bluuebunny/crossref_metadata_2025_split huggingface dataset:
Physical genome analysis of bacteria
Authors: Römling Ute , Grothues Dietmar , Heuer Thomas , Tümmler Burkhard 
Published: May 2005

AbstractPulsed‐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

AbstractMethods 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

AbstractThree 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

AbstractTo 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

AbstractIsoelectric 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

AbstractUsing 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

AbstractA 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

AbstractCyclodextrins 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

Abstract‘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

AbstractSheep 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

AbstractBarley (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

AbstractSeeds 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

AbstractDiscontinuous 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

AbstractFish 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

AbstractThe 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

AbstractThe 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

AbstractTransient 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

AbstractThe 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

AbstractS‐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

AbstractHuman 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.