bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genomic fingerprinting of Yersinia enterocolitica species by degenerate oligonucleotide‐primed polymerase chain reaction
Authors: Sayada Chalom , Picard Bertrand , Elion Jacques , Krishnamoorthy Rajagopal 
Published: May 2005

AbstractA novel version of the polymerase chain reaction (PCR) termed degenerate oligonucleotide‐primed PCR (DOP‐PCR) was used to fingerprint the bacterial genome of Yersinia enterocolitica species. Eight well‐characterized reference strains of Y. enterocolitica (ribotype, serotype, biotype and zymotype) were examined. Optimal experimental conditions for reproducibility, sensitivity and specificity were obtained using a single DOP‐primer. All the strains gave distinct DOP‐PCR profiles composed of 12 to 19 fragments with sizes from 200 to 1.500 bp. Thus, the DOP‐PCR, which has so far been used to fingerprint human, mouse, fruitfly and plant DNA, is also well‐suited to bacterial DNA.
Double‐ and single‐strand conformation polymorphism analysis of point mutations and short tandem repeats
Authors: Barros Francisco , Muñoz‐Barús Ignacio , Lareu Maria V. , Rodriguez‐Calvo Maria S. , Carracedo Angel 
Published: May 2005

AbstractSingle point mutations in small DNA fragments and single unit differences in simple repetitive DNA can be detected as double‐strand conformation polymorphisms using polyacrylamide gel electrophoresis, with and without sodium dodecyl sulfate, even at temperatures as low as 3°C. Changes in a single base are distinguished by means of the analysis of the heteroduplexes, and changes in more than two bases can be distinguished in both homoduplexes and heteroduplexes. Polymerase chain reaction (PCR) conditions can be designed not only to amplify homoduplexes, heteroduplexes and singlestrand DNA at the same time, but also to focus the analysis on either singlestrand conformation polymorphism (SSCP) or double‐strand conformation polymorphism (DSCP). DSCP seems to be advantageous in typing DNA polymorphisms or mutations in loci with few variants, but, because it is necessary to have a simple pattern of all possible combinations of the alleles, it is not as advantageous in typing systems with many variants.
Electroosmosis of polymer solutions in fused silica capillaries
Authors: Bello Michael S. , De Besi Patrizia , Rezzonico Roberta , Righetti Pier Giorgio , Casiraghi Ernestina 
Published: May 2005

AbstractThe classical von Smoluchowski equation predicts that the electroosmotic mobility generated by the wall zeta potential could be suppressed if the viscosity of the solution adjacent to the wall were extremely high. When performing runs in capillaries filled with polymer solutions (2% methyl cellulose solutions with viscosities of 25 cP), however, one consistently finds that the quenching of electroosmotic mobility is substantially less than predicted by the von Smoluchowski relationship. The electroosmotic flow is progressively suppressed with subsequent electrophoretic runs, suggesting a “dynamic coating” of the polymers onto the capillary wall. This progressive reduction of electroosmotic mobility tends to a plateau value which is still substantially higher than the value derived on the basis of the von Smoluchowski relationship. The following explanation is proposed: due to the very high shear rate in the electric double layer, the polymer molecules change their orientation and/or conformation, which lowers the fluid viscosity in this region. A scaling equation for electroosmotic mobility taking into account the non‐Newtonian properties of polymer solutions is derived. It predicts electric field dependence of the electroosmotic mobility as the shear rate in the double layer is proportional to the electric field. Experimental measurements confirm the dependence of the electroosmotic mobility on the electric field.
Characterization of the acute phase serum protein response in pigs
Authors: Lampreave Fermín , González‐Ramón Nieves , Martínez‐Ayensa Silvio , Hernández Miguel‐Angel , Lorenzo Hans‐Kristian , García‐Gil Agustín , Piñeiro Andrés 
Published: May 2005

AbstractAcute inflammation was induced in pigs using a single subcutaneous turpentine injection. The acute phase serum protein response was analyzed using crossed immunoelectrophoresis and immunodiffusion. The concentration of C reactive protein and haptoglobin increases 5–7 times 48 h after the injection, whereas the concentration of an α2‐globulin, named pig major acute phase protein (pig‐MAP), increases at least 15‐fold. A molecular mass of 115 kDa for pig‐MAP was determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. This protein did not crossreact with antisera to human hemopexin, ceruloplasmin, H‐kininogen and complement factor C3. Albumin and α‐lipoprotein were negative acute phase proteins because their concentration significantly decreased during inflammation. Finally, the concentration of α1‐acid glycoprotein, fetuin, α1‐protease inhibitor, transferrin and α2‐macro‐globulins, as well as total proteins, did not change significantly during inflammation.
An informativity index for multilocus DNA fingerprints
Authors: Krawczak Michael , Lubjuhn Thomas 
Published: May 2005

AbstractAn informativity index for multilocus DNA fingerprints, r, was developed. It is based upon the Shannon information that a paternal DNA fingerprint conveys about the offspring phenotype pattern. Both simulation and empirical data reveal that the index r is strongly correlated with the mean log likelihood ratio (paternity vs. nonpaternity) expected in trio cases of true paternity. Since r can be estimated from DNA fingerprints of unrelated individuals in advance, it will provide an easy means to assess the potential utility of a given probe/enzyme combination in kinship testing.
Detection of pectinesterase in polyacrylamide gels
Authors: Alonso Jesús , Rodríguez M. Teresa , Canet Wenceslao 
Published: May 2005

AbstractA rapid and sensitive method of detecting pectinesterase activity following electrophoresis or isoelectric focusing in polyacrylamide gels is described. The method uses ruthenium red and requires no addition of substrate when making the gels, thus obviating direct enzyme‐substrate contact during electrophoresis. Because of its versatility, the method can be used in a wide variety of applications, such as plant and microbial taxonomy, enzyme purification and characterization, or as an analytical method in fresh and processed plant technology.
Analysis of recombinant human tumor necrosis factor beta by capillary electrophoresis
Authors: Yao Yi Ju , Loh Kean Chong , Chung Maxey C. M. , Li Sam F. Y. 
Published: May 2005

AbstractCapillary electrophoresis was used for the analysis of recombinant human tumor necrosis factor beta (rhTNF‐β). Reproducible separation of the protein could be achieved in Polybrene‐coated capillaries. Optimum conditions for separation were achieved with a pH 4.5 sodium acetate buffer. Besides Polybrene‐coated capillaries, other commercially available column and column‐coating reagents were also examined for the separation of rhTNF‐β. Capillary electrophoresis was found to be a potentially useful method for product analysis and process monitoring in recombinant‐DNA technology.
Trapping gel electrophoresis of end‐labeled DNA: An analytical model for mobility and diffusion
Authors: Slater Gary W. , Desruisseaux Claude , Villeneuve Christine , Guo Hong L. , Drouin Guy 
Published: May 2005

AbstractAs shown by Ulanovsky, Drouin and Gilbert (Nature 1990, 343, 190–192), the gel electrophoretic migration of DNA is severely reduced by steric trapping when streptavidin is attached to one end of the polyelectrolyte. We present a model that allows us to calculate both the mobility and the diffusion coefficient, hence the resolution factor of the resulting separation. We compare our results to those of Défontaines and Viovy (Electrophoresis 1993, 14, 8–17) and we show that the averages over the molecular conformations must be done carefully. We also show that trapping increases diffusion substantially and that this makes constant‐field trapping electrophoresis incapable of increasing the number of bases read per sequencing run. Finally, we conclude that severe trapping may lead to highly anomalous transport behavior where one cannot define a velocity or a diffusion constant.
Detection of two hypervariable (ATTTT)n loci in the human genome
Authors: Dürr Christine , Hinney Anke , Luckenbach Christine , Ritter Horst 
Published: May 2005

AbstractObject of this investigation was the isolation of a single‐locus probe from a multi‐locus fingerprint. Individual specific multi‐locus fingerprints in man were generated by using the oligonucleotide probe (ATTTT)5. An isolated (ATTTT)5‐positive DNA fragment was analyzed using polymerase chain reaction (PCR) cycle‐sequencing and nonradioactive direct‐blotting electrophoresis. A digoxigenated oligonucleotide synthesized according to this sequence was used as a single‐locus probe. Two hypervariable loci were detected on Southern blots. Formal genetic investigations for the two loci were performed in order to estimate the allele frequencies. Locus 1 shows an individual‐specific banding pattern with an autosomal‐codominant inheritance and can be used for forensic investigations. Locus 2 also represents a polymorphic pattern, but the inheritance is not according to the Mendelian rules. Probably we have detected a highly mutagenic locus in the human genome.
Temperature gradient gel electrophoresis for analysis of clonal evolution in non‐Hodgkin's lymphoma of the thyroid
Authors: Tiemann Markus , Menke Martin A. O. H. , Asbeck Robert , Wacker Hans‐Heinrich , Parwaresch Reza 
Published: May 2005

AbstractTo analyze the transition of an autoimmune disease into a mucosa‐associated lymphoid tissue‐non‐Hodgkin's lymphoma (MALT‐NHL), we investigated a total of 27 cases of clinically diagnosed autoimmune thyroiditis with lymphoid hyperplasia. Three cases of thyroid hyperplasia served as controls. Monoclonal B cells were detected by studying rearrangement patterns of the hypervariable CDR III regions within the immunoglobulin heavy chain gene locus and the T‐cell receptor γ chain gene (TCRG). We used a seminested polymerase chain reaction (PCR) to demonstrate immunoglobulin rearrangements and a multiplex PCR for TCRG rearrangements. The PCR products were analyzed by temperature gradient gel electrophoresis to expand mixtures of homo‐ and hetero‐duplices within heterogeneous populations of B cells. With this approach we found monoclonality in 14 of the 27 cases of Hashimoto's disease. In a reinvestigation we discovered additional histological and immunohistochemical features of MALT‐NHL in 17 cases. The 14 cases of thyroiditis with clonally expanded B cells clearly demonstrate the transition from autoimmune disease to non‐Hodgkin's lymphoma.
Quantification of oxidative phosphorylation enzymes after blue native electrophoresis and two‐dimensional resolution: Normal complex I protein amounts in Parkinson's disease conflict with reduced catalytic activities
Authors: Schägger Hermann 
Published: May 2005

AbstractBlue native polyacrylamide gel electrophoresis (BN‐PAGE), a method for the isolation of native membrane proteins from biological membranes, was adapted to the isolation of oxidative phosphorylation (OXPHOS) enzymes from milligram amounts of human tissues. Combined with Tricine‐sodium dodecyl sulfate (SDS)‐PAGE in the second dimension, the protein subunits of OXPHOS complexes could be analyzed and quantified. The characteristics of the technique are described and protocols for processing different tissues are provided. The technique was applied for the analysis of defects of OXPHOS complexes in Parkinson's disease. A significant reduction of complex V was observed in one case. Absolutely normal complex I protein amounts were in contrast to reduced catalytic activities of complex I in Parkinson's disease. This discrepancy can be explained by binding of endogenous complex I inhibitors or by alterations of a protein subunit not affecting the assemblage of the complex but modifying the enzymatic properties.
Cyclodextrin modified micellar electrokinetic chromatography for the chiral direct resolution of (+), (−)‐trans‐1,2‐dihydrodiol metabolite of chrysene in vitro activated by rat liver microsome S9 fraction
Authors: Desiderio Claudia , Fanali Salvatore , Sinibaldi Massimo , Polcaro Chiara 
Published: May 2005

AbstractA γ‐cyclodextrin (γ‐CD) modified electrokinetic micellar capillary chromatography (MEKC) method was used for the enantiomer separation of a racemic trans‐1,2‐dihydro‐1,2‐dihydroxy‐chrysene (chry‐trans‐1,2‐diOH) mixture. The chiral resolution was strongly influenced by several important parameters: surfactant concentration and addition of organic modifier to the background electrolyte (BGE). An optimized electrophoretic system was used, consisting of the following conditions: 25 mM phosphate buffer, pH 7.8, 50 mM sodium dodecyl sulfate, 20 mM γ‐CD, 7.4% V/V 2‐propanol as BGE; the applied voltage, 18 kV, corresponded to 37 μA at a constant temperature of 25°C. This electrophoretic method was applied for monitoring the chry‐trans‐1,2‐diOH enantiomer formation in a real sample, obtained from in vitro metabolic activation of chrysene by phenobarbital‐ß‐naphthoflavone‐induced rat microsomes. The (+) and (‐) enantiomers were identified by the racemate and the single enantiomer standard addition method and by spectra comparison with the synthetic compound. Under the experimental conditions used for chrysene activation, the (+) optical isomer was the prevailing form. The CD‐MEKC system showed high reproducibility and selectivity, allowing a fast and interference‐free analysis even of the in vitro metabolic sample extract, without any pretreatment.
Separation of jumper ant (Myrmecia pilosula) venom allergens: A novel group of highly basic proteins
Authors: Donovan Gregory R. , Street Michael D. , Baldo Brian A. 
Published: May 2005

AbstractThe sting of the jumper ant (Myrmecia pilosula) causes severe allergic reactions, including anaphylaxis in sensitized individuals. Two of the major allergens, Myr p I and Myr p II, have been cloned, immunocharacterized and nucleotide‐sequenced and they encode 112 and 75 residue polypeptides, respectively. Both allergens are highly basic proteins having isoelectric point values greater than 10. However, electrophoretic analysis has generated conflicting results as to the actual sizes of the allergens in the native venom. Electrophoretic, immunological and N‐terminal analyses suggested that these allergens undergo extensive post‐translational processing to final forms of 45 and 27 residues, respectively. The results highlight the difficulties in the study of small, basic proteins and polypeptides by electrophoretic techniques.
Expression of the human urokinase‐type plasminogen activator receptor in E. coli and Chinese hamster ovary cells: Purification of the recombinant proteins and generation of polyclonal antibodies in chicken
Authors: Magdolen Viktor , Rettenberger Peter , Lopens Antje , Oi Hidekazu , Lottspeich Friedrich , Kellermann Josef , Creutzburg Sabine , Goretzki Lothar , Weidle Ulrich H. , Wilhelm Olaf , Schmitt Manfred , Graeff Henner 
Published: May 2005

AbstractThe receptor for urokinase‐type plasminogen activator (uPAR) may contribute to the invasive and metastatic capacity of tumor cells by focusing the serine protease urokinase‐type plasminogen activator (uPA) to the cell surface. uPA activates plasminogen to plasmin which in turn degrades extracellular matrix proteins or activates other proteases. Mature uPAR is a heavily glycosylated protein of about 284 amino acids attached to the plasma membrane via a glycosyl‐phosphatidylinositol (GPI) anchor. A set of different polyclonal uPAR antibodies has been generated in order to investigate the role of uPAR in tumor spreading in more detail. For this purpose, uPAR (lacking the GPI anchor) was expressed in E. coli and Chinese hamster ovary (CHO) cells. Recombinant uPAR from E. coli (corresponding to amino acids 1–284 of human uPAR) was expressed with an N‐terminal histidine‐tag insertion and purified by nickel chelate affinity chromatography. Soluble uPAR, synthesized by CHO cells (corresponding to amino acids 1–277 of human uPAR), was isolated by ligand (uPA) affinity chromatography. Expression in E. coli led to a nonglycosylated form of uPAR, whereas uPAR produced by CHO cells seemed to be glycosylated to a similar extent as the naturally occurring human form of uPAR (as analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis). Prior to immunization the N‐termini of the recombinant uPAR variants were determined by amino acid sequence analysis. Polyclonal antibodies were generated in chickens and purified from egg yolk. The reaction patterns of these antibodies were analyzed by Western blot analyses and flow cytofluorometry.
Differentiation of scombroid fish species (tunas, bonitos and mackerels) by isoelectric focusing, titration curve analysis and native polyacrylamide gel electrophoresis of sarcoplasmic proteins
Authors: Rehbein Hartmut 
Published: May 2005

AbstractDifferentiation of scombroid fishes was possible by electrophoresis of sarcoplasmic proteins using either isoelectric focusing (IEF), titration curve analysis, or native polyacrylamide gel electrophoresis (PAGE). By IEF with Phast‐Gels 3–9 species specific patterns, characterized by a few bands in the cathodal part of the gels, were obtained. This type of gel was also used for titration curve analysis. Here, too, the closely related species Thunnus thynnus and T. albacares gave different protein patterns. Native PAGE, native cathodal electrophoreses in Clean Gel 10% with buffer pH 5.5, proved to be a fast and simple method for differentiation of scombroid fishes. As most of the prominent sarcoplasmic proteins of these species have pIs in the neutral or alkaline pH range, they are positively charged at pH 5.5 and move to the cathode.
Diffusion, Joule heating, and band broadening in capillary gel electrophoresis of DNA
Authors: Slater Gary W. , Mayer Pascal , Grossman Paul D. 
Published: May 2005

AbstractWe calculated the longitudinal and transverse diffusion coefficients for a DNA molecule undergoing gel electrophoresis in the limit where it is reptating through a dense polymer matrix. Our results indicate that both diffusion coefficients increase with the electric field intensity. The transverse and longitudinal diffusion coefficients are roughly equal for regular field intensities, but the former dominates for the high field intensities normally used for capillary gel electrophoresis (CE). This has important implications for the optimization of CE. Our results clearly show that the naive use of the zero‐field diffusion constant, the Einstein relation or the longitudinal diffusion constant when calculating the contribution of the parabolic temperature profile to band broadening may lead to large overestimates under typical CE conditions. Finally, we show that the field‐dependent diffusion coefficients may be responsible for the existence of an optimal field intensity for CE, even if Joule heating is neglected.
Phosphorylation of ribosomal protein L30 after herpes simplex virus type 1 infection
Authors: Simonin Denis , Diaz Jean‐Jacques , Kindbeiter Karine , Denoroy Luc , Madjar Jean‐Jacques 
Published: May 2005

AbstractIn addition to an irreversible stimulation of S6 ribosomal protein phosphorylation, there is a modification of a subset of ribosomal proteins by phosphorylation after herpes simplex virus type 1 (HSV‐1) infection. Moreover, in the course of this infection, three additional phosphorylated proteins can be extracted from ribosomes and separated by two‐dimensional electrophoresis (2‐DE) of total ribosomal proteins. One of them exhibits an identical molecular mass to L30, while being more acidic. This protein is phosphorylated on serine residues. The kinetics of appearance of this protein in the ribosomal fraction correlated with a decrease in L30 staining, as shown by 2‐DE. Determination of the N‐terminal amino acid sequence of this extra phosphoprotein and of L30‐derived peptides demonstrated the identity of these two proteins.
Association of protein polymorphism among pepper (Capsicum annuum L.) inbred lines with agronomic performance of their crosses
Authors: Posch Anton , van den Berg Bartel M. , Görg Angelika 
Published: May 2005

AbstractThe genetic variation of 10 morphologically similar pepper (Capsicum annuum L.) inbred lines has been analyzed by two‐dimensional electrophoresis with immobilized pH gradient (IPG‐DALT) of seed proteins. For all pairs of inbred lines genetic distance indices were calculated on the basis of the IPG‐DALT analysis and a genetic tree was constructed. The protein polymorphism data of the 10 inbred lines were integrated into a pepper breeding program to validate the assumption that there is a higher chance of achieving better hybrid performance when the genetic distance between the parents is as great as possible. Field trials were performed in Turkey and consisted of a total of 27 crossings based on 9 inbred lines. Fifteen hybrids exceeded the crop yield of the better parent and 8 additional hybrids exceeded the mean crop yield of both parents. The genetic distance indices of the parental inbred lines based on protein polymorphism data were not significantly correlated to heterosis performance of the experimental hybrids (r = 0.20), indicating that protein polymorphism data add little to the prediction of single cross hybrid performance. Nevertheless, protein polymorphism data support the breeder in being more effective in finding out the best inbred combinations, because most of the less promising crossings can be omitted from the expensive and time‐consuming performance tests. Those hybrids whose distance indices exceeded the mean of all 27 distance values (0.52) were superior to the mean value of all heterosis performances by 19%. Moreover, 11 out of 15 hybrids having distance indices higher than the mean of the 27 crossings exceeded the mean of all heterosis values (304 g), but only 5 hybrids out of 12 showing distance values below the mean exceeded the overall mean of heterosis performance. We conclude that IPG‐DALT can be used as an efficient tool in vegetable hybrid breeding.
From image processing to classification: I. Modeling disturbances of isoelectric focusing patterns
Authors: Jensen Kirsten , Søndergaard Ib , Skovgaard Ib Michael , Nielsen Hans Bruun 
Published: May 2005

AbstractIn order to optimize the conditions for evaluation of isoelectric focusing (IEF) patterns by digital image processing, the sources of error in determination of the pI values were analyzed together with the influence of a varying background. The effects of band distortions, in the spectra of the individual lanes, were examined. In order to minimize the effect of these distortions, optimal conditions for handling IEF patterns by digital image processing were elucidated. The systematic part of the global deformation on the gels was investigated and an algorithm was developed by which it was possible to correct for a part of the individual distortions. The effects of various corrections for lane distortions were illustrated by classification, using different types of discriminant analysis. Finally the background disturbances were examined, and described by a mathematical model.