the, otherwise promising, oligomerisation of N>p's seem as unlikely as that of the 5,-activated nucleotides. We now find that cyanoacetylene, the reagent used for the second stage of nucleobase assembly in the synthesis of C>p, also reverses the effect of the hydrolysis by driving efficient cyclisation of C2,p and C3,p back to C>p. Excess cyanoacetylene also derivatises the nucleobase, but this modification is reversible at neutral pH. These findings significantly strengthen the case for N>p's in a prebiotic synthesis of RNA. [source] Interference from Trace Copper in Electrochemical Investigations Employing Carboxylic Acid Terminated Thiol Modified Gold ElectrodesELECTROANALYSIS, Issue 6 2009Xiaohu Qu Abstract Unexpectedly, electrochemistry at variable chain length carboxylic acid terminated alkylthiol self-assembled monolayers (SAMs) on gold electrodes gives rise to a Faradaic process in buffered aqueous electrolyte solution. In particular, the three-carbon chain length, 3-mercaptopropionic acid (MPA), exhibits a chemically reversible process with a mid-point potential of 175,mV vs. Ag/AgCl under conditions of cyclic voltammetry. This process is associated with the presence of trace (parts per billion) amounts of copper(II) ions present in the chemical reagents used to prepare the aqueous electrolyte and also from the gold electrode itself. The carboxylic acid moiety on the SAM concentrates Cu2+ ions by coordination and this surface confined layer is then reduced. Methods to minimize the interference of Cu2+ ions at carboxylic acid terminated SAM are discussed and caution with respect to the interpretation of protein electrochemistry is recommended when using carboxylic acid functionalized SAMs to provide biocompatible electrochemical transduction surfaces, unless a metal free environment can be obtained. [source] Direct examination of soil for sporangia of Synchytrium endobioticum using chloroform, calcium chloride and zinc sulphate as extraction reagentsEPPO BULLETIN, Issue 1 2005G. C. M. Van Leeuwen Fields infested with Synchytrium endobioticum can be descheduled when the soil is found free from sporangia of S. endobioticum. For direct examination, EPPO Standard PM 3/59 describes a soil extraction technique based on the use of a sieve shaker with six sieves. We compared recovery of sporangia between this (modified) method and an extraction method employed by the Dutch Plant Protection Service (PPS method). Recovery was determined using an inoculum dilution series: 125, 25, 5, 1, 0.2 or 0.04 sporangia per g soil. Extraction reagents used were chloroform and calcium chloride in the method described by EPPO, calcium chloride and zinc sulphate in the PPS method. At 125 sporangia per g soil, the mean density determined for the modified EPPO method was 228 sporangia per g soil when chloroform was used. Using calcium chloride, recovery percentage was higher for the modified EPPO method than for the PPS method (286, 136%, n.s. P < 0.05). The advantage of the modified EPPO method was the larger soil volume to be processed; its disadvantages were use of complex equipment and noxious reagents (chloroform). Both extraction methods showed high variation in recovery between samples, making accurate estimation of sporangial densities in soil awkward. [source] Lipopolysaccharide is a frequent and significant contaminant in microglia-activating factorsGLIA, Issue 1 2008Jonathan R. Weinstein Abstract Lipopolysaccharide (LPS/endotoxin) is a potent immunologic stimulant. Many commercial-grade reagents used in research are not screened for LPS contamination. LPS induces a wide spectrum of proinflammatory responses in microglia, the immune cells of the brain. Recent studies have demonstrated that a broad range of endogenous factors including plasma-derived proteins and bioactive phospholipids can also activate microglia. However, few of these studies have reported either the LPS levels found in the preparations used or the effect of LPS inhibitors such as polymyxin B (PMX) on factor-induced responses. Here, we used the Limulus amoebocyte lysate assay to screen a broad range of commercial- and pharmaceutical-grade proteins, peptides, lipids, and inhibitors commonly used in microglia research for contamination with LPS. We then characterized the ability of PMX to alter a representative set of factor-induced microglial activation parameters including surface antigen expression, metabolic activity/proliferation, and NO/cytokine/chemokine release in both the N9 microglial cell line and primary microglia. Significant levels of LPS contamination were detected in a number of commercial-grade plasma/serum- and nonplasma/serum-derived proteins, phospholipids, and synthetic peptide preparations, but not in pharmaceutical-grade recombinant proteins or pharmacological inhibitors. PMX had a significant inhibitory effect on the microglia-activating potential of a number of commercial-, but not pharmaceutical-grade, protein preparations. Novel PMX-resistant responses to ,2 -macroglobulin and albumin were incidentally observed. Our results indicate that LPS is a frequent and significant contaminant in commercial-grade preparations of previously reported microglia-activating factors. Careful attention to LPS levels and appropriate controls are necessary for future studies in the neuroinflammation field. © 2007 Wiley-Liss, Inc. [source] Quantitative evaluation of the lengths of homobifunctional protein cross-linking reagents used as molecular rulersPROTEIN SCIENCE, Issue 7 2001Nora S. Green Abstract Homobifunctional chemical cross-linking reagents are important tools for functional and structural characterization of proteins. Accurate measures of the lengths of these molecules currently are not available, despite their widespread use. Stochastic dynamics calculations now provide quantitative measures of the lengths, and length dispersions, of 32 widely used molecular rulers. Significant differences from published data have been found. [source] Methylation of acidic moieties in poly(methyl methacrylate-co-methacrylic acid) copolymers for end-group characterization by tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 14 2010Rémi Giordanengo The complete structural characterization of a copolymer composed of methacrylic acid (MAA) and methyl methacrylate (MMA) units was achieved using tandem mass spectrometry. In a first step, collision-induced dissociation (CID) of sodiated MAA-MMA co-oligomers allowed us to determine the co-monomeric composition, the random nature of the copolymer and the sum of the end-group masses. However, dissociation reactions of MAA-based molecules mainly involve the acidic pendant groups, precluding individual characterization of the end groups. Therefore, methylation of all the acrylic acid moieties was performed to transform the MAA-MMA copolymer into a PMMA homopolymer, for which CID mainly proceeds via backbone cleavages. Using trimethylsilyldiazomethane as a derivatization agent, this methylation reaction was shown to be complete without affecting the end groups. Using fragmentation rules established for PMMA polymers together with accurate mass measurements of the product ions and knowledge of reagents used for the studied copolymer synthesis, a structure could be proposed for both end groups and it was found to be consistent with signals obtained in nuclear magnetic resonance spectra. Copyright © 2010 John Wiley & Sons, Ltd. [source] Plasma phospholipids implicated in the matrix effect observed in liquid chromatography/tandem mass spectrometry bioanalysis: evaluation of the use of colloidal silica in combination with divalent or trivalent cations for the selective removal of phospholipids from plasmaRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2008Steven T. Wu The feasibility of the use of colloidal silica in combination with a number of divalent or trivalent cations for the removal of plasma phospholipids was evaluated by sequentially adding the two reagents (i.e., colloidal silica and a cation) directly to blank plasma samples or plasma samples spiked with analytes. Three representative plasma phospholipids were monitored to determine the efficiency of the phospholipids removal under different reagent combinations. The recovery of each spiked analyte was also monitored under each condition in order to determine if any of the analyte was removed along with the phospholipids. By optimizing the amounts of the reagents used and the sequence of the addition of the reagents, quantitative and reproducible removal of the phospholipids was achieved. Using the finally selected lanthanum cation, the removal of phospholipids was achieved with minimal concomitant loss of the ten investigated analytes which were carefully selected to incorporate functional groups that could potentially interact with the added reagents and hence could be removed along with the phospholipids. Copyright © 2008 John Wiley & Sons, Ltd. [source] Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of 4-sulfophenyl isothiocyanate-derivatized peptides on AnchorChipÔ sample supports using the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone and diammonium citrateRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2005Leon P. Oehlers The reagent 4-sulfophenyl isothiocyanate (SPITC) is an effective, stable, and inexpensive alternative to commercially available reagents used in the N-terminal sulfonation of peptides for enhanced postsource decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analyses. However, suppression of ionization of sulfonated peptides due to sample and matrix contaminants such as sodium can be a problem when using prestructured MALDI target sample supports, such as the Bruker Daltonics AnchorChipÔ. We show that use of the salt-tolerant matrix 2,4,6-trihydroxyacetophenone containing diammonium citrate (THAP/DAC) as an alternative to , -cyanohydroxycinnamic acid (HCCA) reduces the need for extensive washing of ZipTip-bound peptides or additional on-target sample clean-up steps. Use of the THAP/DAC matrix results in selective ionization of sulfonated peptides with greater peptide coverage, as well as detection of higher mass derivatized peptides, than was observed for HCCA or THAP alone. The THAP/DAC matrix is quite tolerant of sodium contamination, with SPITC-peptides detectable in preparations containing up to 50,mM NaCl. In addition, THAP/DAC matrix was found to promote efficient PSD fragmentation of sulfonated peptides. We demonstrated the utility of using the THAP/DAC MALDI matrix for peptide sequencing with DNA polymerase , tryptic peptide mixture, as well as tryptic peptides derived from Xiphophorus maculatus brain extract proteins previously separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Copyright © 2005 John Wiley & Sons, Ltd. [source] Case Report: Fatal Apophysomyces elegans Infection Transmitted by Deceased Donor Renal AllograftsAMERICAN JOURNAL OF TRANSPLANTATION, Issue 9 2010B. D. Alexander Two patients developed renal mucormycosis following transplantation of kidneys from the same donor, a near-drowning victim in a motor vehicle crash. Genotypically, indistinguishable strains of Apophysomyces elegans were recovered from both recipients. We investigated the source of the infection including review of medical records, environmental sampling at possible locations of contamination and query for additional cases at other centers. Histopathology of the explanted kidneys revealed extensive vascular invasion by aseptate, fungal hyphae with relative sparing of the renal capsules suggesting a vascular route of contamination. Disseminated infection in the donor could not be definitively established. A. elegans was not recovered from the same lots of reagents used for organ recovery or environmental samples and no other organ transplant-related cases were identified. This investigation suggests either isolated contamination of the organs during recovery or undiagnosed disseminated donor infection following a near-drowning event. Although no changes to current organ recovery or transplant procedures are recommended, public health officials and transplant physicians should consider the possibility of mucormycosis transmitted via organs in the future, particularly for near-drowning events. Attention to aseptic technique during organ recovery and processing is re-emphasized. [source] | ||||||||||||||||