bluuebunny/crossref_metadata_2025 huggingface dataset:
The identification of proliferation and tumour‐induced proteins in human endothelial cells: A possible target for tumour therapy
Authors: Clarke Mark S. F. , West David C.
Published: May 2005

The continued growth and spread of tumours is dependent on the proliferation of the endothelial cells of their vasculature. The presence of proliferation‐ or tumour‐induced surface proteins on these endothelial cells would offer a suitable epitope for monoclonal antibody therapy of tumours. Using cultured human umbilical and capillary endothelial cells, we have stimulated them with simple mitogens and tumour conditioned media and examined the proteins induced by [35S]methionine incorporation and 125I‐surface‐labelling. Two‐dimensional polyacrylamide gel electrophoresis revealed the induction of proliferation and tumour‐related antigens on the surface of the endothelial cells. Subsequent monoclonal antibody studies suggest that tumour specific surface proteins are present on most tumour endothelium.
Quantitation of human leukocyte proteins after silver staining: A study with two‐dimensional electrophoresis
Authors: Giometti Carol S. , Gemmell M. Anne , Tollaksen Sandra L. , Taylor John
Published: May 2005

The quantitative attributes of human leukocyte proteins detected by silver staining two‐dimensional electrophoresis (2‐DE) gels were studied by using computerassisted data analysis. Experiments included (a) analysis of replicate patterns of the same sample, (b) analysis of different dilutions of the same sample, and (c) analysis of samples from different individuals. Over 200 proteins were observed to have coefficients of variation (CV) less than or equal to 15 % when data from replicate patterns were analyzed. In contrast, 8 proteins had CV values of less than or equal to 15 % when data from different samples were analyzed. The dilution experiment showed that a majority of the proteins detected with some consistency (i.e., observed in at least 80 % of the patterns) have a linear relationship between the amount of protein loaded onto a 2‐DE gel and the spot volume in the final 2‐DE pattern. The slope of the curves and the deviation from linearity were found to be quite protein‐specific. These results indicate that optimization of sample purity and minimization of staining protocol variables are required to limit the background quantitative variability between and within 2‐DE runs to a level that will allow detection of quantitative changes indicative of biological responses.
Computer‐aided calculation of the molecular size of nondenatured proteins in pore‐gradient gel electrophoresis
Authors: Rothe Gunter M. , Weidmann Hans
Published: May 2005

A computer program written in Turbo C is described, which uses the two‐step mathematical procedure published recently (Rothe, G. M., Electrophoresis 1988, 9, 307–316) to evaluate the molecular mass, Stokes'radius, spherical radius, and frictional coefficient of nondenatured proteins. The program runs on any IBM‐PC or 100% compatible IBM‐PC, provided the disk operating system MS‐DOS or PC‐DOS 3.0 or later has been installed. Functions that are permanently in use are accessible by menu. Storage and loading of data from disk and help instructions can be called by use of function keys. The program provides several tables into which inserted and calculated data is automatically integrated. Each table can be printed out, provided a printer with IBM character set is connected to the computer.
The master two‐dimensional gel database of human AMA cell proteins: Towards linking protein and genome sequence and mapping information (Update 1991)
Authors: Celis Julio E. , Leffers Henrik , Rasmussen Hanne Holm , Madsen Peder , Honoré Bent , Gesser Borbala , Dejgaard Kurt , Olsen Eydfinnur , Ratz Gitte P. , Lauridsen Jette B. , Basse Bodil , Andersen Annette H. , Walbum Else , Brandstrup Birgitte , Celis Ariana , Puype Magda , Van Damme Josef , Vandekerckhove Joël
Published: May 2005

The master two‐dimensional gel database of human AMA cells currently lists 3801 cellular and secreted proteins, of which 371 cellular polypeptides (306 IEF; 65 NEPHGE) were added to the master images during the last 10 months. These include: (i) very basic and acidic proteins that do not focus under normal running conditions and (ii) low‐abundant proteins that can only be detected after prolonged gel exposure. Annotation categories updated in this version include “protein name”, “antibody against protein”, “cellular localization”, and “microsequenced proteins”. New entries include “human autoantigens” and “cDNAs”. For convenience we have included an alphabetical list of all known proteins recorded in this database. In the long run, the main goal of this database is to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various cellular functions both under physiological and abnormal conditions.
High resolution‐separation of DNA bands by electrophoresis with a long gel in a fluorescence‐detection DNA sequencer
Authors: Nishikawa Tetsuo , Kambara Hideki
Published: May 2005

A high‐resolution separation of DNA bands is achieved by electrophoresis with a long gel in DNA base sequencing using fluorescence detection. We separate 760 and 761 base DNA fragments using the 93 cm migration electrophoresis optimized for the separation of DNA bands. A T7 DNA polymerase and an Mn++ buffer are used in sequencing reactions to obtain fluorescence peaks of uniform strength, and the peak areas in the spectrum are used for recognizing the peak number in a cluster of successive peaks. This method is successfully applied to the DNA fragment spectrum obtained by 93 cm migration electrophoresis, which results in a single‐band differentiation of bands of 1040 base DNA.
Sequencing by hybridization: Towards an automated sequencing of one million M13 clones arrayed on membranes
Authors: Drmanac Radoje , Drmanac Snezana , Labat Ivan , Crkvenjakov Radomir , Vicentic Aleksandra , Gemmell Anne
Published: May 2005

An immediately applicable variant of the sequencing by hybridization (SBH) method is under development with the capacity to determine up to 100 million base pairs per year. The proposed method comprises six steps: (i) arraying genomic or cDNA M13 clones in 864‐well plates (wells of 2 mm); (ii) preparation of DNA samples for spotting by growth of the M13 clones or by polymerase chain reaction (PCR) of the inserts using standard 96‐well plates, or plates having as many as 864 correspondingly smaller wells; (iii) robotic spotting of 13 824 samples on an 8 × 12 cm nylon membrane, or correspondingly more, on up to 6 times larger filters, by offset printing with a 96 or 864 0.4 mm pin device; (iv) hybridization of dotted samples with 200–2000 32P‐labeled probes comprising 16–256 10‐mers having a common 8‐mer, 7‐mer, or 6‐mer in the middle (20 probes per day, each hybridized with 250 000 dots); (v) scoring hybridization signals of 5 million sampleprobe pairs per day using storage phosphor plates; and (vi) computing clone order and partial‐to‐complete DNA sequences using various heuristic algorithms. Genome sequencing based on a combination of this method and gel sequencing techniques may be significantly more economical than gel methods alone.
High‐throughput automated DNA sequencing facility with fluorescent labels at the European Molecular Biology Laboratory
Authors: Ansorge Wilhelm , Voss Hartmut , Wiemann Stefan , Schwager Christian , Sproat Brian , Zimmermann Jürgen , Stegemann Josef , Erfle Holger , Hewitt Neil , Rupp Thomas
Published: May 2005

One of the aims of the facility is to develop and push the automated on‐line DNA sequencing gel technology to its limit in sequence throughput, which may be somewhere around 100 kilobases of sequence per device per day. Key new developments were initiated and applied in operation on the European Molecular Biology Laboratory (EMBL) automated sequencer and its commercial version A.L.F. (Pharmacia). Sequencing speed was increased by a factor of 10–20, up to 1500 bases per hour per clone on ultrathin (about 100 μm) gels, while the resolution and reading length were extended to 1000 bases on gels with 50 cm separation length, using fluorescein‐15‐*dATP as the internal label. With our sequencing strategy, closing about 40% of the sequence with “walking” primers and F‐15‐*dATP as internal label, we sequenced both strands of a cosmid insert of 38.5 kb in length, each strand twice, in only 430 sequencing reactions and with average reading of 380 bases per reaction.
Towards a fingerprint method covering the immunological genome
Authors: Gomolka Maria , Schwaiger Werner , Weyers Eva , Epplen Cornelia , Buitkamp Johannes , Epplen Jörg T.
Published: May 2005

The principle of a novel typing techniques is described for screening with high efficiency the “immunological genome” by using various intronic and exonic oligonucleotide hybridization probes simultaneously or subsequently.
Chromosomal localization of the HYP2‐gene in Saccharomyces cerevisiae and use of pulsed‐field gel electrophoresis for detection of irregular recombination events in gene disruption experiments
Authors: Wöhl Thorsten , Baur Monika , Friedl Anna A. , Lottspeich Friedrich
Published: May 2005

In the hypusine‐containing protein (HP), a specific lysine residue is modified by spermidine to form the unusual amino acid hypusine (4‐amino‐2‐hydroxybutyl‐lysine). The HP has been designated as an eucaryotic translation initiation factor—eIF‐5A—because of its stimulating effect in the methionyl‐puromycin in vitro assay. Nevertheless, the precise function of this protein remains to be elucidated. In the yeast Saccharomyces cerevisiae two genes, HYP1 and HYP2, coding for two different forms of the HP, are present. The HYP1‐gene is identical to the AN B1‐gene and has already been localized on chromosome X. However, the chromosomal localization of the HYP2‐gene has not been elucidated. By using pulsed‐field gel electrophoresis (PFGE) and subsequent Southern blotting, we determined the localization of the HYP2‐gene to chromosome V. Furthermore, PFGE was used for the detection of irregular recombination events, such as misintegration or integration into a duplicated gene, and in gene disruption experiments using haploid and diploid yeast cells. The obtained data support the critical role of the HP for cell viability.
Plasma protein map: An update by microsequencing
Authors: Hughes Graham J. , Frutiger Séverine , Paquet Nicole , Ravier Florence , Pasquali Christian , Sanchez Jean‐Charles , James Richard , Tissot Jean‐Daniel , Bjellqvist Bengt , Hochstrasser Denis F.
Published: May 2005

The reference plasma protein map, obtained with immobilized pH gradients in the first dimension of two‐dimensional electrophoresis, is presented. By microsequencing, more than 40 polypeptide chains were identified. The new polypeptides and previously known proteins are listed in a table and labeled on the protein map, thus providing an update of the human plasma two‐dimensional gel database.
Using two‐dimensional gel electrophoresis to study immune response against intracellular bacterial infection
Authors: Kovarova Hana , Stulik Jiri , Macela Ales , Lefkovits Ivan , Skrabkova Zuzana
Published: May 2005

Principal component analysis was applied to two‐dimensional (2‐D) gel electrophoresis patterns, obtained in various phases of infection. Untreated controls could be satisfactorily differentiated from patterns after infection on days 3 and 7 whereas day 10 of infection was grouped with the controls. Comparison of host cellular protein patterns could help to classify in vivo developing infection without requiring any so‐called immune marker functions. Immunoaffinity separation of infected cells treated with detergent, followed by 2‐D electrophoresis of negative as well as positive eluates, did not reveal radiolabeled bacterial protein antigens.
Two‐dimensional electrophoresis analysis of human serum proteins during the acute‐phase response
Authors: Bini Luca , Magi Barbara , Cellesi Carla , Rossolini Aldo , Pallini Vitaliano
Published: May 2005

The serum of patients with meningitis, due to infection by Haemophilus influenzae type b, was analyzed. Several known acute‐phase proteins were separated by two‐dimensional electrophoresis and estimated quantitatively. In addition, hitherto undescribed reactants were recognized. Gels were calibrated and relevant spots related to master spot numbers in the human serum protein database.
Preincubation with cysteine prevents modification of sulfhydryl groups in proteins by unreacted acrylamide in a gel
Authors: Chiari Marcella , Righetti Pier Giorgio , Negri Armando , Ceciliani Fabrizio , Ronchi Severino
Published: May 2005

It has been found that the double bond of free, unreacted acrylamide in a gel can react with a free ‐SH group of proteins, forming a cysteinyl‐S‐propionamide adduct. When β‐lactoglobulin was incubated at concentration levels lower than those of free acrylamide, left after polymerizing a 5% T, 4% C gel (barely 12 mM), under anaerobic conditions in 0.1 M borate, pH 9.5, for 1 h and then the tryptic digests analyzed by high performance liquid chromatography (HPLC), two new peptides were detected. The two new peaks were recovered and sequenced by the Edman degradation procedure. They correspond to the sequence from Leu‐149 to lle‐162. Residue No. 160 was found to be a cysteinyl‐S‐propionamide reaction product. Interestingly, only this residue, out of a total of 5 Cys residues, had reacted. No other amino acids (including ‐NH2 terminus and free ‐NH2 in Lys) reacted with free acrylamide. The addition of free acrylamide to the ‐SH group could be completely inhibited if: (i) the gel was extensively washed prior to sample application, or (ii) the gel was incubated for 1 h in 100 mM free Cys.
Chicory seed lot variety identification by leucine‐aminopeptidase and esterase zymogram analysis
Authors: Baes Peter , Van Cutsem Pierre
Published: May 2005

A method of identifying closely related chicory varieties has been developed using native polyacrylamide gel electrophoresis and subsequent leucine aminopeptidase and esterase staining of bulked seed sample extracts. The variety zymograms are analyzed according to a mobility‐density scheme. This quick, easy and low‐cost method can be a useful tool for chicory breeders.
Mouse liver protein database: A catalog of proteins detected by two‐dimensional gel electrophoresis
Authors: Giometti Carol S. , Taylor John , Tollaksen Sandra L.
Published: May 2005

Alterations in the abundance or structure of mouse liver proteins are being studied using two‐dimensional gel electrophoresis (2‐DE) to build a database of protein changes correlating with exposure to ionizing radiation or toxic chemicals. Thus far, studies have included the analysis of proteins from the offspring of exposed parents or from the exposed individuals themselves. In order to characterize and identify proteins found altered by such exposures, sex‐ and strain‐related differences in protein patterns have been analyzed, and the subcellular locations of a large portion of the mapped proteins have been determined. As part of these studies, data are collected and stored using a variety of computer hardware and software tools that allow the accumulation of information on the origin of samples, gel identification, experiment description, and protein similarities and differences. This accumulation of information constitutes the mouse liver protein database. Relational database software is used to tie the different facets of the data‐base together so that the results of a variety of experiments can be compared and interrelated. The database optimizes the information obtained from 2‐DE gel sets by allowing use of the data for many purposes, including monitoring of gel resolution to ensure the collection of high quality data and correlation of protein effects induced by different agents. This first edition of the Argonne National Laboratory mouse liver protein database lays the foundation for future work and communication that should elucidate the significance of observed protein effects as possible markers of exposure to toxic agents.
Comparison of different staining methods for polyvinylidene difluoride membranes
Authors: Christiansen Jesper , Houen Gunnar
Published: May 2005

Several new staining methods for polyvinylidene difluoride membranes, including mercurochrome, silver and dimethylaminoazobenzene isothiocyanate staining were compared with Coomassie Brilliant Blue and gold staining. Of these, Coomassie was most versatile and completely compatible with ensuing microsequencing, immunostaining or other visualization methods, while gold and silver staining were more sensitive. Mercurochrome allows selective detection of sulfhydryl‐containing proteins while dimethylaminoazobenzene isothiocyanate staining may allow quantitation of sequenceable protein.
Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients
Authors: Bischoff Rainer , Roecklin Dominique , Roitsch Carolyn
Published: May 2005

Isoelectric focusing in immobilized pH gradients (IEF‐IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion‐exchange and reversed‐phase chromatography proved to be homogeneous with regard to its isoelectric point (pI). In addition, the theoretical pI, calculated on the basis of the primary structure, corresponded precisely to the measured pI of 4.30. IEF‐IPG was further employed to follow the stability of recombinant hirudin at pH 9, indicating that deamidation occurred under these conditions. A variant of recombinant human α1‐antitrypsin (AAT) (389 amino acids, one cysteine residue) expressed in Escherichia coli and purified by anion‐exchange, metal chelate and hydrophobic‐interaction chromatography appeared to be homogeneous by polyacrylamide gel electrophoresis under reducing and denaturing conditions as well as by various high performance liquid chromatography methods. However, some heterogeneity was detected by IEF‐IPG between pH 5–6. The measured pI values of 5.43–5.58 were slightly lower than the calculated pI based on the primary structure (5.72). This indicated deamidations of Asn or Gln residues. A recombinant Schistosoma mansoni parasite antigen, p28 (210 amino acids, one cysteine residue) obtained after intracellular expression in Saccharomyces cerevisiae and affinity purification on glutathione agarose was analyzed by IEF‐IPG in a pH 7.3–8.3 gradient. It appeared to be heterogeneous with regard to its pI, with the major component having a pI of 7.81 compared to the calculated value of 7.17. N‐Terminal amino acid sequencing as well as amino acid composition analysis were performed on the separated forms of p28 after electroblotting showing the feasibility of combining IEF‐IPG with subsequent analytical methods to obtain structural information.
Production of mitogen‐contamination free alginates with variable ratios of mannuronic acid to guluronic acid by free flow electrophoresis
Authors: Zimmermann Ulrich , Klöck Gerd , Federlin Konrad , Hannig Kurt , Kowalski Matthias , Bretzel Reinhard G. , Horcher Andrea , Entenmann Heike , Sieber Ulrike , Zekorn Tobias
Published: May 2005

Commercial alginates consisting of variable homopolymeric regions of β‐D‐mannuronic acid and α‐L‐guluronic acid, interspaced with regions of alternating blocks, are potent stimulators of macrophages and lymphocytes. Therefore, inflammatory reactions and fibrotic overgrowth of the beads result if Langerhans islets are encapsulated in raw alginate hydrogel beads (cross‐linked with divalent cations). The result is random failure of the islets some time after transplantation. Analysis of raw alginates by using free flow electrophoresis demonstrated that commercial alginates contained at least 10–20 fractions (characterized by different electrophoretic mobilities) which showed mitogenic activity. These fractions could be quantitatively separated from the alginic acids by free flow electrophoresis on a preparative scale. The purified alginates cross‐linked with Ca2+ ions exhibited no mitogenic reactions as proved by an in vitro assay. In addition, examination of purified Ba2+ alginate beads implanted intraperitoneally in rats or mice for three weeks showed no fibrotic overgrowth in contrast to implants made from unpurified alginate.
High resolution two‐dimensional mapping of tissue‐specific polypeptides in the desert locust, Schistocerca gregaria
Authors: Annadurai Ramasamy Sambasivam , Oberthür Walter , Remboldm Heinz
Published: May 2005

High resolution two‐dimensional gel electrophoresis (2‐DE) using immobilized pH gradients was used to map the tissue‐specific polypeptides of the desert locust, Schisiocerca gregaria. Highly specific comprehensive 2‐DE reference maps (“master gels”) were developed for the brain, corpus cardiacum, subesophageal ganglion, and hemolymph. The polypeptides were well resolved within the pH 4–7 range in the first dimension and within the 14–94 kDa molecular mass range in the second dimension.
Photopolymerization of polyacrylamide gels with methylene blue
Authors: Lyubimova Tatyana , Caglio Silvia , Gelfi Cecilia , Righetti Pier Giorgio , Rabilloud Thierry
Published: May 2005

Photopolymerization of polyacrylamide gels in the presence of methylene blue (100 μM) and a redox couple (1 mM sodium toluenesulfinate, a reducer, and 50 μM dipheriyliodonium chloride, an oxidizer) has been investigated. The gel point, i.e. the time needed for onset of gelation upon illumination, has been found to lengthen progressively at lower temperatures and at lower light intensities. If the three catalysts are progressively diluted, the gel point does not vary for a threefold dilution, but gelation is greatly hampered below a 1:5 dilution of the three effectors. Photobleaching has been assessed as a function of liquid layer thickness (from 0.5 to 2 mm), of a progressive dye dilution (down to a fourfold dilution) and as a function of temperature. A maximum of elastic modulus is located in correspondence to a minimum of permeability (both situated at 5% cross‐linker). It is found that methylene blue‐activated polymerization produces polyacrylamide gels with elastic properties which are higher than in persulfate‐activated gels, so far the most popular matrices for electrokinetic separations. Due to the ease of preparation, the full control of all experimental parameters, and the lack of oxidizing power of this catalyst system (as opposed to the strong oxidation power of persulfate catalysis), methylene blue catalysis is advocated as a valid alternative to other redox systems.
Theoretical studies on the mobility‐shift behavior of binary protein‐DNA complexes
Authors: Cann John R.
Published: May 2005

The theory of mass transport coupled to reversible interactions under chemical kinetic control forms the basis for computer simulation of the electrophoretic mobility‐shift behavior of binary protein‐DNA complexes. Several systems have been modeled in terms of either (i) specific binding of a protein molecule to a single site on the DNA molecule; (ii) cooperative binding to two or three sites; (iii) noncooperative binding to two sites, both of which bind protein with equal affinity; (iv) statistical binding to multiple sites having identical intrinsic binding constants; or (v) protein‐induced DNA loop formation. Both models (iii) and (v) embody the concept of reversible isomerization of protein‐DNA complexes. The resulting simulations have provided fundamental information concerning (i) the factors governing the electrophoretic persistence and separation of protein‐DNA complexes; (ii) the shape of experimental mobility‐shift patterns; (iii) the generation of the protein‐DNA ladder upon titration, for example, of the 203‐base pair operator with lac repressor; and (iv) the theoretical bases for quantitative interpretation of the patterns in terms of thermodynamic and kinetic parameters. The practical implications of these findings are discussed.
Peptide dot immunoassay and immunoblotting: Electroblotting from aluminum thin‐layer chromatography plates and isoelectric focusing gels to activated nitrocellulose
Authors: Lauritzen Edgar , Másson Már , Rubin Inger , Bjerrum Ole J. , Holm Arne
Published: May 2005

Nitrocellulose membrane was preactivated with divinyl sulfone, and a spacer of 1,6‐diaminohexane was coupled to the membrane which was functionalized by glutaraldehyde, leaving a reactive carbonyl group. The peptides were coupled to the carbonyl by the side chain and terminal amino groups. The octapeptide angiotensin II (sequence: DRVYIHPF) and peptide analogs containing 6–10 amino acid residues were dotted directly onto the matrix at 45°C for 15 min and detected by specific antisera, which were raised in rabbits against angiotensin I and II, respectively. They were visualized by peroxidase‐coupled anti‐rabbit IgG antibodies. The detection limit for synthetic angiotensin II was 500 fg per cm2 (= 500 amol per cm2) and for the decapeptide angiotensin I (sequence: DRVYIHPFHL) it was 500 pg per cm2 (= 400 fmol per cm2). Separation of synthetic angiotensin analogs by high performance thin‐layer chromatography on silica coated aluminum plates was followed by electroblotting onto activated nitrocellulose and detection with specific antibodies, showing a sensitivity of 100 fg and 1 pg for angiotensin II and angiotensin I, respectively. Isoelectric focusing in agarose using Ampholine carrier ampholytes and immunoblotting with specific antisera displayed a lower sensitivity for angiotensin II and angiotensin I of 2 ng and 20 ng, respectively. The isoelectric focusing and immunoblotting technique was applied for separation of angiotensin I and II and related peptides in serum, where synthetic angiotensin I was degraded in the presence of 1 mM phenylmethylsulfonyl fluoride and 10 mM ethylenediaminetetraacetic acid. The versatility of dot immunobinding on activated nitrocellulose was shown with two sera from patients infected with human immunodeficiency virus, HIV‐1 and HIV‐2, by using pH 10.2 in incubation media, resulting in a low background. These sera were bound specifically to either one of the closely related HIV‐1 and HIV‐2 peptide antigens from the two viruses.
Chemical activation of nitrocellulose membranes for peptide antigen‐antibody binding studies: Direct substitution of the nitrate group with diaminoalkane
Authors: Másson Már , Lauritzen Edgar , Holm Arne
Published: May 2005

A method to covalently link peptide and proteins, through a diaminoalkane spacer to nitrocellulose membrane was developed for immunochemical applications. Initially the nitrocellulose membrane was modified by covalent incorporation of diaminoalkane spacers without any prior activation. The incorporation was shown primarily to involve α‐elimination of the nitrate groups, and an imine was formed between the carbonyl group on the membrane and the diaminoalkane. The rate of incorporation increased exponentially with the length of the diaminoalkane as determined by a ninhydrin colorometric reaction, which was developed for the study. More than 200 nmole diamine per mg nitrocellulose could be incorporated, but less than 11 nmol/mg (63 nmol/cm2) was chosen in order to retain the strength of the membrane. The primary amino groups of the modified membrane was glutaraldehyde activated and the octapeptide, angiotensin II, was covalently bound. A dot immunoassay was performed where specific anti‐angiotensin II antibodies reacted with the peptide and was visualized by peroxidase coupled secondary antibodies. The results were quantified by video densitometry above 0.005 μg All per cm2. The immunoassay showed improved detection of the peptide on the activated as compared to unactivated membrane as well as increased retention of radiolabeled [125I]angiotensin II.
Western blot as a tool in the diagnosis of Lyme borreliosis
Authors: Zöller Lothar , Cremer Josef , Faulde Michael
Published: May 2005

Borrelia burgdorferi is the causative agent of Lyme borreliosis, a multisystem disorder, which can mimic numerous immune disorders and inflammatory diseases. Laboratory diagnosis of Borrelia infection relies on immunodiagnostic assays, which, however, are hampered by unsatisfactory specificity. The Western blot technique has been employed to analyze the humoral immune response in Lyme borreliosis and is used as a serodiagnostic confirmation test. The most important immunodominant proteins of Borrelia burgdorferi are the 94 kDa, 60 kDa, 41 kDa (flagellin), 34 kDa (Osp B), 31 kDa (Osp A), 30 kDa, 21 kDa (Osp C), and 17/18 kDa proteins. Whereas the 60 kDa, 41 kDa, and 34 kDa constituents reveal a marked cross‐antigenicity with other spirochetes and even more distantly related bacteria, antibodies against the 94 kDa, 31 kDa and 21 kDa proteins are largely species‐specific. The early immune response in Lyme borreliosis is triggered mainly by the flagellin. In the later stage a wide range of immunogenic proteins is involved, with the 94 kDa antigen being the best marker for late immune response. If the Western blot is used for diagnostic purposes the differences between early and late‐stage immunogenicity of Borrelia proteins must be taken into account. Interpretation criteria for blot positivity in early‐stage borreliosis are primarily based on the presence of the 21 kDa band and the semiquantitatively recorded intensity of the 41 kDa band. In the diagnosis of late‐stage infection, blot positivity relies on the presence of the 94 kDa, 39 kDa, 31 kDa, 30 kDa and 21 kDa bands.
A new buffering system and its use in electrophoresis and isoelectric focusing
Authors: Bier Milan , Ostrem James , Marquez Rodolfo B.
Published: May 2005

A novel buffer system with rather unique properties particularly well suited for preparative isoelectric focusing is described. The system was developed in response to the long standing need for pH gradients formulated using inexpensive components of known chemical composition. The system is binary consisting of buffer pairs covering the pH range of 3 to 10, in increments of less than 1 pH. The components are all readily available, nontoxic, and biologically acceptable, and are selected on the basis of their dissociation constants. The requirement for each buffer pair is that there be an overlapping pH zone where both components are largely non‐ionized, having a ratio of neutral species to total buffer concentration between about 0.90 and 0.99. This ratio is defined as the ‘electrophoretic reserve capacity’ (ERC). Within the above ERC range a stable pH gradient is obtained, assuring sharply resolved protein focusing. Buffer parameters, mainly pH, buffering capacity, ERC, transport numbers, and conductiviy, can be easily calculated and the buffer composition adjusted to meet requirements. All components are used at high concentration, typically of the order of up to 100 mM, yielding buffering capacities and conductivities analog to those customary in electrophoresis.
Analysis of scaling methods to minimize experimental variations in two‐dimensional electrophoresis quantitative data: Application to the comparison of maize inbred lines
Authors: Burstin Judith , Zivy Michel , de Vienne Dominique , Damerval Catherine
Published: May 2005

The analysis of two‐dimensional (2‐D) electrophoresis quantitative data from a design involving 21 maize genotypes revealed a significant experimental variation. In order to minimize this variation, we investigated the possible causes and found that it was essentially due to global effects, affecting all the spots in a gel in a similar way, and occurring during the 2‐D run/staining procedure. Three scaling methods to discard these experimental variations were, analyzed: the linear scaling method, a method based on principal component analyis, and a combined method that unites the advantages of both of the former. Comparing these three methods, we found that they led to consistent results with regard to the factor under study, i.e. the genetic factor in our case. However, the combined scaling method was the most efficient in reducing experimental variations.
SWISS‐2DPAGE: A database of two‐dimensional gel electrophoresis images
Authors: Appel Ron D. , Sanchez Jean‐Charles , Bairoch Amos , Golaz Olivier , Miu Marina , Vargas J. Reynaldo , Hochstrasser Denis F.
Published: May 2005

This publication presents the SWISS‐2DPAGE database which gathers data on proteins identified on various two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) maps. Each SWISS‐2DPAGE entry contains data on one protein, including mapping procedures, physiological and pathological data and bibliographical references, as well as several 2‐D PAGE images showing the protein location. Links are also provided to other databases such as SWISS‐PROT, EMBL, PROSITE and OMIM. The database has been set up on a server which may be accessed from any computer connected to the internet and it also makes it possible to display the theoretical location of proteins, the positions of which are not yet known on the 2‐D PAGE.
Application of two‐dimensional gel analysis to identification and characterization of tyrosine phosphorylated substrates for growth factor receptors
Authors: Hansen Klaus , Møller Jesper V.
Published: May 2005

The technique of two‐dimensional gel electrophoresis was used for analysis of tyrosine phosphorylated polypeptide substrates after epidermal growth factor (EGF)‐induced stimulation of receptor tyrosine kinase activity in a brush border fraction of human placental syncytiotrofoblast cells. After incubation with [γ32P]ATP, followed by autoradiography of the gels, 35 phosphorylated components were detected, of which 8 were strongly tyrosine phosphorylated by EGF. Using a more sensitive assay with phosphotyrosine‐specific antibody, an additional 12 polypeptide components were found to be strongly tyrosine phosphorylated by EGF. A number of the phosphorylated substrates could be aligned with components in a protein catalog of the human brush border membrane fraction that was characterized by glycoprotein staining, Triton X‐114 fractionation, immunoreaction with specific antibodies, and comigration with 35S‐labeled AMA (transformed human amnion) cells. Identified components, stimulated by EGF, in addition to well‐recognized substrates (calpactin II, ezrin, EGF receptor) included β‐tubulin and serum albumin, while other cytoskeletal proteins and alkaline phosphatase were excluded as substrates. A notable feature of the catalog was that a number of glycoproteins were present in both the membrane and cytoskeletal fraction, suggesting involvement in membrane/cytoskeletal interactions. The data demonstrate the feasibility of using two‐dimensional gel electrophoresis in a global way to identify target substrates for tyrosine kinase activity. In addition they suggest that many of these are located in the vicinity of tyrosine kinase at the membrane/cytoskeletal border at a location which is probably involved, at the molecular level, in morphological changes of the plasma membrane associated with cell proliferation.
Degradation of fibrinogen and fibrin by plasmin and nonplasmin proteases in the chronic subdural hematoma: Evaluation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immunoblot
Authors: Nomura Sadahiro , Kashiwagi Shiro , Ito Haruhide , Mimura Yusuke , Nakamura Kazuyuki
Published: May 2005

To characterize local hyperfibrinolysis in the pathogenesis of chronic subdural hematoma (CSDH), fibrin degradation products (FDPs) were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) to identify each FDP fragment (fragments HMW, YY, DY and DD) by immunoblotting. The electrophoretic patterns of FDP in 40 hematomas from 33 patients could be categorized into 3 patterns; (i) type 1, the sharp DY and DD bands; (ii) type 2, the broad DY and DD bands; and (iii) type 3, the sharp DY and DD with two bands with higher molecular weights than fragment DY and DD. The broadening of DY and DD bands was reproduced by incubating the sample containing sharp DD and DY bands with the hemolysed peripheral blood cells. These results indicated that the hyperfibrinolysis in CSDH could be characterized by the constant and incomplete proteolysis of fibrinogen and fibrin by plasmin and further degradation of FDPs by non‐plasmin proteases released from hemolysed blood cells.
Two‐dimensional electrophoresis as an aid in the analysis of the clonality of immunoglobulins
Authors: Tissot Jean‐Daniel , Schneider Philippe , Hohlfeld Patrick , Spertini François , Hochstrasser Denis F. , Duchosal Michel A.
Published: May 2005

In this paper, we summarize our five‐year observation of two‐dimensional polyacrylamide gel electrophoretic (2‐D PAGE) analysis of immunoglobulin (Ig) light (L) chain patterns on serum/plasma and/or purified human Ig, and compare this technique with agarose electrophoresis and/or immunofixation examination. Polyclonal Ig L chains were seen as large “fuzzy” areas with several zones of high density. The majority (71%) of the monoclonal Ig L chains of monoclonal gammopathy detected by conventional electrophoresis appeared as a single large and well‐defined spot on 2‐D PAGE analysis, with the remaining appearing as multiple spots. The presence of oligoclonal Ig, reflected by multiple spots in 2‐D PAGE, and several bands in immunofixation, was observed in 5 of 26 patients after allogeneic bone marrow transplantation and in 5 patients with tumors. In the majority (77%) of hypergamma‐globulinemia, L chains appeared as a wide spread of small and well‐defined spots in 2‐D PAGE analysis. This pattern suggested oligoclonal Ig‐secreting B cell clone expansion, and corresponding abnormalities were not detected with immunofixation. 2‐D PAGE analysis also detected oligoclonal Ig expansions whereas conventional electrophoretic examination was normal in 10 additional patients after bone marrow transplantation, and in 6 of 10 immunocompetent patients with acute severe infections. Analysis of the Ig L chain pattern of a severe combined immune‐deficient mouse populated with human peripheral blood leukocytes confirmed the skewed human Ig production in the model. In summary, 2‐D PAGE appears to be a sensitive tool for the analysis of Ig diversity in various clinical situations.
Colloidal carriers for intravenous drug targeting: Plasma protein adsorption patterns on surface‐modified latex particles evaluated by two‐dimensional polyacrylamide gel electrophoresis
Authors: Blunk Torsten , Hochstrasser Denis F. , Sanchez Jean‐Charles , Müller Bernd W. , Müller Rainer H.
Published: May 2005

Targeting to specific sites of the body via colloidal carriers is sought in order to reduce drug side effects. The adsorption of plasma proteins on intravenously injected particles is regarded as the key factor in explaining their organ distribution: total bound protein, or, more likely, the presence of specific proteins and their conformation, are expected to influence macrophage uptake. Polystyrene beads, 60 nm in diameter, were used as model carriers; their surface was differentially modified by adsorption of increasingly hydrophilic block copolymers, poloxamers 184, 188 and 407. After incubation in plasma, the patterns of protein adsorption onto coated beads were analyzed by high‐resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). The behavior of some representative proteins was monitored, including albumin, fibrinogen, IgG, factor B and the apolipoproteins, A‐I, A‐IV, C‐III, E and J. The more hydrophobic the particles, the larger the total amount of bound protein. However, this correlation was not valid for all of the analyzed protein species, which proves that it is insufficient to look only at physico‐chemical data to predict organ distribution. On the contrary, it is essential to use 2‐D PAGE to establish the correlation between adsorbed proteins and carrier behavior in vivo.
Electrophoretic detection of chitinase isoenzymes using the PhastSystem
Authors: McBride Jeffrey D. , Stubberfield Colin R. , Hayes David J.
Published: May 2005

Zymographic assays are described for the detection of chitinase isoenzymes following isoelectric focusing. Method 1 used a polyacrylamide overlay gel containing glycol chitin. Following hydrolysis, visualisation of isoenzymes was achieved by fluorescent counter‐staining of the gel with Fluorescent Brightener 28. Method 2 used a cellulose acetate membrane overlay which incorporates 4‐methylumbelliferyl substrates, hydrolysis of which released a fluorescent product (4‐methylumbelliferone). The pIs of chitinase isoenzymes were estimated by using coloured pI markers. A significant time advantage was obtained over previous methods. The method was used to demonstrate chitinase activity in control rat lungs and in rat lungs infected with the pathogen Pneumocystis carinii.
Concanavalin A crossed affinoimmunoelectrophoretic analysis of the major pig serum proteins during fetal development
Authors: Lampreave Fermín , Alava María A. , Piñeiro Andrés
Published: May 2005

The interaction between concanavalin A (Con A) and α‐fetoprotein (AFP), transferrin (TF), fetuin, α1‐antitrypsin (AT) and α1‐acid glycoprotein (AGP) has been analyzed by crossed affinoimmunoelectrophoresis (CAIE) in fetal extracts or sera, from 26‐day‐old porcine fetuses to birth, and in adult pigs. Most of the TF and AFP (100 and 85–90%, respectively) reacted with Con A during the entire developmental period. AGP showed both two reactive and one nonreactive Con A isoforms, whose proportions change greatly during development. In younger fetuses 100% of the protein was Con A‐nonreactive. This variant represented 64% in 50‐day‐old fetuses, 80% in newborn pigs and 20% in adult sera. Fetuin and AT showed a maximum of three Con A‐reactive microforms and one Con A‐nonreactive microform, which was always a minor form. These microforms were detected mainly in young fetuses. Although the proportion of Con A‐reactive variants of fetuin and AT changes during fetal development, the predominant microform was always that with intermediate affinity against Con A. The same microform was also predominant in adult AT, whereas the more reactive microform in respect to Con A predominates in adult fetuin.
Capillary electrophoresis of DNA restriction fragments with solutions of entangled polymers
Authors: Kleemiß Maria H. , Gilges Martin , Schomburg Gerhard
Published: May 2005

Separation of DNA restriction fragments in dilute buffer solutions of network forming polymers such as linear polyacrylamide (PAA), hydroxyethyl cellulose (HEC) and polyvinylalcohol (PVA) in phosphate buffer were investigated. PVA in buffers already became inhomogeneous after a few separations with resultant deterioration of resolution. Hydroxylic polymers, capable of forming suitable networks in buffers, are strongly adsorbed to fused silica surfaces suppressing the electroosmotic flow. These polymers could be applied as surface modifiers in capillaries, filled with buffer media containing other polymers such as PAA: Suppression of the electroosmotic flow by adsorbed HEC at pH 7 was slightly more effective than with PVA but the former coating was less stable due to weaker binding to the fused silica surface. Good separations of restriction fragments could be achieved with solutions of PAA or HEC as separation media after surface pretreatment of fused silica capillaries by either PVA rinsing or coating according to Hjertén [41].
Analysis of lipoproteins with analytical capillary isotachophoresis
Authors: Schmitz Gerd , Möllers Christoph
Published: May 2005

An analytical free flow capillary isotachophoresis procedure, with a discontinuous electrolyte system, for the detailed analysis of lipoproteins in human body fluids has been developed. The technique is based on prestaining whole serum lipoproteins with a lipophilic dye before separation. Human serum lipoproteins are separated into 14 well‐characterized subfractions according to their electrophoretic mobility. High density lipoproteins (fraction 1 to 6) are separated into three major subpopulations, the fast migrating high density lipoprotein (HDL) subpopulation, containing mainly apo AI and phosphatidylcholine, the subpopulation with intermediate mobility, consisting of particles rich in apo AII, apo E, and C apolipoproteins, and the slowly migrating HDL subfraction, containing mainly particles rich in apo AI, apo AIV, and lecithin: cholesterol acyltransferase (LCAT) activity. The apo B containing lipoproteins (fraction 7 to 14) can be subdivided into four major functional groups. The first represents chylomicron derived particles and large triglyceride‐rich very low density lipoproteins (VLDL). The second group consists of small VLDL and intermediate density lipoprotein (IDL) particles, anf the third and fourth group represent the low density lipoproteins. The isotachophoretic analysis of human serum samples obtained from patients with hyperlipoproteinemias is compatible with the classification according to the Frederickson phenotypes and reflects the respective biochemical abnormalities. Furthermore, several genetic disorders of lipid and lipoprotein metabolism like HDL deficiency syndromes, familial LCAT deficiency, Fish eye disease, hypobetalipoproteinemia and abetalipoproteinemia can be well characterized by analytical capillary iso tachophoresis. In addition to patient analysis we investigated the influence of lipid lowering drugs on the lipoprotein subfraction distribution during therapy with analytical capillary isotachophoresis.
Investigation of drug metabolism using capillary electrophoresis with photodiode array detection and online mass spectrometry equipped with an array detector
Authors: Tomlinson Andy J. , Benson Linda M. , Johnson Kenneth L. , Naylor Stephen
Published: May 2005

The application of capillary electrophoresis (CE) with photodiode array detection (DAD) and on‐line CE‐mass spectrometry (CE‐MS) equipped with a position and time resolved (PATRICtm) focal plane detector for analysis of both in vitro and in vivo drug metabolism is demonstrated. Separation of metabolites derived from the neuroleptic drug haloperidol, by CE, using a simple, volatile run buffer containing 50 mM ammonium acetate with 10% methanol and 1% acetic acid is reported. The potential utility of CE‐DAD for screening drug metabolite mixtures derived from hepatic microsomal incubations is demonstrated for haloperidal (HAL). Also the potential problems associated with using this technology to screen human urine samples for HAL metabolites is discussed. Furthermore, the usefulness of CE‐MS and CE‐electrospray ionization skimmer collision induced dissociation‐MS (CE‐ESI‐CID‐MS) in identification and structure elucidation of HAL metabolites derived from both a guinea pig hepatic microsomal incubation and urine from a patient treated with 0.5 mg/day of HAL is shown. The utility of such an approach in the general area of clinical pharmacology is also discussed.
Neutral and anionic cyclodextrins in capillary zone electrophoresis: Enantiomeric separation of ephedrine and related compounds
Authors: Dette Christoph , Ebel Siegfried , Terabe Shigeru
Published: May 2005

Two methods for the enantiomeric separation of chiral ephedrine alkaloids (ephedrine, methylephedrine, methylpseudoephedrine and norephedrine) by capillary zone electrophoresis in uncoated capillaries were developed. Both methods were optimized to more than 100 000 theoretical plate numbers. The first method used a neutral cyclodextrin (CD) derivative: heptakis (2,6–di‐O‐methyl)‐β‐cyclodextrin at an acidic pH of 2.5 (20 mM phosphate buffer) at an 18 mM concentration. The second used a newly developed acidic CD derivative, the tetrakis[6‐O‐(4‐sulfobutyl)]‐β‐cyclodextrin sodium salt. The benefits of this new reagent for the chiral separation are a wide range of basic pH available for the enantiomeric resolution. It is also useful for other cases, e.g. preventing adsorption without additives.