bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum amyloid A protein in very low density – and high density lipoproteins during the course of acute myocardial infarction
Authors: Feussner Giso , Schuster Markus , Ziegler Reinhard
Published: May 2005

In a preceding paper [1] we reported on the detection and characterization of human serum amyloid A protein (SAA) in very low density lipoproteins (VLDL) and high density lipoproteins (HDL) of patients after acute myocardial infarction. Here we describe the time course of the occurrence of SAA in VLDL and HDL in the postinfarction period. SAA reached its maximum in VLDL d HDL approximately 53 h after the acute event. At the peak of the acute‐phase response, SAA comprised as much as 38% of the total apoproteins of VLDL and HDL. SAA appeared at the same points in time and with nearly the same concentrations in VLDL and HDL. We conclude that SAA is not exchanged in plasma between lipoproteins of different densities and that this protein is secreted on its own by hepatocytes and not as a part of an already constituted lipoprotein particle.
Determining consanguinity by oligonucleotide fingerprinting with (GTG)5/(CAC)5
Authors: Pöche Hubert , Peters Christian , Wrobel Gudrun , Schneider Volkmar , Epplen Jörg T.
Published: May 2005

Simple tandemly organized (GTG)n/(CAC)n sequences are spread throughout the human chromosomes. The most informative DNA fingerprints for the testing of pedigrees and/or paternity were obtained with the simple triplet repeat probe (GTG)5 or its complement (CAC)5. These hypervariable simple‐repeat fragments are stably inherited in a Mendelian fashion. Using these highly discriminating probes, all human individuals could, theoretically, be differentiated, except for genetically identical monozygotic twins. Examples from actual case work are reported and pertinent advantages of this methodology are discussed.
Effect of hair dyes and bleach on the hair protein patterns as revealed by isoelectric focusing
Authors: Nagai Atsushi , Komoriya Hideou , Bunai Yasuo , Yamada Sadao , Jiang Xiuyan , Ohya Isao
Published: May 2005

The effect of hair dyes. i.e., temporary, semi‐permanent, or permanent hair dyes, or hair bleach on the isoelectric focusing (IEF) hair protein patterns was studied. A permanent hair dye (metallic, alkaline oxidative, or acidic oxidative) and hair bleach induced changes in the IEF hair protein patterns and in the intensity of hair protein bands. The changes in the IEF patterns, caused by the alkaline oxidative dye or the bleach, are considered to result from the combined effect of an alkaline agent and an oxidative agent in the alkaline oxidative dye and in the hair bleach.
A practicable two‐dimensional electrophoresis of urinary proteins as a useful tool in medical diagnosis
Authors: Lapin Alexander , Feigl Walter
Published: May 2005

Practical experience with a rapid two‐dimensional electrophoresis technique for routine analysis of urinary proteins is discussed. The method consists of cellulose acetate electrophoresis in combination with sodium dodecyl sulfate (SDS) electrophoresis, performed together with Coomassie Blue gel staining on “PhastSystem”. Over 400 analyses were performed within the time of two years. Most patients were from nephrological, urological and kidney‐transplant departments. Some of them were from obstetric, pediatric or oncological departments. A systematic discussion and evaluation of the two‐dimensional protein pattern with typical examples is outlined.
Analysis of circulating immune complexes isolated from plasma, cerebrospinal fluid and urine
Authors: Wiederkehr Felix , Büeler Martin R. , Vonderschmitt Dieter J.
Published: May 2005

The protein nature of soluble immune complexes (IC) from fresh plasma, cerebrospinal fluid (CSF), and urine was studied by combining several analytical and biochemical techniques. In plasma and CSF, free immunoglobulins G were separated from larger IC by gel filtration with a fast protein liquid chromatographic system. In urine, IC were separated by precipitation with polyethylene glycol. IC were further purified by protein‐A and protein‐G affinity chromatography and analyzed by two‐dimensional gel electrophoresis. Apart from plasma samples from healthy donors, IC from cases with macrocreatine kinase type 1 and multiple sclerosis were analyzed. For CSF two cases of multiple sclerosis and for urine one case with urinary tract infection are shown. The method can be used for the examination of IC of unknown protein composition in body fluids.
A comparative analysis of alterations in protein expression after activation or human immunodeficiency virus, type 1 infection of human CD4+ T cells
Authors: Van de Keere Fabienne , Willard‐Gallo Karen E.
Published: May 2005

We have previously described an in vitro model for studying human immunodeficiency virus, type 1 (HIV‐1) infection in CD4+ T cells [1]. This model employs the WE17/10 cell line, which loses expression of its T cell receptor/CD3 (TCR/CD3) after several months of productive infection. We have used this model to analyze the synthesis and posttranslational modification of viral and cellular proteins after HIV‐1 infection and to determine the relationship of these changes to TCR/CD3 expression. Mainly we observe positive changes in protein expression after infection. A phosphoprotein, referred to as WH:1, appears in infected cells that still express their TCR/CD3 complex, and its persistence is linked to the presence of the complex. We examined whether loss of the TCR/CD3 complex could be associated with alterations in the T cell activation pathway as a result of infection. We used T cell activators and inhibitors to determine whether there were common elements between the two events. Quantitative enhancement in one spot, Cs:1, occurred after both Cyclosporin A treatment of uninfected cells and HIV‐1 infection of untreated cells. Taken altogether, these data suggest that a correlation exists between negative regulation of late events in the T cell activation pathway and down regulation of the TCR/CD3 complex after HIV‐1 infection.
Analysis of limiting factors of DNA band separation by a DNA sequencer using fluorescence detection
Authors: Nishikawa Tetsuo , Kambara Hideki
Published: May 2005

The factors affecting the electrophoretic separation of DNA bands in DNA base sequencing using fluorescence detection are analyzed. All the factors contributing to DNA band spacing and band width are evaluated; DNA diffusion and thermal effects on gels are the main considerations. The dependence of the gel's electrical resistivity on gel temperature and the variation of temperature over gel thickness are associated with a broadening of DNA band width. As a result of the analyses the maximum separable base number is represented as a function of various electrophoretic variables. The best separations are possible with an electric field strength corresponding to gel thickness. The maximum separable base number increases as the gel thickness decreases. It also increases as the migration distance increases, but it becomes saturated and has an upper limit when the migration distance is long. This upper limit increases as gel thickness decreases. DNA fragments with 600 and 601 bases can be completely separated from each other under optimum conditions for a 0.2 mm thick gel plate. Furthermore, using the band spacing information, under the same conditions, 750 bases could be assigned separately.
Capillary zone electrophoresis for the quantitation of oligosaccharides formed through the action of chitinase
Authors: Lee Kyung‐Bok , Kim Yeong‐Shik , Linhardt Robert J.
Published: May 2005

Capillary zone electrophoresis with fluorescence detection was used to analyze the products formed by chitinase acting on N‐acetylchitooligosaccharide‐fluorescent conjugates. Six oligosaccharides of the structure [N‐acetylglucosamine(1→4)]n (where n = 1–6) were conjugated to 7‐amino‐1,3‐naphthalene disulfonic acid by reductive amination. Each oligosaccharide‐fluorescent conjugate was purified by preparative gradient polyacrylamide gel electrophoresis, semi‐dry electrotransfer to a positively‐charged nylon membrane and recovered by washing the membrane with salt solution. The products formed by treating each oligosaccharide‐fluorescent conjugate with chitinase were analyzed by capillary zone electrophoresis. The chitinase treatment hexasaccharide‐fluorescent conjugate was also examined kinetically to study the action pattern of this enzyme.
Increased selectivity in the detection of glycoproteins on nitrocellulose membranes by washing with sodium hydroxide solution
Authors: Kondo Maki , Harada Hiroshi , Sunada Saburo , Yamaguchi Takashi
Published: May 2005

We increased selectivity in the detection of glycoproteins on nitrocellulose membranes by introducing a washing step using sodium hydroxide solution. Glycoproteins on the nitrocellulose membrane were first oxidized by sodium periodate; biotin hydrazide was then coupled to the aldehyde groups generated in the sugar moiety of the glycoproteins. The membrane was washed twice using sodium hydroxide solution, and avidin‐horseradish peroxidase was then coupled to the remaining biotin. This system allows the detection of nanograms of glycoproteins on nitrocellulose membranes, and its specificity allows the clear distinction of glycoproteins from the nonglycosylated protein of bovine serum albumin.
The rat liver epithelial (RLE) cell protein database
Authors: Wirth Peter J. , Luo Lin‐di , Fujimoto Yoshinori , Bisgaard Hanne C. , Olson Arthur D.
Published: May 2005

Computer databases of rat liver epithelial (RLE) cellular polypeptides have been established using high resolution two‐dimensional gel electrophoresis and computer‐assisted analysis. Databases have been constructed utilizing both [35S]methionine‐ and [32P]orthophosphate‐labeled as well as silver‐stained polypeptides from normal RLE cells. The RLE database, which contains both qualitative and quantitative annotations, includes experiments with normal, chemically and oncogene transformed as well as spontaneously transformed cell lines. A total of 2537 [35S]methionine‐labeled polypeptides from whole cell lysates (1920 acidic and 617 basic, separated in the first dimension using isoelectric focusing and nonequilibrium pH gradient electrophoresis, respectively) were analyzed and databases constructed using the Elsie 5 gel analysis system. To increase the “viewing window” and hence the usefulness of the RLE database, subcellular fractionation of whole cell preparations was performed and high resolution two‐dimensional maps of the individual subcellular components were constructed. Databases representing 1229 cytosolic, 1539 acidic and 674 basic nuclear, 1746 membrane‐associated, 415 mitochondrial, 773 in vitro translated and 350 phosphoproteins were established from these maps. The RLE databases contain the Elsie 5 identification number, protein name (if known), molecular weight and pI information, quantitative and spot shape data, and specific information regarding transformation‐sensitive, growth‐related (exponentially proliferating versus confluent) cell populations as well as those polypeptides modulated by specific growth factors. The RLE databases represent initial efforts toward the establishment of comprehensive databases of rat liver proteins and serve as a vital resource for on‐going as well as future studies regarding the regulation of growth and differentiation as well as transformation of RLE cells.
Immobilized metal ion affinity electrophoresis: A preliminary report
Authors: Goubran‐Botros Hany , Vijayalakshmi Mookambeswaran A.
Published: May 2005

The ligand “Sepharose‐IDA‐Cu(II)” was entrapped into an agarose gel used for afinity electrophoresis. The binding of three closely related proteins, namely α‐chymotrypsinogen A, α‐chymotrypsin, and α‐chymotrypsin inactivated with disopropyl fluorophosphate (DIFP) to the affinity gel, was investigated. When the protein having affinity for the ligand was run in the presence of small amounts of the ligand, the retention of the protein by the ligand caused “tailing” of the sample. This pattern was changed in the presence of increasing amounts of the ligand, leading to a “rocket” shape due to the stronger binding of the protein to the chelated metal ligand entrapped in the gel. The degree of retardation in the gel with the ligand is an expression of the affinity between the protein and the ligand. The migration distance of α‐chymotrypsin and α‐chymotrypsin treated with DIFP at a given concentration of the ligand is linearly related to the protein amount deposited on the gel. The dissociation constant for the tested proteins were calculated from the Bøg‐Hansen‐Takeo plot. The difference in the affinity strength of these structurally related proteins towards the ligand suggests the involvement of the surface topography of histidine residues on their binding to the ligand.
Irreversible trapping of DNA during crossed‐field gel electrophoresis
Authors: Viovy Jean‐Louis , Miomandre Fabien , Miquel Marie‐Cecile , Caron François , Sor Frédéric
Published: May 2005

Using an original protocol with a rotating gel electrophoresis apparatus, it is shown that duplex DNA undergoing crossed‐field electrophoresis in agarose gets trapped in the gel when the field is increased above a threshold value which decreases with the chain length and depends on the angle between the fields in a non‐monotonous manner. This trapping is irreversible, i.e. once trapped at a high field strength, chains are unable to resume their motion when the field is returned to a lower value at which they moved prior to trapping. A model of trapping by “tight knots” is proposed. It predicts a trapping threshold proportional to the inverse square of the electric field, in qualitative agreement with the data. The implications of our results for the separation of large DNA molecules are discussed.
Use of cyclodextrins for the enantioselective separation of ergot alkaloids by capillary zone electrophoresis
Authors: Fanali Salvatore , Flieger Mirko , Steinerova Nadezda , Nardi Annalise
Published: May 2005

Ergot alkaloid enantiomer derivatives were resolved using capillary zone electrophoresis. The effect of cyclodextrins, added to the background electrolyte, on the migration time and the resolution was studied. Good separation for epimericergot alkaloid derivatives was also obtained using phosphate buffer at pH 2.5. Separation was improved by supplementing the background electrolyte with 30 mM of γ‐cyclodextrin. Good resolution of racemic ergot alkaloid derivatives in their enantiomers was achieved in a background electrolyte containing either β‐cyclodextrin or its derivative, or γ‐cyclodextrin.
Traveling‐wave dielectrophoresis of microparticles
Authors: Hagedorn Rolf , Fuhr Günter , Müller Torsten , Gimsa Jan
Published: May 2005

The traveling‐wave‐induced linear transfer of dielectric particles like living cells and artificial objects of microscopic dimensions is analyzed. It is shown that the electrode geometries must correspond to particle sizes to allow an effective manipulation of particles immersed in weakly electrolytic solutions by high frequency traveling waves. The theoretical model elaborated in this paper is in good agreement with experimental results obtained in microfabricated chambers of linearly arranged electrodes. It explains the behavior of homogeneous cellulose spheres as well as that of membrane‐covered pine polls. The traveling‐wave‐driven electrodes are described by a superposition of time‐dependent point charges. Subsequently, each of these point charges has to be considered as polarizing the dielectric particle and interacting with the polarized particle. This results in forces which effectively translocate the particle.
Stability of capillary gels for automated sequencing of DNA
Authors: Swerdlow Harold , Dew‐Jager Kerry E. , Brady Kevin , Grey Ronda , Dovichi Norman J. , Gesteland Raymond
Published: May 2005

Recent interest in capillary gel electrophoresis has been fueled by the Human Genome Project and other large‐scale sequencing projects. Advances in gel polymerization techniques and detector design have enabled sequencing of DNA directly in capillaries. Efforts to exploit this technology have been hampered by problems with the reproducibility and stability of gels. Gel instability manifests itself during electrophoresis as a decrease in the current passing through the capillary under a constant voltage. Upon subsequent microscopic examination, bubbles are often visible at or near the injection (cathodic) end of the capillary gel. Gels have been prepared with the polyacrylamide matrix covalently attached to the silica walls of the capillary. These gels, although more stable, still suffer from problems with bubbles. The use of actual DNA sequencing samples also adversely affects gel stability. We examined the mechanisms underlying these disruptive processes by employing polyacrylamide gel‐filled capillaries in which the gel was not attached to the capillary wall. Three sources of gel instability were identified. Bubbles occurring in the absence of sample introduction were attributed to electroosmotic force; replacing the denaturant urea with formamide was shown to reduce the frequency of these bubbles. The slow, steady decline in current through capillary sequencing gels interferes with the ability to detect other gel problems. This phenomenon was shown to be a result of ionic depletion at the gel‐liquid interface. The decline was ameliorated by adding denaturant and acrylamide monomers to the buffer reservoirs. Sample‐induced problems were shown to be due to the presence of template DNA; elimination of the template allowed sample loading to occur without complications. The improved samples permitted multiple consecutive DNA sequencing runs on the same capillary.
Compensation for mobility inequalities between lanes computed from band signals in on‐line fluorescence DNA sequencing
Authors: Fujii Hidehiko , Kashiwagi Katsuya
Published: May 2005

Among on‐line fluorescence DNA sequencing systems, the four‐lane method exhibits the potential for reporting an erroneous sequence due to nonuniform mobility of the DNA fragments migrating among the four lanes. This error is manifest in phenomenon commonly called smiling. This paper presents a computational algorithm which compensates for the mobility inequalities between lanes using signal data obtained from the shorter DNA fragments forming the faster migrating bands. The program mainly consists of two routines: (i) calculation of calibration coefficients (mobility ratios between lanes), and (ii) examination of the coefficients by applying them to a later domain of the same signals. Both routines are connected with several feed‐back branches for recalculation. Homology analysis of final sequences has shown that the accuracy rate is maximized with this algorithm and any ambiguous result can be assigned to the residual error inherent in the band identification method used.
Movement of fluorescence pattern after photobleaching: An accelerated procedure for DNA electrophoretic mobility analysis
Authors: Chu Benjamin , Wang Zhulun
Published: May 2005

A new approach which is compatible with many of the existing procedures for the analysis of DNA species in gel electrophoresis is being demonstrated. It takes advantage of fluorescence photobleaching in order to create a sharp boundary between the stained and the (partially) photobleached DNA. By arbitrarily creating a stained DNA band of narrower width, the sensitivity to detect (averaged) DNA band movements has been increased. This feature permits measurements of time‐dependent electrophoretic mobility over very short time periods. The approach can be used to shorten the running time of gel electrophoresis experiment and to increase the resolution because of the sharper boundary and narrower band width. With faster running time, diffusion of both DNA and dye in the gel also becomes less serious. Movement of fluorescence pattern after photobleaching also permits measurements of localized motions when the gel pores are small in comparison with DNA sizes. Experiments demonstrating some aspects of the proposed technique, as well as the anticipated limitations, are presented and discussed.
Molecular sieving of lambda phage DNA in polyacrylamide solutions as a function of the molecular weight of the polymer
Authors: Pulyaeva Helena , Wheeler David , Garner Mark M. , Chrambach Andreas
Published: May 2005

Electrophoresis of lambda phage DNA was carried out in solutions at various concentrations of uncrosslinked polyacrylamide of 0.6, 1, 5 and 9 × 106 molecular weight (Mw) with narrow Mw distribution. By inspection of mobilities in the various concentration ranges, it appears that mobilities decrease, and retardation increases, with increasing Mw. The relation between electrophoretic retardation and the Mw of the polymer was also interpreted (i) in the manner previously applied to nonlinear Ferguson plots and compatible with the Ogston model; and (ii) empirically, on the basis of the first derivatives of the functions describing the Ferguson plots at the polymer concentrations used. Interpretation (i) shows that the retardation increases linearly in the order of 0.6, 1, 5 and 9 × 106 Mw of polyacrylamide. Interpretation (ii) shows a nonlinear increase of retardation in the Mw range 5 to 9 × 106, and a decrease in retardation as Mw is raised from 0.6 to 5.0 × 106. Hypothetically, interpretation (ii) can be explained mechanistically by a progressive change, as the polymer size is increased, from a collision with the surface of the polymer fiber to one occurring after permeation in the interior of a randomcoiled fiber. Interpretation (i) may fail to detect that change due to the large difference between DNA mobility in solutions of the smallest polymer and the free mobility. DNA peak detection in all of the four size classes of polyacrylamide in solution is limited to relatively narrow ranges of polymer concentration. For the electrophoresis of lambda DNA, polyacrylamide of 5 × 10−6 Mw represents an optimum due to a compromise between small mobility differences at various concentrations of the polymer with Mw 0.6 × 106, either little retardation (interpretation (i)) or insufficient responsiveness of the Ferguson plot to concentration changes (interpretation (ii)) in presence of the polymer of 1 × 106 Mw and an excessively narrow available concentration range at 9 × 106 Mw. A highly disperse solution of polyacrylamide of 18 × 106 molecular weight retarded lambda DNA only slightly. Retardation decreased upon ultrafiltration of the polymer solutions, presumably through the decrease of their concentrations or adsorption of the largest chains within the size distribution.
Advances in DNA electrophoresis in polymer solutions
Authors: Tietz Dietmar , Aldroubi Akram , Pulyaeva Helena , Guszczynski Tadeusz , Garner Mark M. , Chrambach Andreas
Published: May 2005

DNA electrophoresis in gels and solutions of agarose and polyacrylamide was objectively evaluated with regard to separation efficiency at optimal polymer concentrations. In application to DNA fragments, polyacrylamide gels were superior for separating fragments of less than 7800 bp, and agarose gels are the best choice for larger fragments. Agarose solutions are nearly as good as polyacrylamide gels for small DNA (< 300 bp). Agarose solutions have a higher efficiency than polyacrylamide solutions for DNA of less than 1200 bp. Separation efficiency sharply decreases with increasing length of DNA. Retardation in polyacrylamide solutions was found to depend on polymer length in a biphasic fashion. The choice of resolving polymer concentrations depends on the progressive stretching of DNA in proportion to polymer concentration. The rate of that stretching appears higher in polyacrylamide solution than in gels or in liquid or gelled agarose. Application of polymer solutions to capillary electrophoresis raises further problems concerning agarose plugs, DNA interactions with the polymers, operation at low field strength and long durations as well as detection sensitivity.
Physical genome analysis of bacteria
Authors: Römling Ute , Grothues Dietmar , Heuer Thomas , Tümmler Burkhard
Published: May 2005

Pulsed‐field gel electrophoresis (PFGE) is a general analytical tool to separate large DNA molecules and may therefore be applied to problems from all areas of bacteriology. The genome size of bacteria covers the range of 0.6 to 10 megabase pairs. For genome fingerprinting, the bacterial chromosome is cleaved with a restriction endonuclease that gives a resolvable and informative number of five to one hundred fragments on the PFGE gel. Restriction enzymes are chosen according to GC content, degree of methylation, and codon usage of the respective bacterial genus. Macrorestriction fingerprinting allows the identification of bacterial strains and the distinction between related and unrelated strains. If fragment patterns of several restriction digestions are quantitatively evaluated, strains can be classified according to genetic relatedness at the level of genus, species, and biovar. In particular, members of a clonal lineage can be uncovered. Hence, any problem from applied, environmental, and clinical microbiology may be addressed by PFGE restriction analysis where the spatiotemporal spread of a bacterial clone is of interest. In bacterial genomics, PFGE is employed for the top‐down construction of macrorestriction maps of the chromosome which yields data about genome organization, mobile genetic elements, and the arrangement of gene loci and gene families. The genomic diversity of a bacterial species is elucidated by comparative chromosome mapping. Map positions of restriction sites and gene loci of interest serve as landmarks to assess the extent of gross chromosomal modification, namely insertions, deletions and inversions. Intra‐ and interspecies comparisons of genome organization provide insights into the structure and diversity of bacterial populations and the phylogeny of bacterial taxa.
Separation of peptides on Empore™ thin‐layer chromatography sheets and blotting onto polyvinylidene difluoride membranes with subsequent gas phase sequencing
Authors: Reisinger Peter W. M. , Kleinschmidt Traute , Welsch Ulrich
Published: May 2005

Methods for the separation of peptides on a new type of thin‐layer chromatography (TLC) sheet and blotting onto polyvinylidene difluoride (PVDF) membranes with subsequent gas phase sequencing are described. For validation, the A and B chain of insulin were chromatographed on EmporeTM TLC sheets and either extracted or blotted onto PVDF membranes. The advantages and disadvantages of thin‐layer chromatography on Empore sheets versus high performance liquid chromatography (HPLC) are discussed, along with the possibility of combining the two methods. In addition, TLC was combined with electrophoresis (finger‐printing) for the separation of complex peptide mixtures. Blotting from TLC sheets onto PVDFmembranes was performed in two ways: contact diffusion and electrophoretic transfer. In our experiments electroblotting was more effective. Amino acid sequence determination of the B chain of insulin was possible both after extraction from the TLC sheet and after blotting onto PVDF membranes. In the former case, liquid phase sequencing and, in the latter case, gas phase sequencing was performed. The possibility to blot from TLC sheets onto membranes, e.g. PVDF, may prove useful in many fields, for example in biochemistry, and in molecular and cell biology.
Simple repeats are not found more abundantly at G/C‐rich regions
Authors: Melmer Georg
Published: May 2005

Three hundred and fifty cosmids from chromosome 7, previously analyzed for the presence of rare‐cutting restriction enzyme sites, were analyzed for the presence of microsatellite sequences [(CA)n or (CT)n repeats]. Of these, 147 cosmids were found to contain at least one (CA)n repeat unit and 51 cosmids contained at least one (CT)n repeat unit. No evidence was found for the prevalence of microsatellite repeat units in the vicinity of rare‐cutter restriction enzyme sites.
Genetic heterogeneity of prostatic carcinoma‐derived cell lines as emphasized by DNA fingerprinting
Authors: Bettink Stephanie , Wullich Bernd , Christmann Alexander , Zwergel Thomas , Zang Klaus‐Dieter , Unteregger Gerhard
Published: May 2005

To investigate genetic heterogeneity during in vitro cultivation of human prostatic carcinomas following radical prostatectomy we performed DNA fingerprinting using the digoxygenin‐labeled probes (GACA)4 and (GTG)5. DNA was isolated from fresh material stemming from different areas within one tumor and from cell cultures of the same material. The patterns which were obtained by the nucleolar organizer region (NOR)‐specific probe (GACA)4 exhibit only a few prominent low molecular mass bands and no differences were observed between any of the tumors analyzed so far. Changes in the fingerprint pattern occurred between cell cultures derived from different areas within one tumor when the DNA was cleaved by HaeIII and signals detected with the (GTG)5 probe. The “area‐specific” pattern was stable during several subcultivations of these cell lines, indicating genetic stability of these prostatic carcinoma cells in vitro. Thus individual cell lines derived from radical prostatectomy seem to represent a biological system very close to the situation in vivo.
Evaluation of isoelectric focusing runs as specific resistance/volthour plots
Authors: Köst Hans‐Peter
Published: May 2005

Isoelectric focusing (IEF) runs, e.g. on ultrathin gels, are characterized by an extensive change of gel electric parameters, caused by the rearrangement of carrier ampholyte components from a uniform distribution to a highly ordered pH gradient. A particularly important parameter is the specific resistance ϱ [Ohm*cm] which has been determined in polymerization mixtures (with and without carrier ampholytes) and in 125× 0.15 mm ultrathin gels, pH 3–10 with 5 %T, 3 %C, 5 % Servalyt carrier ampholytes, pH 3–10. The starting specific resistance ϱ of ultrathin IEF gels, calculated from the geometric gel dimensions and electric current values (V, mA), is in agreement with the data determined directly in 30 mL samples of polymerization mixtures by using a conductivity meter. Electric specific conductivity/Volthour (Vh) plots proved to be a valuable tool for the evaluation of gel systems with and without protein samples during IEP runs. These plots are usually S‐shaped, indicating that the key part of pH gradient formation takes places in a relatively short time. A “good” ultrahin gel, after a short lag phase, shows a rapid increase in specific resistance due to a rapid pH gradient formation and a slope of about 18 Ohm*cm/Vh. IEF is finished in about 3000 Vh. After prolonged gel storage, and especially in partially dried gels, the electrical parameters approach equilibrium only slowly, as indicated by the relatively shallow slope (8.9 Ohm*cm/Vh). Accordingly, separations need more than 4000 Vh. In gels with salt containing samples, after normal IEF for about 1750 Vh, cracks appear, often followed by sparking at about 2700 Vh, accompanied by a sharp (often intermittent) drop in specific resistance.
Reduced chemical and radioactive liquid waste during electrophoresis using polymerized electrode gels
Authors: Kleine Bernhard , Löffler Gerhard , Kaufmann Heidrun , Scheipers Peter , Schickle Hanspeter , Westermeier Reiner , Bessler Wolfgang G.
Published: May 2005

Using polyacrylamide electrode gels during horizontal sodium lauryl sulfate‐polyacrylamide electrophoresis of radiolabeled proteins instead of electrode papers reaching into electrode buffer reservoirs, silver staining is improved and we reduce chemical and radioactive liquid waste during electrophoresis.
Purification to single isoforms of a secreted epidermal growth factor receptor in a multicompartment electrolyzer with isoelectric membranes
Authors: Wenisch Elisabeth , Righetti Pier Giorgio , Weber Wolfgang
Published: May 2005

A purified, size‐homogeneous (100 kDa), desialylated form of a truncated, soluble form of the epidermal growth factor receptor secreted by A431 human tumor cells has been found, by isoelectric focusing in immobilized pH gradients, to consist of two major isoforms (with pIs of 6.96 and 6.71), one intermediate form (pI 6.45) and a number (> 10) of minor components. The two major components have been purified to charge homogeneity by isoelectric focusing in amulticompartment electrolyzer with buffering isoelectric membranes having the following pI values: 5.90, 6.63, 6.76, 6.92, 7.05 and 7.35. Such single pI species are presently used for attempts at crystal growing.
Enantiomers resolution in capillary zone electrophoresis by using cyclodextrins
Authors: Schutzner Wolfgang , Fanali Salvatore
Published: May 2005

Cyclodextrins added to the background electrolyte are shown to be useful for the resolution of racemic compounds in their enantiomers. Several parameters have to be controlled in order to achieve resolution, e.g., cyclodextrin type, concentration, analyte shape, as well as column temperature. The resolution of nor‐epinephrine, epinephrine and isoproterenol in their enantiomers decreased by increasing the column temperature. Octopamine and ketamine have been resolved by supporting the background electrolyte with 2, 6‐di‐O‐methyl‐β‐cyclodextrin. In spite of the stronger inclusion‐complex of ketamine than octopamine with the modified cyclodextrin its resolution was not satisfactory.
Capillary electrophoresis of macromolecules in ‘syrupy’ solutions: Facts and misfacts
Authors: Chiari Marcella , Nesi Marina , Fazio Marco , Righetti Pier Giorgio
Published: May 2005

‘Syrupy’ solutions of liquid linear polyacrylamide (⩾ 10%T, 0%C) appear to be excellent for fractionation of oligonucleotides and potentially, for DNA sequencing. For such analyses, the silica wall must be coated by covalently bound strings of polyacrylamide; otherwise, the electroosmotic flow will slowly pump out the viscous electrolyte solution. Due to the enormous viscosity (100 Pa s for an 8 %T solution) the polymer strings must be prepared in situ, by filling the capillary with the appropriate monomer solution. The reaction, however, cannot be driven to better than 80–85% conversion: in 10%T, the concentration of unreacted monomers will thus be 200–300 mM. This will give a substantial background absorbance (even at 254 nm) and leave a huge amount of potentially harmful reacting species in the background electrolyte. A chemical scavenging method is proposed here: after polymerization, a 100 mM solution of cysteine is driven in from the cathode and allowed to react for up to 10 h. At the end of the reaction period, the excess cysteine and its acrylamido adduct are driven out electrophoretically and the column is reconstituted with its normal background electrolyte. Columns thus preconditioned have been found to perform extremely well and to last as long as the inner coating (and the linear polymer filling) will last. No ‘carry over’ from run to run was experienced.
Electrophoretic analysis of electrically trained skeletal muscle
Authors: O'Brien Geraldine A. , Corbett Joseph M. , Dunn Michael J. , Cumming Debbie V. E. , May Andrew J. , Yacoub Magdi H.
Published: May 2005

Sheep latissimus dorsi muscle was electrically trained, thereby inducing fast‐to‐slow fibre‐type transformation. Using a combination of one‐ and two‐dimensional gel electrophoresis techniques with computer analysis, we have analysed altered expression of contractile protein isoforms at protein and mRNA level over a time course of electrical training extending to 5 months. Myosin heavy chain and regulatory myosin light chain analysis showed predominant expression of their slow isoforms (86% and 92%, respectively) after 3 months of training. At the same time point, however, tropomyosin analysis revealed that the slow isoform of the α‐subunit accounted for 64% of the total α expression. Troponin T isoform switching proceeded more slowly over the same time course than tropomyosin and the thick filament proteins studied. Troponin T analysis revealed 5 fast and 2 slow isoforms in the sheep, of which the second slow isoform only became clearly visible after 5 months' training. At this time point the two slow isoforms were more predominant than their fast counterparts. This suggests that a wide heterogeneity of fast and slow isoform combinations are possible in the thin filament of skeletal muscle.
Application of sequential extraction procedures and glycoprotein blotting for the characterization of the 2‐D polypeptide patterns of barley seed proteins
Authors: Weiss Walter , Postel Wilhelm , Görg Angelika
Published: May 2005

Barley (Hordeum vulgare L.) proteins were sequentially extracted from ground seeds with Tris‐HCl buffer, 55% 2‐propanol, 55% 2‐propanol containing 1% dithiothreitol, and 6 M urea containing 2 % Nonidet P‐40 and 1 % dithiothreitol. The protein composition of these solubility fractions was then analyzed by high resolution two‐dimensional gel electrophoresis with immobilized pH gradient 4–9 in the first dimension, followed by silver staining and glycoprotein blotting, respectively, for a more detailed characterization of the two‐dimensional polypeptide pattern of barley seed proteins.
Qualitative and quantitative changes in barley seed protein patterns during the malting process analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with respect to malting quality
Authors: Weiss Walter , Postel Wilhelm , Görg Angelika
Published: May 2005

Seeds of two barley cultivars, similar in total protein content and malt extract yield but different in their final attenuation values, were malted. Samples taken at daily intervals during the malting process were extracted sequentially with Tris‐HCl buffer, aqueous 2‐propanol, aqueous 2‐propanol containing 0.5% dithiothreitol, and 4 M urea, containing 0.5% dithiothreitol and 1% Nonidet P‐40. The protein composition of these extracts was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and computer densitometry to determine whether differences observed in the rate or extent of protein modification are related to the malting quality character final attenuation. It was found that, common to both cultivars, the albumin and globulin proteins were relatively resistant to proteolysis, whereas the hordeins suffered a dramatic breakdown during malting, with the D hordein being degraded most rapidly, followed by the B and C hordeins. Besides these similarities, differences between both cultivars were observed in the relative rates of D hordein degradation, as this rate was considerably higher in the cultivar with high malting quality. Similar, but much less distinct kinetics were seen with certain B hordeins. Since a possible relationship might exist between the rate of proteolysis of the D hordeins and the character final attenuation, we analyzed a larger number of barley cultivars with different final attenuation values with a simplified technique. For the ten cultivars examined, differences during germination were again seen in the rates of modification of the D hordeins. However, significant correlations between the D hordein breakdown and final attenuation values were not obtained, so that we propose that there exists at best a loose correlation between the relative rate of proteolysis of these proteins and the malting quality character final attenuation.
Discontinuous electrophoresis of β‐caseins for the determination of bovine caseins in milk and dairy products
Authors: Mayer Walter , Hörtner Hermann
Published: May 2005

Discontinuous acidic anodic polyacrylamide gel electrophoresis enables the separation of bovine β‐caseins from those of ovine and caprine. Interfering protein bands as a consequence of ripening or processing have not been detected. After evaluation of the stained gel by laser densitometry, quantification was performed with calibration standards on the same gel by the ratio of the peak areas from bovine to ovine and bovine to caprine, respectively. Thus, independence from the extractability of proteins affected by denaturation and ripening (which might in some cases raise the limit of detection) is achieved. The range of quantification extends from 5 to approximately 70% bovine casein in relation to total casein.
Fish species identification by peptide mapping of the myosin heavy chain
Authors: Rehbein Hartmut
Published: May 2005

Fish species of seafood made of washed fish flesh (e.g. imitation crab meat from surimi) were identified by peptide mapping of the myosin heavy chain (MHC). In the first step the MHC was separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in 7.5% T gel, followed by a second SDS‐PAGE in a 15% T gel with proteolytic digestion of the MHC in the gel. The resulting peptides gave species specific patterns.
Polyethyleneglycol methacrylate 200 as an electrophoresis matrix in hydroorganic solvents
Authors: Zewert Thomas , Harrington Michael
Published: May 2005

The properties of gels composed of poly(polyethyleneglycol methacrylate) and copolymers of polyethyleneglycol methacrylate and acrylamide were studied. These novel electrophoresis matrices are amphigels (swellable in water and organic solvents) that have unprecedented organic solvent compatibility. Hydrophobic proteins which are poorly solubilized in aqueous detergent systems (e.g., zein) are well‐resolved in these gels with hydro‐organic solvents. This is especially relevant for isoelectric focusing, to avoid using either ionic detergents that may interfere with the focusing, or urea that may cause carbamylation of proteins. Variations of crosslinker, buffer, and solvent systems in these gels were explored.
Investigation of the electroosmotic flow effect on the efficiency of capillary electrophoresis
Authors: Andreev Victor P. , Lisin E. E.
Published: May 2005

The influence of the electroosmotic flow profile on the efficiency and resolution of capillary electrophoresis is studied. The mathematical model is formulated and the set of equations is solved numerically. The results of the analysis are applicable to a wide range of buffer concentrations and capillary diameters. The temperature dependence of electrophoretic mobility, viscosity and thermal conductivity and the dependence of electrical conductivity on temperature and ion concentration in the double layer are taken into account. It is shown that there exists a region of buffer concentrations and capillary diameters where the influence of the electroosmotic flow profile on the efficiency and resolution is much greater than that of the temperature dependence of the electrophoretic mobility. The results are especially essential for small buffer concentrations or capillary diameters comparable with the double electrical layer thickness.
Computer simulation of transient states in capillary zone electrophoresis and isotachophoresis
Authors: Ermakov Sergey V. , Mazhorova Olga S. , Zhukov Michael Y.
Published: May 2005

Transient states in the evolution of electrophoretic systems comprising aqueous solutions of weak monovalent acids and bases are simulated. The mathematical model is based on the system of nonstationary partial differential equations, expressing the mass and charge conservation laws while assuming local chemical equilibrium. It was implemented using a high resolution finite‐difference algorithm, which correctly predicted the behavior of the concentration, pH and conductivity fields at low computational expense. Both the regular and the irregular modes of separation in capillary zone electrophoresis and isotachophoresis are considered. It is shown that the results of separation, particularly zone order, strongly depend on pH distribution. Simulation data as well as simple analytical assessments may help to predict and correctly interpret the experimental results.
The human keratinocyte two‐dimensional gel protein database (update 1992): Towards an integrated approach to the study of cell proliferation, differentiation and skin diseases
Authors: Celis Julio E. , Rasmussen Hanne Holm , Madsen Peder , Leffers Henrik , Honoré Bent , Dejgaard Kurt , Gesser Borbala , Olsen Eydfinnur , Gromov Paul , Hoffmann Hans J. , Nielsen Morten , Celis Ariana , Basse Bodil , Lauridsen Jette B. , Ratz Gitte P. , Nielsen Helle , Andersen Annette H. , Walbum Else , Kjærgaard Inge , Puype Magda , Van Damme Josef , Vandekerckhove Joël
Published: May 2005

The master two‐dimensional gel database of human keratinocytes currently lists 2980 cellular proteins (2098 isoelectric focusing, IEF; and 882 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to posttranslational modifications. About 20% of all recorded proteins have been identified (protein name, organelle components, etc.) and they are listed in alphabetical order together with their Mr, pI, cellular localization and credit to the investigator(s) that aided in the identification. Also, we have listed 145 microsequenced proteins that are recorded in this database. As an aid in localizing the polypeptides we have included blow‐ups of the master images (IEF, NEPHGE) displaying all the protein numbers. In the long run, the master keratinocyte database is expected 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 keratinocyte functions both in health and disease.
S‐Carboxymethylation of proteins transferred onto polyvinylidene difluoride membranes followed by in situ protease digestion and amino acid microsequencing
Authors: Iwamatsu Akihiro
Published: May 2005

S‐Carboxymethylation of immobilized proteins, separated by gel electrophoresis and transblotted to polyvinylidene difluoride (PVDF) membranes is described. In situ protease digestion was used to produce peptide fragments in high yields that were suitable for microsequencing. These fragments could also be used to produce peptide maps, obtained by reverse‐phase high performance liquid chromatography (HPLC), that exhibited excellent reproducibility and were comparable to results obtained from digestions in solution. This technique makes it possible to identify Cys residues during Edman degradation. Approximately 70% of the amino acid residues from a protein were determined using only 50–200 pmol. Sufficient information for production of oligonucleotide probes was obtained using 10–50 pmol of material applied to a polyacrylamide gel.
Five new alleles of plasma Zn‐alpha 2‐glycoprotein variants phenotyped by isoelectric focusing and immunoblotting in twelve populations
Authors: Nakayashiki Nori , Umetsu Kazuo , Yuasa Isao , Suenaga Kazuyuki , Omoto Keiichi , Ishida Takafumi , Misawa Shogo , Katsura Syusaku
Published: May 2005

Human Zn‐alpha 2‐glycoprotein (ZAG) in plasma samples from twelve populations was tested by immunoblotting after polyacrylamide gel isoelectric focusing. Eleven ZAG phenotypes produced by one common and nine rare alleles, including five new ones (ZAG*6–ZAG*10), were detected. Additionally, an application of separator IEF with N′‐2‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid (HEPES) was found to be useful for discriminating the rare ZAG 7 band.
Tissue‐specific variation of pea mitochondrial polypeptides detected by computerized image analysis of two‐dimensional electrophoresis gels
Authors: Humphery‐Smith Ian , des Francs‐Small Catherine Colas , Ambart‐Bretteville Françoise , Remy René
Published: May 2005

A tissue‐specific variation of a total of 152 mitochondria‐associated polypeptides is detailed for etiolated leaves, epicotyls and roots from the pea, Pisum satisum L., using computerized image analysis. These organ systems possess, respectively, 128, 100, and 96 mitochondria‐associated polypeptides with 38, 8, and 10, respectively, being unique to each. Seventy‐one polypeptides were observed to be present in all gels, but some of these also underwent a variety of quantifiable changes. Two polypeptides were identified that varied similarly to a polypeptide of interest (W), known to be abundant in pea epicotyls and potato tubers. It is postulated that these latter three polypeptides are therefore probably related functionally.