bluuebunny/crossref_metadata_2025 huggingface dataset:
Construction of HSC‐2DPAGE: A two‐dimensional gel electrophoresis database of heart proteins
Authors: Evans Guy , Wheele Colin H. , Corbett Joseph M. , Dunn Michael J.
Published: May 2005

The dissemination of information relating to the characterisation of proteins from two‐dimensional electrophoresis (2‐DE) gel databases is essential for their effective utilisation in the study of protein expression in cell biology. Since the inception of the World Wide Web and the pioneering development of SWISS‐2DPAGE as a tool for retrieving information on proteins separated by 2‐DE, the Internet has become the method of choice for disseminating and accessing information on 2‐DE protein databases. At Harefield we have established HSC‐2DPAGE which is an advanced interface for accessing protein databases relating to heart disease. The Web site currently includes databases of proteins from human, dog and rat ventricular tissue and a human endothelial cell line. The databases are searchable individually or as a whole by remote keyword searches. Each database is represented by both synthetic (computer generated) and real (scanned gel) clickable images upon which characterised protein spots are highlighted by hyperlinked symbols. The database conforms to all the rules proposed for federated 2‐DE protein databases and individual protein entries are linked to other protein databases such as SWISS‐PROT by active cross‐references. This paper describes the construction of HSC‐2DPAGE, its maintenance, and access via the Internet.
Acute‐phase proteins in perinatal human plasma
Authors: Liberatori Sabrina , Bini Luca , De Felice Claudio , Magi Barbara , Marzocchi Barbara , Raggiaschi Roberto , Pallini Vitaliano , Bracci Rodolfo
Published: May 2005

Plasma from eight newborns (4 pre‐term and 4 full‐term) with early‐onset (<72 h) sepsis and six apparently healthy controls was analyzed. The presence of spots identified as haptoglobin and serum amyloid A protein was the electrophoretic result most consistently associated with disease. Time course monitoring showed rises, peaks and declines of spot intensity as expected for acute‐phase proteins induced by transient stimuli. Haptoglobin β chains appear to be undersialated in pre‐term newborns, whereas post‐translational modifications of α chains and serum amyloid A protein are similar to those observed in adults. The undersialation of β chain and occurrence of α chain phenotypes different from those found in maternal serum indicate that perinatal haptoglobin originates from neonatal synthesis.
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

Employing 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

Results 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

Cell 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

In 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

The 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

Evidence 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 cancer

Presented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996.

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Combined immunostaining and Coomassie Brilliant Blue staining of polyvinylidene difluoride membranes without organic solvent
Authors: Houen Gunnar , Bruun Leif , Barkholt Vibeke
Published: May 2005

A 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

The 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

The 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

Bakers' 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, respectively

Presented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996

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Comparison of alkylglycoside surfactants in enantioseparation by capillary electrophoresis
Authors: Mechref Yehia , El Rassi Ziad
Published: May 2005

Three 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

We 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

A 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

The 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.
The serine proteinase inhibitory proteins of the chondrodystrophoid (beagle) and non‐chondrodystrophoid (greyhound) canine intervertebral disc
Authors: Melrose James , Taylor Thomas K. F. , Ghosh Peter
Published: May 2005

Trypsin inhibitory proteins of low buoyant density (ρ ≤ 1.35 g/mL) fractions were prepared by CsCl density gradient ultracentrifugation of 4 M guanidinium hydrochloride extracts of lumbar beagle and greyhound annulus fibrosus and nucleus pulposus from animals aged 1 to 6 years. Affinity blotting with biotinylated trypsin was used to identify active trypsin inhibitory protein species; these species were also identified immunologically by Western blotting using antibodies against bovine pancreatic trypsin inhibitor (BPTI), and human inter‐α‐trypsin inhibitor (ITI). None of the trypsin inhibitory species evident in Western blots were reactive with anti‐human α1‐proteinase inhibitor (α‐1‐PI), α2‐macroglobulin or secretory leucocyte proteinase inhibitor. The greyhound intervertebral disc samples generally had higher levels of active trypsin inhibitor species per unit weight of tissue extracted, and a more extensive range of inhibitor species. Inhibitor species of 30, 32, 34 kDa were identified in both beagle and greyhound intervertebral disc samples; these species were generally most prominent in the annulus fibrosus samples. In contrast, the nucleus pulposus samples contained relatively large trypsin inhibitor species; the anti‐BPTI detected an inhibitor species of ∼ 85–90 kDa; anti‐ITI detected species of 120–250 kDa; biotinylated trypsin detected species of 60–110 kDa. A small molecular mass trypsin inhibitor species of 6 kDa, which was of similar mobility to BPTI, was also detected in annulus fibrosus samples; however, this species did not react with anti‐BPTI.
Electrophoretic characterisation of fractions collected from gluten protein extracts subjected to size‐exclusion high‐performance liquid chromatography
Authors: Larroque Oscar R. , Gianibelli Maria C. , Batey Ian L. , Macritchie Fin
Published: May 2005

The electrophoretic analysis by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE; reduced and unreduced) of fractions, collected from a size exclusion‐high performance liquid chromatography (SE‐HPLC) separation of gluten proteins using a column with pore size of around 400Å, showed clear resolution for the seven elution ranges studied in two Australian bread wheat lines. Polymeric proteins — high molecular weight (HMW) glutenin subunits, low molecular weight (LMW) glutenin subunits, HMW albumins and some modified ω‐gliadins — appeared exclusively in the region within the first peak of the chromatogram (fractions 1 to 5), the limit being a region that resolved as a small peak before the large peak of gliadins and where some ω‐gliadins eluted. A larger proportion of HMW glutenin subunits and B subunits contributed to polymer formation of higher molecular weight. The polymer size decreased as the proportion of the other protein components increased.
Two‐dimensional gel electrophoresis of actin‐binding proteins isolated by affinity chromatography from human skeletal muscle
Authors: Coumans Joëlle V. F. , Humphery‐Smith Ian , dos Remedios Cristobal G.
Published: May 2005

In muscle cells actin exists as a mixture of monomeric (G‐actin) and filamentous actin (F‐actin) and ionic conditions strongly favor the formation of F‐actin. The existence of unpolymerized actin depends, among other factors, on proteins that bind to G‐actin, the so‐called G‐actin‐binding proteins (G‐ABPs). We have coupled monomeric actin to divinylsulphone‐activated agarose (Mini‐Leak) to isolate G‐ABPs in human skeletal muscle. Eluted proteins were analyzed by two‐dimensional gel electrophoresis (2‐DE), which shows that some proteins are selectively retained. Deoxyribonuclease I (DNase I) is known to bind residues at the “pointed end” of actin (subdomains 2 and 4) with a high affinity. When DNase I is bound to the actin Mini‐Leak before applying the skeletal muscle extract, the 2‐DE gels of the eluted proteins reveals differences when compared to gels of proteins eluted from actin‐Mini‐Leak and DNase I‐Mini‐Leak affinity columns. This strategy should detect ABPs which bind to sites other than the DNase I‐binding site and some may prove to be novel.
Applications of consensus polymerase chain reaction with subsequent electrophoretic distinction of amplificates
Authors: Krause Günter , Oduncu Fuat , Kaltstein Anja , Pusl Thomas , Pachmann Katharina , Gerzer Rupert
Published: May 2005

Conserved sequences within gene families permit the design of consensus primers that match several members of a given class of homologous genes. Polymerase chain reaction (PCR) products obtained with such consensus primers were characterized by restriction mapping or single‐strand conformation polymorphism (SSCP) analysis, using precast polyacrylamide minigels and automated silver staining. Examples for the electrophoretic distinction of consensus amplificates are presented in the fields of guanylyl cyclase expression studies and in the determination of B‐cell clonality in human blood samples. Guanylyl cyclase expression in inner ear tissues of guinea pigs was investigated by reverse transcription PCR using consensus primers with specificity for the subclass of particulate guanylyl cyclases. The resulting PCR products were assigned to three representatives of this group by restriction mapping. The consensus PCR approach enabled the detection of an unexpected receptor type, namely guanylyl cyclase C, in the inner ear. The distinction by SSCP analysis of denatured consensus amplificates was appropriate for the identification of clone‐specifically rearranged immunoglobulin heavy chain genes of B‐lymphocytes. Genomic DNA isolated from blood samples of leukemia patients served as the template for the consensus amplification of clone‐specific VDJ rearrangements. Rapid distinction and re‐identification of consensus PCR products was achieved by SSCP analysis for regular antigen receptor rearrangements and for t(14; 18) translocations. The potential of these procedures for detecting leukemia or lymphoma clones when monitoring minimal residual disease was assessed.

Presented at the “Elektrophorese Forum '96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996.
Multistructure sequencing of N‐linked fetuin glycans by capillary gel electrophoresis and enzyme matrix digestion
Authors: Guttman András
Published: May 2005

Oligosaccharide sequencing by specific enzymatic digestion of N‐linked fetuin glycans using a carefully designed exoglycosidase matrix in conjunction with separation of the combined digests by high performance capillary gel electrophoresis is described. Due to the extremely high separation power and excellent reproducibility of capillary gel electrophoresis, in most instances no isolation of the individual oligosaccharides is necessary, enabling multistructure sequencing from a released glycan pool. By comparing the positions of the separated exoglycosidase digest fragments to maltooligosaccharides of known size, relative migration times and migration shifts are easily calculated. Hence, the particular sequence of each oligosaccharide in a released glycan pool can be proposed with high confidence. Additionally, with the use of high sensitivity laser‐induced fluorescence detection, complete sequence information can be attained from picomolar amounts of purified glycoproteins.
Separation of human plasma/serum proteins by capillary isoelectric focusing in the absence of denaturing agents
Authors: Manabe Takashi , Iwasaki Aiko , Miyamoto Hiromitsu
Published: May 2005

Human plasma/serum proteins were separated by capillary isoelectric focusing in the absence of denaturing agents. Proteins focused in a fused silica capillary were mobilized by replacing the catholyte sodium hydroxide to acetic acid. The performance of the separation of human plasma proteins has been examined by changing the duration of the step of isoelectric focusing, carrier ampholyte concentration, and plasma protein concentration. The separation patterns of plasma proteins were compared with those obtained by micro two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) run in the absence of denaturants in order to locate the major plasma proteins on the capillary electropherograms. Using the established electrophoretic conditions and the results of peak identification, proteins in the sera of IgG myeloma patients were analyzed by capillary isoelectric focusing. The advantages of capillary isoelectric focusing in plasma protein analysis compared with 2‐D PAGE are discussed.
Proteome analysis of Spiroplasma melliferum (A56) and protein characterisation across species boundaries
Authors: Cordwell Stuart J. , Basseal David J. , Humphery‐Smith Ian
Published: May 2005

Spiroplasma melliferum (Class: Mollicutes) is a wall‐less, helical bacterium with a genome of approximately 1460 kbp encoding 800–1000 gene‐products. A two‐dimensional electrophoresis gel reference map of S. melliferum was produced by Phoretix 2‐D gel software analysis of eight high quality gels. The reference map showed 456 silver‐stained and replicated protein spots. 156 proteins (34% of visible protein spots) from S. melliferum were further characterised by one, or a combination, of the following: amino acid analysis, peptide‐mass fingerprinting via matrix assisted laser desorption ionisation‐time of light (MALDI‐TOF) mass spectrometry, and N‐terminal protein microsequencing. Proteins with close relationship to those previously determined from other species were identified across species barriers. Thus, this study represents the first larger‐scale analysis of a proteome based upon the attribution of predominantly ‘unique numerical parameters’ for protein characterisation across species boundaries, as opposed to a sequence‐based approach. This approach allowed all database entries to be screened for homology, as is currently the case for studies based on nucleic acid or protein sequence information. Several proteins studied from this organism were identified as hypothetical, or having no close homolog already present in the databases. Geneproducts from major families such as glycolysis, translation, transcription, cellular processes, energy metabolism and protein synthesis were identified. Several gene‐products characterised in S. melliferum were not previously found in studies of the entire Mycoplasma genitalium and Mycoplasma pneumoniae (both closely related Mollicutes) genomes. The presence of such geneproducts in S. melliferum is discussed in terms of genome size as compared with the smallest known free‐living organisms. Finally, the levels of expression of S. melliferum gene‐products were determined with respect to total optical itensity associated with all visible proteins expressed in exponentially grown cells.
Proteome studies of Saccharomyces cerevisiae: Identification and characterization of abundant proteins
Authors: Garrels James I. , McLaughlin Calvin S. , Warner Jonathan R. , Futcher Bruce , Latter Gerald I. , Kobayashi Ryuji , Schwender Brian , Volpe Tom , Anderson Damon S. , Mesquita‐Fuentes Rodrigo , Payne William E.
Published: May 2005

Two‐dimensional (2‐D) gel electrophoresis can now be coupled with protein identification techniques and genome sequence information for direct detection, identification, and characterization of large numbers of proteins from microbial organisms. 2‐D electrophoresis, and new protein identification techniques such as amino acid composition, are proteome research techniques in that they allow direct characterization of many proteins at the same time. Another new tool important for yeast proteome research is the Yeast Protein Database (YPD), which provides the sequence‐derived protein properties needed for spot identification and tabulations of the currently known properties of the yeast proteins. Studies presented here extend the yeast 2‐D protein map to 169 identified spots based upon the recent completion of the yeast genome sequence, and they show that methods of spot identification based on predicted isoelectric point, predicted molecular mass, and determination of partial amino acid composition from radiolabeled gels are powerful enough for the identification of at least 80% of the spots representing abundant proteins. Comparison of proteins predicted by YPD to be detectable on 2‐D gels based on calculated molecular mass, isoelectric point and codon bias (a predictor of abundance) with proteins identified in this study suggests that many glycoproteins and integral membrane proteins are missing from the 2‐D gel patterns. Using the 2‐D gel map and the information available in YDP, 2‐D gel experiments were analyzed to characterize the yeast proteins associated with: (i) an environmental change (heat shock), (ii) a temperature‐sensitive mutation (the prp2 mRNA splicing mutant), (iii) a mutation affecting post‐translational modification (N‐terminal acetylation), and (iv) a purified subcellular fraction (the ribosomal proteins). The methods used here should allow future extension of these studies to many more proteins of the yeast proteome.
Osmoresponsive proteins and functional assessment strategies in Saccharomyces cerevisiae
Authors: Blomberg Anders
Published: May 2005

Cells respond to increased external osmolarities by enhanced accumulation of compatible solutes. In yeast‐cells, mainly exemplified by Saccharomyces cerevisiae, the premier compatible solute is the polyhydroxy‐alcohol glycerol, the production of which is accompanied by overall metabolic changes. By applying two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) coupled to computerized image quantification, a large body of valuable physiological information relating to this stress‐adaptation has been gathered. One of the presumed key‐enzymes in the production of glycerol in the cell is glycerol 3‐phosphate dehydrogenase encoded by the GPD1 gene. The amount of this protein is enhanced during saline stress, and from 2‐D analysis linked to microsequencing it became apparent that the osmo‐regulated form contained a putative presequence. Sequence analysis of another salt‐induced spot in the 2‐D pattern revealed identity to a gene, YER062c, with previously unknown function. Biochemical characterization of this protein, including standard purification via chromatography and subsequent activity/specificity measurements, identified this salt‐regulated protein as the missing protein/gene in glycerol production, namely the glycerol 3‐phosphatase. The sequence of another salt regulated protein resolved in the 2‐D gel revealed identity to a bacterial dihydroxyacetone kinase, thus indicating salt induced glycerol dissimilation. Comparing Northern data to the 2‐D generated expression pattern revealed a strong correlation, indicating mainly regulation at the transcriptional level. In addition, altered expression during saline growth of some of the glycolytic enzymes was also apparent. Signalling mutants, either in the cAMP‐dependent protein kinase A pathway or in a protein kinase cascade, have been analyzed during osmotic stress via 2‐D PAGE, grouping proteins/genes apparently regulated via similar mechanismus. Proteome analysis has proven invaluable in the unravelling of the molecular physiology of yeast cells during adaptation and growth under osmotic stress, identifying vital components not selected by purely genetic approaches.
Amplification of anonymous DNA fragments using pairs of long primers generates reproducible DNA fingerprints that are sensitive to genetic variation
Authors: Gillings Michael , Holley Marita
Published: May 2005

The reproducibility and potential applications of anonymous amplification protocols can be improved by using pairs of primers, each of 18 to 24 bases, to replace the single 8 to 10 base primers normally used in randomly amplified polymorphic DNA (RAPD) or DNA amplification fingerprinting (DAF) methods. Amplification using large primer pairs (LP‐RAPD) generates 5 to 30 bands that can be resolved on standard agarose gels. Complex fingerprints can be readily generated from viruses, bacteria, fungi, plants, invertebrates and vertebrates. We also present evidence that a number of polymerase chain reaction (PCR) methods, including those based on the use of enterobacterial repetitive intergenic consensus (ERIC‐PCR) or microsatellite primed (MP‐PCR) sequence, may in essence operate by the same mechanism as LP‐RAPD. Using standard LP‐RAPD protocols, reproducible fingerprints can be generated from a single specimen using different thermocyclers, regardless of the mechanism used for thermocycling (air‐cooled, Peltier effect, or robotic arm). LP‐RAPD is sensitive to intraspecific and interspecific genetic variation, demonstrated here by analysis of mites and apple cultivars. Approximately 50% of LP‐RAPD products are expected to have different primers at either end. Polymorphic bands with this arrangement can be recovered from the gel and directly sequenced using the LP‐RAPD primers themselves. The efficiency of sequencing is improved by the length of the LP‐RAPD primers. This method has the potential to allow the production of allele‐specific species markers in less than two days.
Ancient and modern mitochondrial DNA sequences and the colonization of the pacific
Authors: Hagelberg Erika
Published: May 2005

Mitochondrial DNA (mtDNA) is a valuable tool for the study of recent human evolution because it is easy to analyse, is inherited uniparentally and has a relatively rapid rate of evolution. mtDNA analysis has been used extensively for the elucidation of the pattern of migrations of human populations. Several studies have focused on the Pacific because Polynesia was settled by humans for the first time relatively recently and there is a wealth of archaeological and linguistic data to complement genetic data on the region. Results of mtDNA analyses on modern‐day Pacific populations indicate reduced genetic variability, and suggest that the Polynesians descend from people who migrated relatively recently from island Southeast Asia and that a population bottleneck occurred during the settlement of the central Pacific. Several informative polymorphisms have been identified in the hypervariable control region of mtDNA in modern‐day Pacific populations that are helpful in tracing the ancestral affinities of these people. Studies of these mtDNA polymorphisms in ancient bones of prehistoric Pacific islanders indicate that the proto‐Polynesian colonizers may have descended from the early settlers of island Melanesia. Although fraught with technical difficulties, studies of ancient DNA can provide valuable evidence on the genetic affinities of past peoples.
Applications of microsatellite‐based Y chromosome haplotyping
Authors: Kayser Manfred , De Knijff Peter , Dieltjes Patrick , Krawczak Michael , Nagy Marion , Zerjal Tatiana , Pandya Arpita , Tyler‐Smith Chris , Roewer Lutz
Published: May 2005

Y‐chromosomal microsatellites have been investigated for the purposes of application to male identification, population genetics and population history. With nine markers, every male in a German population sample (n = 70) could be identified by an individual‐specific Y microsatellite haplotype. The analysis of 474 unrelated males of nine human populations with seven markers revealed 301 different Y haplotypes. The analysis of molecular variance (AMOVA) approach was used to detect male population characteristics of Y microsatellite haplotypes. With pairwise comparisons of inter‐population variance, most of the populations could be distinguished significantly. Sixty individuals from different male populations in Asia and Northern Europe carrying a novel Y‐chromosomal T→C transition show reduced microsatellite variability together with haplotype similarities. Microsatellite data suggest that the mutation occurred recently in Asia, supporting the hypothesis of Asian ancestry of some northern European populations.
Capillary electrophoresis of DNA for molecular diagnostics
Authors: Righetti Pier Giorgio , Gelfi Cecilia
Published: May 2005

A number of applications of capillary zone electrophoresis (CZE) in sieving liquid polymers (notably linear polyacrylamides and cellulose) for the analysis of polymerase chain reaction (PCR) products of clinically relevant, diagnostic DNA, are reviewed. The fields covered are: human genetics, quantitative gene dosage, microbiology and virology, forensic medicine and therapeutic DNA (notably, antisense nucleotides). Some unique, novel developments are highlighted, such as: (i) nonisocratic CZE, i.e., temperature‐programmed CZE for detection of DNA point mutations; (ii) the synthesis of novel N‐substituted acrylamides, offering extreme resistance to alkaline hydrolysis coupled to high hydrophilicity. In the field of denaturing gradient gel electrophoresis (DGGE), as routinely performed in gel slabs, a novel methodology is described in CZE: double‐gradient DGGE. In this technique, two gradients are simultaneously applied along the migration direction: a chemical (or thermal) denaturing gradient, for partially unwinding homo‐ and hetero‐duplexes of DNA, and a porosity gradient, for recompacting diffuse bands melting over a broader range of denaturing conditions. It is thus demonstrated that chemical gradients, in addition to temperature gradients, can be easily implemented even in a capillary format.
Capillary electrophoresis as a clinical tool for the analysis of protein in serum and other body fluids
Authors: Oda Robert P. , Clark Raynell , Katzmann Jerry A. , Landers James P.
Published: May 2005

Most electrophoretic analyses of proteins in the clinical laboratory are currently carried out by electrophoresis in acrylamide or agarose gels. This labor‐intensive method is now being challenged by capillary electrophoresis (CE) because of its potential for automated, rapid, high efficiency separations. The automatability of CE itself, combined with its amenability to interfacing with other robotized functions, positions this technology perfectly for the analysis of proteins in physiological matrices, such as serum, urine, and cerebrospinal fluid. The focus of this overview is to familiarize the clinical scientist with the research demonstrating the applicability of the technology to the clinical protein laboratory.