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Reverse-phase HPLC (reverse-phase + hplc)
Terms modified by Reverse-phase HPLC Selected AbstractsWeak agonist self-peptides promote selection and tuning of virus-specific T cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2003Samuel Abstract Recent progress has begun to define the interactions and signaling pathways that are triggered during positive selection. To identify and further examine self-peptides that can mediate positive selection, we searched a protein-database to find peptides that have minimal homology with the viral peptide (p33) that activates a defined P14 transgenic TCR. We identified four peptides that could bind the restriction element H-2Db and induce proliferation of P14 transgenic splenocytes at high concentration. Two of the four peptides (DBM and RPP) were able to positively select thevirus-specific TCR in fetal thymic organ culture but were unable to induce clonal deletion. Reverse-phase HPLC and mass spectrometry demonstrated that these peptides were presented by H-2Db molecules on thymic epithelial cell lines. We also examined whether the selecting ligands altered T cell responsiveness in vitro. DBM-selected T cells lost their ability to respond to the positively selecting ligand DBM, whereas RPP-selected T cells only retainrd their ability to respond to high concentrations of RPP. These results demonstrate that self-peptides that mediate positiveselection can differentially "tune" the activation threshold of T cells and alter the functional repertoire of T cells. [source] Oxime Carbonates: Novel Reagents for the Introduction of Fmoc and Alloc Protecting Groups, Free of Side ReactionsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 17 2010Sherine N. Khattab Abstract Fmoc and Alloc protecting groups represent a consistent alternative to classical Boc protection in peptide chemistry. The former was established in the last decades as the ,-amino protecting group of choice, whereas the latter allows a fully orthogonal protection strategy with Fmoc and Boc. Usually, the introduction of the Fmoc and Alloc moieties takes place through their halogenoformates, azides, or activated carbonates. This rather simple reaction is accompanied by several side reactions, specially the formation of Fmoc/Alloc dipeptides and even tripeptides. The present work describes new promising Fmoc/Alloc-oxime reagents, which are easy to prepare, stable, and highly reactive crystalline materials that afford almost contaminant-free Fmoc/Alloc-amino acids in high yields by following a conventional procedure. Amongst the Fmoc-oxime derivatives, the N -hydroxypicolinimidoyl cyanide derivative (N -{[(9H-fluoren-9-yl)methoxy]carbonyloxy}picolinimidoyl cyanide) gave the best results for the preparation of Fmoc-Gly-OH, which is the most predisposed to give side reactions. The same Alloc-oxime analogue afforded the preparation of Alloc-Gly-OH in good yield, purity, and extremely low dipeptide formation, as analyzed by reverse-phase HPLC and NMR spectroscopy. [source] Biosynthesis and transcriptional analysis of thurincin H, a tandem repeated bacteriocin genetic locus, produced by Bacillus thuringiensis SF361FEMS MICROBIOLOGY LETTERS, Issue 2 2009Hyungjae Lee Abstract Thurincin H, a bacteriocin produced by Bacillus thuringiensis SF361 isolated from honey, strongly inhibited the growth of Bacillus cereus F4552. The bacteriocin was purified by 65% ammonium sulfate precipitation of the culture supernatant, followed by octyl-sepharose CL-4B and reverse-phase HPLC. The molecular mass of the bacteriocin was determined to be 3139.51 Da and the 14 amino acids of the bacteriocin at the N-terminus were identified. The complete amino acid sequence of mature thurincin H was deduced from three structural genes, thnA1, thnA2, and thnA3 found in tandem repeats on the chromosome, all of which encode for the same bacteriocin, thurincin H. The genetic determinants for thurincin H biosynthesis consist of 10 ORFs, including three thurincin H structural genes. Northern hybridization elucidated that the transcription of all three bacteriocin structural genes was regulated by a putative promoter located upstream of thnA1. [source] Expression of glutathione transferase isoenzymes in the human H295R adrenal cell line and the effect of forskolinJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2002Tuula Stark Abstract In previous studies in our laboratory (L. Mankowitz, L. Staffas, M. Bakke, and J. Lund, Biochem J, 1995, 305, 111,118; L. Staffas, L. Mankowitz, M. Söderström, A. Blanck, I. Porsch-Hällström, C. Sundberg, B. Mannervik, B. Olin, J. Rydström, and J.W. DePierre, Biochem J, 1992, 286, 65,72) isoenzymes of GST, primarily of the , class, have been shown to be downregulated by adrenocorticotropic hormone (ACTH) in rat and mouse adrenal cells. In the present investigation the human adrenal H295R cell line (W.E. Rainey, I.M. Bird, and J.I. Mason, Mol Cell Endocrinol, 1994, 100, 45,50) was examined in a similar manner. Analysis by reverse-phase HPLC revealed that these cells express four isoenzymes of GST, i.e., A1, A2, P1, and M4, as well as another unidentified protein that was retained by our affinity column (elution time of 32 min) and, thus, presumably binds glutathione. Among these forms, A1 was present at the highest level. Upon addition of forskolin (an activator of adenylate cyclase which has been shown previously to mimic the effect of ACTH on adrenal cells) to the culture medium, the level of A1 decreased approximately 70% by forskolin, whereas the levels of the other isoenzymes were slightly increased, and that of the unknown form doubled. Thus, the influence of ACTH on expression of GST isoenzymes in this human adrenal cell line differs from that in rat and mouse adrenal cells. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:169,173, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10034 [source] Cyclic guanosine monophosphate phosphodiesterase activity in human gingival carcinomaJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 4 2003Giuseppe Spoto Abstract Background:, Cyclic guanosine monophosphate (cGMP) is an essential second messenger metabolized by phosphodiesterases (PDEs). Objectives:, We looked for a possible correlation of PDE activities in human oral squamous cell carcinoma (OSCC) with and without lymph node metastases. Materials and methods:, The analysis of phosphodiesterase activity and the cGMP assay were done by reverse-phase HPLC on samples of fresh or frozen gingival tissues. Analysis of cGMP was confirmed with the enzyme-linked immunoabsorption assay. Results and conclusions:, cGMP PDE activity was 34.92 ± 7.17 SD, 12.89 ± 4.43 SD, and 35.88 ± 8.76 SD (nmols/mg of protein), respectively, in controls, samples without lymph node involvement (N,), and specimens with lymph node metastases (N+). cGMP values were 1.97 ± 0.63 SD, 3.30 ± 1.47 SD, and 3.49 ± 1.47 SD (nmols/mg of protein). Our data support the hypothesis of a role for cGMP and PDE in the progression of OSCC. [source] Elimination and exchange of trifluoroacetate counter-ion from cationic peptides: a critical evaluation of different approachesJOURNAL OF PEPTIDE SCIENCE, Issue 3 2008Stéphane Roux Abstract Most synthesized peptides are nowadays produced using solid-phase procedures. Due to cleavage and purification conditions, they are mainly obtained in the presence of trifluoroacetic acid (TFA) and, for cationic peptides, as trifluoroacetate (TF-acetate) salts. However, TF-acetate interferes with physicochemical characterizations using infrared spectroscopy and might significantly affect the in vivo studies. Thus, TF-acetate exchange by another counter-ion is often required. Up to now, the classical procedure has consisted of freeze-drying the peptide several times in the presence of an excess of a stronger acid than TFA (pKa ,0): generally HCl (pKa = , 7). This approach means that working at pH < 1 can induce peptide degradation. We therefore tested three different approaches to exchange the tightly bound TF-acetate counter-ion from the dicationic octapeptide lanreotide: (i) reverse-phase HPLC, (ii) ion-exchange resin, and (iii) deprotonation/reprotonation cycle of the amino groups. The first two approaches allow the partial to almost complete exchange of the TF-acetate counter-ion by another ion from an acid weaker than TFA, such as acetic acid (pKa = 4.5), and the third requires a basic solution that permits the complete removal of TF-acetate counter-ion. The efficiency of these three procedures was tested and compared by using different analytical techniques such as 19F-NMR, 1H-NMR and attenuated total reflectance Fourier transformed infrared spectroscopy (ATR FT-IR). We also show that ATR-IR can be used to monitor the TFA removal. The counter-ion exchange procedures described in this study are easy to carry out, fast, harmless and reproducible. Moreover, two of them offer the very interesting possibility of exchanging the initial TF-acetate by any other counter-ion. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] In-vitro transcutaneous delivery of tamoxifen and ,-linolenic acid from borage oil containing ethanol and 1,8-cineoleJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 11 2004Suzanna Ho The objective of this study was to examine the effects of ethanol and 1,8-cineole on the transcutaneous delivery of tamoxifen and ,-linolenic acid (GLA) as a two-pronged anti-breast cancer therapy. Formulations containing tamoxifen and varying concentrations of borage oil (,25% GLA), 1,8-cineole and ethanol were prepared and the simultaneous permeation of tamoxifen and GLA determined across full-thickness pig skin using Franz-type diffusion cells over 48 h. Analysis of tamoxifen and GLA (as methyl ester) were by reverse-phase HPLC. The highest flux of tamoxifen of 488.2 ± 191 times 10,3 ,g cm,2 h,1 was observed with a formulation containing 20% 1,8-cineole and 20% ethanol. The same formulation also provided the greatest flux of GLA, 830.6 times 10,3 ,g cm,2 h,1. The findings from this work demonstrate the ability of 1,8-cineole and ethanol to enhance the in-vitro permeation of tamoxifen and GLA across the skin and support the plausibility of simultaneously delivering tamoxifen and GLA transcutaneously as a two-pronged anti-breast cancer system. [source] Detection of N -nitroso-bile acids at 285 nm in reverse-phase HPLCJOURNAL OF SEPARATION SCIENCE, JSS, Issue 15 2008Yoshio Araki Abstract In the present study, we developed a novel, simple, and specific detection method using an RP-HPLC at UV 285 nm for the separation and quantification of N -nitroso-bile acids. First, we found that N -nitroso-bile acids have a specific spectrophotometric absorbance at 285 nm. Using this 285 nm detection system, we could especially measure N -nitroso-bile acids, even in co-existence of non- N -nitroso-bile acids. Next, we observed the decomposition of N -nitroso-glychocholate under alkaline, acidic, and neutral conditions. N -nitroso-glychocholate rapidly decomposed under alkaline conditions (pH 9) (t1/2 = 0.96 h), but remained fairly stable under acidic (pH 2) (t1/2 = 12.8 h) and neutral (pH 7) (t1/2 = 7.8 h) conditions. This study is the first report, which simply and specifically analyzes N -nitroso-bile acids using an RP-HPLC system. [source] HPLC purification and re-evaluation of chemical identity of two circular bacteriocins, gassericin A and reutericin 6LETTERS IN APPLIED MICROBIOLOGY, Issue 4 2010K. Arakawa Abstract Aim:, The study aimed for the complete purification and recharacterization of the highly hydrophobic circular bacteriocins, gassericin A and reutericin 6. Methods and Results:, Gassericin A and reutericin 6 were purified to homogeneity using previously described method and reverse-phase HPLC with an octyl column and eluents of aqueous acetonitrile and 2-propanol. Mass analysis, N-terminal sequencing and bacteriocin assay of the HPLC-purified bacteriocins showed the two bacteriocins had identical seamless circular structures with the same m/z value (5651) of [M + H]+ and both had the same specific activity. d/l- amino acid composition analysis using two distinct methods with the chiral fluorescent derivatization reagents (+)-1-(9-fluorenyl)ethyl chloroformate and o -phthalaldehyde/N -acetyl- l -cystein revealed neither gassericin A nor reutericin 6 contained d -alanine residues contrary to our previous results. Conclusion:, Purified gassericin A and reutericin 6 are chemically identical circular molecules containing no d -alanine residues. Significance and Impact of the Study:, The HPLC conditions developed in this study will facilitate advanced purification and correct characterization of other highly hydrophobic bacteriocins. [source] Role of minor determinants of amoxicillin in the diagnosis of immediate allergic reactions to amoxicillinALLERGY, Issue 5 2010M. J. Torres To cite this article: Torres MJ, Ariza A, Fernández J, Moreno E, Laguna JJ, Montañez MI, Ruiz-Sanchez AJ, Blanca M. Role of minor determinants of amoxicillin in the diagnosis of immediate allergic reactions to amoxicillin. Allergy 2010; 65: 590,596. Abstract Background:, Skin testing of subjects with immediate hypersensitivity to amoxicillin is performed using major and minor determinants of benzylpenicillin plus amoxicillin. However, sensitivity is not optimal, and other determinants need to be considered. We assessed the sensitivity of stable, well-characterized minor determinants of amoxicillin in subjects with immediate allergic reactions to amoxicillin to improve skin test sensitivity. Methods:, Amoxicillin, amoxicilloic acid, and diketopiperazine were prepared and characterized by reverse-phase HPLC, tested in vivo by skin testing and in vitro by basophil activation test and RAST inhibition assay. Results:, Patients with immediate hypersensitivity to amoxicillin were selected: Group A (n = 32), skin test positive just to amoxicillin; Group B (n = 19), skin test positive to benzylpenicillin determinants; Group C (n = 10), skin test negative and amoxicillin drug provocation test positive. In Group A, 27 subjects (81.8%) were skin test positive to amoxicillin, ten (30.3%) to amoxicilloic acid, two (6.1%) to diketopiperacine, and six (18.2%) negative. In Group B, nine (50%) were positive to amoxicillin, eight (42.1%) to amoxicilloic acid, none to diketopiperacine, and nine (50%) negative. In Group C, skin tests were negative. BAT was positive to amoxicillin in 26 patients (50.9%), to amoxicilloic acid in 15 (29.1%), and diketopiperazine in four (7.8%). RAST inhibition studies showed > 50% inhibition in all sera, with the highest concentration of amoxicillin and amoxicilloic acid. Conclusions:, The combination of minor determinants of amoxicillin, amoxicilloic acid, and diketopiperazine seems to be of no greater value than the use of amoxicillin alone. Further efforts are needed to find new structures to improve sensitivity in the diagnosis of immediate hypersensitivity to betalactams. [source] Biomanufacturing process analytical technology (PAT) application for downstream processing: Using dissolved oxygen as an indicator of product quality for a protein refolding reactionBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Shelly A. Pizarro Abstract Process analytical technology (PAT) is an initiative from the US FDA combining analytical and statistical tools to improve manufacturing operations and ensure regulatory compliance. This work describes the use of a continuous monitoring system for a protein refolding reaction to provide consistency in product quality and process performance across batches. A small-scale bioreactor (3,L) is used to understand the impact of aeration for refolding recombinant human vascular endothelial growth factor (rhVEGF) in a reducing environment. A reverse-phase HPLC assay is used to assess product quality. The goal in understanding the oxygen needs of the reaction and its impact to quality, is to make a product that is efficiently refolded to its native and active form with minimum oxidative degradation from batch to batch. Because this refolding process is heavily dependent on oxygen, the % dissolved oxygen (DO) profile is explored as a PAT tool to regulate process performance at commercial manufacturing scale. A dynamic gassing out approach using constant mass transfer (kLa) is used for scale-up of the aeration parameters to manufacturing scale tanks (2,000,L, 15,000,L). The resulting DO profiles of the refolding reaction show similar trends across scales and these are analyzed using rpHPLC. The desired product quality attributes are then achieved through alternating air and nitrogen sparging triggered by changes in the monitored DO profile. This approach mitigates the impact of differences in equipment or feedstock components between runs, and is directly inline with the key goal of PAT to "actively manage process variability using a knowledge-based approach." Biotechnol. Bioeng. 2009; 104: 340,351 © 2009 Wiley Periodicals, Inc. [source] Chenoalbicin, a Novel Cinnamic Acid Amide Alkaloid from Chenopodium albumCHEMISTRY & BIODIVERSITY, Issue 10 2004Francesca Cutillo The roots of Chenopodium album were infused in MeOH, and the extract was partitioned between AcOEt and H2O. AcOEt-Soluble material was subjected to different silica-gel column chromatographies and then purified by reverse-phase HPLC to afford a new cinnamic acid amide alkaloid as a racemic mixture. The new compound, named chenoalbicin (1), was characterized by extensive spectroscopic investigation, especially 1D and 2D NMR spectroscopy. Its effects on the germination and growth of Lactuca sativa L. has been studied. The results are reported as percentage differences of germination, root elongation, and shoot elongation from the control at concentrations ranging from 10,4 to 10,7,M. [source] Stereoselective pharmacokinetics of clausenamide enantiomers and their major metabolites after single intravenous and oral administration to ratsCHIRALITY, Issue 8 2003Chuan Jiang Zhu Abstract The pharmacokinetics of clausenamide (CLA) enantiomers and their metabolites were investigated in Wistar rat. After intravenous and oral administration at a dose of 80 and 160 mg/kg each enantiomer, plasma concentrations of (,)- or (+)-CLA and its major metabolites were simultaneously determined by reverse-phase HPLC with UV detection. Notably, stereoselective differences in pharmacokinetics were found. The mean plasma levels of (+)-CLA were higher at almost all time points than those of (,)-CLA. (+)-CLA also exhibited greater tmax, Cmax, t1/2,, AUC0,12h, and AUC0,, and smaller CL (or CL/F) and Vd (or Vd/F), than its antipode. The (+)/(,) isomer ratios for t1/2,, tmax, AUC0,12 h, and AUC0,,, which ranged from 1.26 to 2.08. The ratio for CL (or CL/F) was about 0.5, and there were significant differences in these values between CLA enantiomers (P < 0.05), implying that the absorption, distribution, and elimination of (,)-CLA were more rapid than those of (+)-CLA. Similar findings for (,)-7-OH-CLA, the major metabolite of (,)-CLA, and (+)-4-OH-CLA, the major metabolite of (+)-CLA, can be also seen in rat plasma. The contributing factors for the differences in stereoselective pharmacokinetics of CLA enantiomers appeared to be involved in their different plasma protein binding, first-pass metabolism and interaction with CYP enzymes, especially with their metabolizing enzyme CYP 3A isoforms. Chirality 15:668,673, 2003. © 2003 Wiley-Liss, Inc. [source] Lipid-transfer proteins as potential plant panallergens: cross-reactivity among proteins of Artemisia pollen, Castanea nut and Rosaceae fruits, with different IgE-binding capacitiesCLINICAL & EXPERIMENTAL ALLERGY, Issue 10 2000A. DÍaz-Perales Background Lipid-transfer proteins (LTPs), but not Bet v 1 homologues, have been identified as major allergens of apple and peach in the Rosaceae fruit-allergic population in the Mediterranean area. Many of these patients show cosensitization to mugwort pollen. LTPs have an ubiquitous distribution in tissues of many plant species, and have been proposed as a novel type of plant panallergens. Objective We sought to isolate LTPs from Artemisia pollen and from a plant food not belonging to the Rosaceae family, such as chestnut nut, and to compare their amino acid sequences and IgE-binding capacities with those of apple and peach LTPs. Methods Allergens (LTPs) were isolated by different chromatographic methods (gel-filtration, ion exchange and/or reverse-phase HPLC), and characterized by N-terminal amino acid sequencing and MALDI analysis. Specific IgE-quantification and immunodetection, as well as immunoblot and ELISA inhibition assays, were carried out using sera from patients allergic to both apple and peach. Results Purified LTPs from Artemisia pollen and from chestnut seed showed molecular masses about 9 700d, and 43,50% sequence identity with the equivalent allergens of apple and peach in the first 30 N-terminal residues, which comprise about one third of the total amino acid sequence. A similar degree of sequence identity (50%) was found between the Artemisia and chestnut proteins. Both isolated LTPs bound specific IgE of sera from Rosaceae fruits allergic patients. However, substantially lower values of specific IgE-binding and maximum ELISA inhibition percentages were obtained for Artemisia and chestnut LTPs when compared to those from apple and peach. Conclusion LTPs from Artemisia pollen and chestnut crossreact with allergens (LTPs) of Rosaceae fruits, but significant differences in specific IgE-binding capacities were observed among members of the plant LTP family. Thus, further studies are needed to evaluate the clinical significance of the observed cross-reactivities of plant LTPs. [source] | ||||||||||||||||