bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two‐dimensional gel electrophoresis of Caenorhabditis elegans homogenates and identification of protein spots by microsequencing
Authors: Bini Luca , Heid Hans , Liberatori Sabrina , Geier Gebhard , Pallini Vitaliano , Zwilling Robert 
Published: May 2005

AbstractEmploying isoelectric focusing on immobilized pH gradients followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) we have obtained a map of C. elegans proteins, from a mixed culture containing all developmental stages, presenting over 2000 spots within the window of isoelectric points (pI) 3.5–9 and a molecular mass of 10–200 kDa. Edman microsequencing yielded successful results in 12 out of 24 analyzed spots. All but one of the N‐terminal sequences retrieved C. elegans sequences in cosmid and/or expressed sequence tag clones. Structurally related protein sequences found in data banks included enzymes in energy metabolism (cytochrome oxydase, ATP synthase, enolase), a fatty acid‐binding protein, a translationally controlled tumor protein, an unknown C. elegans protein, an acidic ribosomal protein, a titin‐like protein, a G‐protein β chain, cyclophilin, and cathepsin D. Experimental determination of N‐termini allowed us to define sites of signal cleavage providing further information on the physiological role of the newly found C. elegans proteins. This report demonstrates the possibility of two‐dimensional gel electrophoresis and Edman microsequencing in the elucidation of C. elegans proteome.
Analysis of polypeptide expression in benign and malignant human breast lesions
Authors: Franzén Bo , Linder Stig , Alaiya Ayodele A. , Eriksson Elina , Fujioka Kaoru , Bergman Ann‐Charlotte , Jörnvall Hans , Auer Gert 
Published: May 2005

AbstractResults of two‐dimensional electrophoresis (2‐DE) analyses of human breast carcinoma are described. Tumor cells were extracted and purified from breast carcinomas with different proliferative indeces and degrees of genomic stability. Cells purified from fibroadenoma tissue served as controls for benign cells. The following results were observed: (i) Analysis of samples from different areas of the same tumor showed a high degree of similarity in the pattern of polypeptide expression. Similarly, analysis of two tumors and their metastases revealed similar 2‐DE profiles. (ii) In contrast, large variations were observed between different lesions with comparable histological characteristics. Larger differences in polypeptide expression were observed between potentially highly malignant carcinomas compared to comparisons of less malignant lesions. These differences were in the same order of magnitude as those observed comparing a breast carcinoma to a lung carcinoma. (iii) The levels of all cytokeratin forms resolved (CK7, CK8, CK15, and CK18) were significantly lower in carcinomas compared to fibroadenomas. (iv) The levels of high molecular weight tropomyosins (1–3) were lower in carcinomas compared to fibroadenomas. The expression of tropomyosin‐1 was found to be 1.7‐fold higher in primary tumors with metastatic spread to axillar lymph nodes compared to primary tumors with no evidence of metastasis (p < 0.05). (v) The expression of proliferating cell nuclear antigen (PCNA) and some members of the stress protein family (pHSP60, HSP90, and calreticulin) were higher in carcinomas. We conclude that malignant progression of breast carcinomas results in large heterogeneity in polypeptide expression between different tumors, but that some common themes such as decreased expression of cytokeratin and tropomyosin polypeptides can be discerned.
Simultaneous analysis of cyclin and oncogene expression using multiple monoclonal antibody immunoblots
Authors: Sanchez Jean‐Charles , Wirth Peter , Jaccoud Sylviane , Appel Ron D. , Sarto Cecilia , Wilkins Marc R. , Hochstrasser Denis F. 
Published: May 2005

AbstractCell dysfunction or dysregulation in cancer generally results from complex gene interactions, numerous cellular events and environmental influences which modify gene expression or post‐translational protein modifications. Genetic analysis in itself cannot always predict or diagnose multigenic diseases. The major technical difficulty is thus to detect, identify and measure simultaneously the expression of several genes and the post‐translational modifications of their products. In order to progress to this direction, this paper describes a simple immunoblot method using several monoclonal antibodies to simultaneously analyze oncogene expression and cell cycle specific checkpoints in patient solid biopsies and transformed cell lines. One mg of normal human liver biopsy and HEPG2 (hepatoblastoma‐derived cell line) protein samples have been separated by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were stained with amido black, scanned and tested separately with the nine monoclonal antibodies p53, c‐myc, PCNA, MEK1, pan‐ras, Cip1, Cdc2, Kip1, and TCTP. The nine antibodies of interest were then combined to form a mixture, and simultaneously used as the primary antibodies. This antibody mixture simultaneously detected the nine proteins of interest or both samples and it demonstrated the extensive expression changes and the presence of various isoforms most likely due to post‐translational modifications of gene products.
A protein expression database for the molecular pharmacology of cancer
Authors: Myers Timothy G. , Anderson N. Leigh , Waltham Mark , Li Guang , Buolamwini John K. , Scudiero Dominic A. , Paull Kenneth D. , Sausville Edward A. , Weinstein John N. 
Published: May 2005

AbstractIn the last six years, the Developmental Therapeutics Program (DTP) of the US National Cancer Institute (NCI) has screened over 60 000 chemical compounds and a larger number of natural product extracts for their ability to inhibit growth of 60 different cancer cell lines representing different organs of origin. Whereas inhibition of the growth of one cancer cell type gives no information on drug specificity, the relative growth inhibitory activities against 60 different cells constitute patterns that encode detailed information on mechanisms of action and resistance (as reviewed in Boyd and Paull, Drug Devel. Res. 1995, 34, 19–109 and Weinstein et al., Science 1997, 275, 343–349). In order to correlate the patterns of activity with properties of the cells, we and other laboratories are characterizing the cells with respect to a large number of factors at the DNA, mRNA, and protein levels. As part of that effort, we have developed a two‐dimensional gel electrophoresis (2‐DE) protein expression database covering all 60 cell types (Buolamwini et al., submitted). Here we present analyses of the correlations among protein spots (i) in terms of their patterns of expression and (ii) in terms of their apparent relationships to the pharmacology of a set of 3989 screened compounds. The correlations tend to be stronger for the latter than for the former, suggesting that the spots have more robust signatures in terms of the pharmacology than in terms of expression levels. Links to pertinent databases and tools of analysis will be updated progressively at http://www.nci.nih.gov/intra/lmp/jnwbio.htm and http://epnwsl.ncifcrf.gov:2345/dis3d/dtp.html.
Construction of a directory of tobacco plasma membrane proteins by combined two‐dimensional gel electrophoresis and protein sequencing
Authors: Rouquié David , Peltier Jean‐Benoǐt , Marquis‐Mansion Monique , Tournaire Colette , Doumas Patrick , Rossignol Michel 
Published: May 2005

AbstractThe polypeptide pattern of the plasma membrane from tobacco was studied by two‐dimensional gel electrophoresis. When using classical carrier ampholyte isoelectric focusing/sodium dodecyl sulfate – polyacrylamide gel electrophoresis (IEF/SDS—PAGE) approximately 400 polypeptide spots were detected after silver staining and computer analysis using the QUEST software. This resolution was sufficient to assess physiological effects such as changes in a phytohormone concentration. By using pH 4–8 immobilized pH gradient (IPG)‐IEF and 10%T SDS‐PAGE gels, approximately 600 polypeptides, corresponding to ca. 80% of the total population expected, were resolved. This cross‐section of the plasma membrane polypeptide population was mainly constituted by low or intermediate molecular mass (25 to 45 kDa) and acidic (5.2 < pI < 6.1) polypeptides. After sample application by in‐gel rehydration, large amounts of plasma membrane protein (between 5 mg and 10 mg protein) were analyzed using IPG‐IEF, and N‐terminal protein sequencing was performed for polypeptides collected from one gel. Internal protein sequences were also obtained. Nearly all protein sequences corresponded to unidentified proteins but several of them matched translated sequences from unidentified plant expressed sequence tags (ESTs). It is concluded that the combined use of IPG‐IEF gels and in‐gel rehydration allows, in the case of plant membrane protein, both analytical and micropreparative separations with an efficiency comparable to that demonstrated for soluble proteins. Finally, it is suggested that a systematic investigation of plant plasma membrane polypeptides is feasible and would constitute a source of new and plant‐specific genes.
Mutational analysis of the genes encoding urokinase‐type plasminogen activator (uPA) and its inhibitor PAI‐1 in advanced ovarian cancer
Authors: Türkmen Beyhan , Schmitt Manfred , Schmalfeldt Barbara , Trommler Paul , Hell Wolfgang , Creutzburg Sabine , Graeff Henner , Magdolen Viktor 
Published: May 2005

AbstractEvidence has accumulated that urokinase‐type plasminogen activator (uPA), its inhibitor (PAI‐1) and receptor (uPAR) are involved in tumor invasion and metastasis. We analyzed the DNA sequences encoding these factors to see if they are altered in the ovarian cancer cell lines OV‐MZ‐6, OV‐MZ‐19, and OVCAR‐3. In the uPA‐encoding cDNA derived from OV‐MZ‐6 cells (but not in the uPA‐cDNA from OVCAR‐3 and OV‐MZ‐19), a so‐far unknown mutation was identified in codon 121, resulting in a proline to leucine exchange. This exchange creates an AluI restriction site making restriction fragment length polymorphism (RFLP) analyses possible. Previously published PAI‐1 sequences pointed to a variation of amino acid 15 of the PAI‐1 signal sequence representing either threonine or alanine, which was confirmed in the present study. The uPAR cDNAs of all three cell lines encoded the published wild‐type sequence. In order to elucidate the possible role of the Pro121Leu exchange in uPA and the Ala/Thr variants in the signal sequence of PAI‐1 in the development and/or progression of human ovarian cancer, we studied the presence of these mutants or variants in a series of 22 ovarian cancer tissues. In addition to the wild‐type sequence, the Pro121Leu exchange in the uPA sequence was detected in 10 out of 22 tumor tissues; 11 tumors carried exclusively the Pro121 allele; in one case exclusively the Leu121 allele was detected. In 18/22 tumors, triplet 15 in the signal sequence of PAI‐1 encoded alanine, four DNAs contained both the Ala and the Thr allele. Furthermore, we analyzed another known common single‐base‐pair insertion/deletion polymorphism (ins/del allele) found in the promoter region of the PAI‐1 gene and thought to be of functional importance in regulating PAI‐1 gene expression. The PAI‐1 ins‐allele was found in 3/22, the del‐allele in 6/22 and both alleles in 13/22 ovarian cancer tissues. In genomic DNA isolated from peripheral blood of 23 healthy donors, we observed similar allele frequencies of the three polymorphisms as found in the 22 ovarian carcinomas. Taken together, these results suggest that the polymorphisms observed in the uPA and PAI‐1 genes may not be linked to ovarian cancerPresented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996.
.
Combined immunostaining and Coomassie Brilliant Blue staining of polyvinylidene difluoride membranes without organic solvent
Authors: Houen Gunnar , Bruun Leif , Barkholt Vibeke 
Published: May 2005

AbstractA method for staining proteins on polyvinylidene difluoride membranes without using organic solvent is described. The method uses preblocking of the membrane with either Tween 20 or polyethylene glycol followed by staining with 0.01% Coomassie Brilliant Blue. No destaining of the membrane is needed afterwards. Preblocking with polyethylene glycol is compatible with microsequencing while Tween 20 leads to very low initial yields. Preblocking with Tween 20 has the additional advantage of allowing immunostaining followed by Coomassie Brilliant Blue staining for total protein on the same membrane.
Temperature‐programmed capillary electrophoresis for the analysis of high‐melting point mutants in thalassemias
Authors: Gelfi Cecilia , Righetti Pier Giorgio , Travi Maurizio , Fattore Silvia 
Published: May 2005

AbstractThe behavior of different sieving polymers for unambiguous determination of point mutations in genomic DNA, based on electrophoresis in thin capillaries, is evaluated. High melters from thalassemia patients are separated by exploiting the principle of denaturing gradient gel electrophoresis, in fact, of its variant utilizing temperature gradients (TGGE), along the migration path, encompassing the melting points of both homo‐ and heteroduplex, polymerase chain reaction (PCR)‐amplified DNA fragments. Unlike TGGE, where the temperature gradient exists along the separation space, the denaturing temperature gradient in the fused‐silica capillaries is time‐programmed, so as to reach the Tm's of all species under analysis prior to electrophoretic transport past the detector window. The DNA fragments are injected in a capillary maintained (by combined chemical and thermal means) just below the expected Tm values. The ΔT applied is rather minute (1–1.5°C) and the temperature gradient quite shallow (e.g., 0.05°C/min). The denaturing thermal gradient is generated internally, via Joule heat produced by voltage ramps. This method is applied to the analysis of the most common point mutations in thalassemias, characterized by being high melters (in the temperature range of 60–62°C) in presence of 6 M urea. Point mutants are fully resolved into a spectrum of four bands only when poly(N‐acryloylaminopropanol) and hydroxyethylcellulose are used. However, the former offers the best separation capability at such high temperatures.
Effect of zwitterionic surfactants on the separation of proteins by capillary electrophoresis
Authors: Gong Bu Yi , Ho John W. 
Published: May 2005

AbstractThe effect of zwitterionic surfactants on capillary electrophoretic separation of proteins was investigated using an uncoated silica capillary column and different buffers containing zwitterionic surfactants. The effects of N‐alkyl‐N,N‐dimethyl ammonio‐1‐propane‐sulfonates on the electrophoretic mobility of three different proteins, namely albumin, lysozyme and myoglobin, were examined. The addition of N‐alkyl‐N,N‐dimethylammonio‐1‐propane sulfonate in phosphate saline buffer is important in minimizing protein‐capillary wall interactions, and facilitated an efficient electrophoretic mobility of myoglobin and lysozyme. The separation efficiency of proteins also depends on the injection pressure for the control of migration time and the peak sharpness. The electrophoretic conditions were applied to evaluate the separation of lysozyme, myoglobin and albumin. The zwitterion surfactants can form a dynamic coating on the capillary surface, thereby reducing the number of adsorption sites to which proteins may adsorb.
Identification and characterization of wheat grain albumin/globulin allergens
Authors: Weiss Walter , Huber Gerd , Engel Karl‐Heinz , Pethran Angelika , Dunn Michael J. , Gooley Andrew A. , Görg Angelika 
Published: May 2005

AbstractBakers' asthma, an immediate‐type allergic response to the inhalation of cereal flours, is an important occupational disease among workers of the baking and milling industries, and the salt‐soluble proteins of wheat and rye flour dust are considered the most relevant allergens. In order to identify and characterize the major IgE‐binding proteins, the polypeptide composition of the albumin/globulin protein fraction obtained from different cultivars was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and high‐resolution two‐dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG‐Dalt), followed by immunoblotting with sera from asthmatic bakers. Relevant allergens were isolated by micropreparative IPG‐Dalt and blotting onto polyvinylidenedifluoride membranes and identified by amino acid composition analysis or N‐terminal amino acid sequence analysis. SDS‐PAGE, IPG‐Dalt, and immunoblotting demonstrated that the sera of the bakers allergic to flour contained IgE antibodies which bound to numerous albumin/globulin polypeptides in the 70, 55, 35, 26–28, and 14–18 kDa areas. More detailed investigations using IPG‐Dalt revealed cultivar‐specific differences in IgE‐binding. It was also demonstrated that the majority of the allergens were not single polypeptide spots, but consisted of up to ten isoforms of similar molecular mass but different isoelectric points. Amino acid composition analysis and N‐terminal amino acid sequence analysis, which were performed for nine allergens located in the 14–18, 26–28, and 35 kDa areas, revealed homologies to amylase/protease inhibitors, acyl‐CoA oxidase and fructose‐bisphosphate‐aldolase from wheat, barley, maize, and rice, respectivelyPresented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996
.
Comparison of alkylglycoside surfactants in enantioseparation by capillary electrophoresis
Authors: Mechref Yehia , El Rassi Ziad 
Published: May 2005

AbstractThree alkylglycoside surfactants, namely n‐octyl‐β‐D‐glucopyranoside (OG), n‐nonyl‐β‐D‐glucopyranoside (NG), and n‐octyl‐β‐D‐maltopyranoside (OM), were compared in the enantiomeric separation of dansyl amino acids, binaphthyl phosphate, bupivacaine and warfarin. While only OM exhibited an enantioselectivity toward warfarin, bupivacaine, and dansyl tryptophan, all three surfactants were effective in the enantiomeric resolution of napthyl phosphate and other dansyl amino acids. With the exception of naphthyl phosphate, which could be resolved enantiomerically with OM at surfactant concentrations below the CMC, all solutes required surfactant concentrations greater than the CMC value. This was attributed to the strong hydrophobic association of napthyl phosphate with the OM monomers and to the presence of maltoside residue in the OM surfactant. In general, the optimum surfactant concentration needed for maximum enantiomeric resolution was an inverse function of the hydrophobic character of the solute. Under a given set of conditions, the enantiomeric resolution exhibited by the alkylglycoside surfactants was largely influenced by the extent and loci of solute solubilization into the micelle, and by the nature of the chiral sugar head group of the surfactant.
Central composite design in the chiral analysis of amphetamines by capillary electrophoresis
Authors: Varesio Emmanuel , Gauvrit Jean‐Yves , Longeray Rémi , Lantéri Pierre , Veuthey Jean‐Luc 
Published: May 2005

AbstractWe have studied a central composite design for the chiral separation of amphetamines using capillary electrophoresis. Five variables,i.e., buffer concentration, pH, chiral selector concentration, temperature, and applied voltage, were investigated. Enantiomeric resolutions as well as analysis time and generated power were established as responses for each experiment. A model of each response was obtained by multiple regression of a quadratic‐degree mathematical expression. From the models, we determined the most favorable conditions for the chiral separation by optimizing the resolution and setting the other responses at threshold values. Results were compared with a previous study in which a systematic investigation of the operating parameters was carried out. In order to visualize the robustness of the method, response surfaces were drawn for the significant variables. We have concluded that experimental designs offer a rapid means of optimizing several variables and provide an efficient test for the robustness of the analytical method.
Enantioresolution of disopyramide by capillary affinity electrokinetic chromatography with human α1‐acid glycoprotein (AGP) as chiral selector applying a partial filling technique
Authors: Amini Ahmad , Pettersson Curt , Westerlund Douglas 
Published: May 2005

AbstractA method using α1‐acid glycoprotein (AGP) as chiral selector for disopyramide by means of affinity electrokinetic chromatography has been developed. In order to avoid UV absorbance interferences, less than the effective length of the capillary was filled with the chiral selector. The electrophoretic conditions were chosen to give opposite migration directions for the chiral selector and the analyte; AGP migrated away from the detector. Enantiomers of disopyramide were separated on a methylcellulose‐coated capillary with 20 cm length to the detector. The enantioresolution of the solute was affected by the concentration of the chiral selector, the plug length of the selector in the capillary, and the applied voltage. Resolution factors and migration times decreased with reduction of the plug length, while the efficiency of the separation system and peak performance were improved by decreasing the separation zone. A special feature of the technique is an enhanced selectivity due to increasing separation of the enantiomers when the fastest has migrated from the selector zone, while the second one still is retained. Equations relating selectivity and resolution with the difference in effective plug lengths between the two enantiomers are developed. Optimized conditions yielding complete resolution, requiring an 0.75 mM AGP plug of only 4.5 cm effective length, also gave high efficiencies (about 400 000 plates/m) for both enantiomer peaks.
Enantioseparation of local anaesthetic drugs by capillary zone electrophoresis with cyclodextrins as chiral selectors using a partial filling technique
Authors: Amini Ahmad , Paulsen‐Sörman Ulla 
Published: May 2005

AbstractThe enantiomers of prilocaine were successfully resolved with α‐cyclodextrin, and those of mepivacaine and bupivacaine, with methyl‐β‐cyclodextrin as chiral selectors, by means of capillary zone electrophoresis (CZE) employing a partial filling technique. By this separation mode, a discontinous separation zone is formed in the capillary. Prior to application of the actual drug substance, the capillary is partially filled with the separation solution. During the enantioseparation both ends of the capillary are dipped into the running buffer solution, i.e., without chiral selector. The consumption of chiral selector is thus very low, less than a microliter per run. The repeatibility of the electrophoretic mobility of the enantiomers was better than 1.2% relative standard deviation (RSD). The effect of the length of the separation zone on the resolution of the enantiomers was studied. The application time of the chiral selector, instead of the selector concentration, was varied in order to improve and regulate the enantioresolution and reduce consumption of the chiral selector as much as possible. It was found that the enantioseparations were directly affected by the length of the separation zone, and there was a minimal plug length where complete enantioresolution was achieved.