bluuebunny/crossref_metadata_2025_split huggingface dataset:
Capillary zone electrophoresis of DNA fragments in a novel polymer network: Poly(N‐acryloylaminoethoxyethanol)
Authors: Chiari Marcella , Nesi Marina , Righetti Pier Giorgio 
Published: May 2005

AbstractMigration of DNA fragments in the 51 to 21 226 bp size range has been investigated by capillary zone electrophoresis in entangled solutions of linear poly(N‐acryloylaminoethoxyethanol) from 8 to 16%. Plots of log mobility vs. log size (in base pairs, bp) clearly evidence three different migration regimes: according to Ogston (i.e. as spherical globules) up to 200 bp, reptation without stretching up to 3–4000 bp and reptation with partial stretching for larger fragments. Guidelines on the % polymer to be used for optimum resolution can be obtained from plots of peak spacing (in seconds per base pair) vs. % of polymer in solution and from standard plots of peak resolution vs. % polymer. An optimum linear poly(N‐acryloylaminoethoxyethanol) concentration, allowing for good resolution of most fragments, from small to large, is found in the range 10% to 12% polymer (as opposed to conventional polyacrylamide, where a sharp maximum is obtained at 6% polymer). It is hypothesized that in situ polymerization allows for formation of a large distribution of polymer sizes, thus facilitating simultaneous separation of short and long DNA fragments based on the principle that shorter polyacrylamide chains will sieve shorter DNA fragments and vice versa. Due to the fact that the novel monomer is 500 times more resistant to hydrolysis and more hydrophilic than acrylamide, its polymeric solutions allow repeated sample injections (> 35) with minimal absolute standard deviations (of the order of ± 3%). No carryover nor sample precipitation at the injection port is experienced with the new matrix composed of poly(N‐acryloylaminoethoxyethanol).
Purification of human recombinant superoxide dismutase by isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes
Authors: Wenisch Elisabeth , Vorauer Karola , Jungbauer Alois , Katinger Hermann , Righetti Pier Giorgio 
Published: May 2005

AbstractHuman recombinant superoxide dismutase (SOD), purified to homogeneity, is resolved by both conventional isoelectric focusing and immobilized pH gradients into three bands, with isoelectric points (p/s) in the pH range 4.8 to 5.1, the pI 4.80 form representing the minor component. Due to the fact that this enzyme is expressed in E. coli, N‐terminal acetylation or glycosylation should be ruled out. When purified by small‐scale preparative isoelectric focusing in immobilized pH gradient gels, it was found that, upon subsequent analysis, the pI 5.07 form would band in the same position, but the intermediate pI 4.92 band would split into the upper (pI 5.07) and the lower (pI 4.80) species, in nearly the same amounts, whereas the lowest pI component would always generate both the intermediate and upper forms. Enzymatic essays pointed out that these three isoforms had nearly the same specific activity, slightly higher than that of the starting material. Metal analysis indicated that all three forms contained the same metal/protein ratio, approaching the value Cu2Zn2‐SOD, as reported in the literature. Circular dichroism spectra of the pI 4.80 and 5.07 forms showed the same profile in the 190–240 nm range, but marked differences in the 250–350 nm region. Treatment with EDTA produces 1–2 additional, slightly higher pI isoforms, whereas treatment with KCN generates a number of higher pI components, reaching pI values as high as pH 7, with nearly complete disappearance of the three major SOD isoforms. It is concluded that these three isoforms could represent interconvertible species, the highest pI component representing the most stable conformer.
Sample preparation of human tumors prior to two‐dimensional electrophoresis of proteins
Authors: Franzén Bo , Hirano Takashi , Okuzawa Ken , Uryu Kazuto , Alaiya Ayodele A. , Linder Stig , Auer Gert 
Published: May 2005

AbstractSample preparation procedures are critical for obtaining high quality two‐dimensional electrophoresis (2‐DE) separations from clinical tumor materials. In this communication, we describe procedures which we have found satisfactory for preparing various tumor samples for 2‐DE. By these procedures, tumor cells are enriched from serum proteins and contaminating stromal cells. Tumors can be prepared with good reproducibility to obtain high resolution using these techniques.
High resolution one‐dimensional electrophoretic separation and partial characterisation of human head hair proteins
Authors: Khawar Sitwat Lubna , Watson Kenneth , Jones Graham L. 
Published: May 2005

AbstractA reproducible, rapid procedure for the extraction, labelling and separation of human hair proteins by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) has been developed. Hair proteins were extracted in 8 M urea, containing 0.2 M mercaptoethanol, followed by sonication. Extracts were neutralised with Tris and incubated with either labelled (14C) or unlabelled iodoacetic acid to S‐carboxymethylate cysteine groups. Proteins were separated on 12.5% SDS‐polyacrylamide gels and gels stained with Coomassie Brilliant Blue and/or silver nitrate to reveal major protein bands. Gels were then treated with a fluorographic agent, dried and autoradiographed to reveal major sites of S‐carboxymethylation. A given gel was scanned by laser densitometry after Coomassie and/or silver stain to quantitate the protein content of each major protein zone. An autoradiogram of the same gel was scanned to estimate the cysteine content of each major zone. In this way it was possible to partially characterise rapidly and reproducibly many different protein zones in different individual samples on one gel at the same time. By calculating the ratio of autoradiograph absorbance to Coomassie Blue absorbance, protein zones could be assigned to four different categories, viz: very high cysteine (VHC) proteins, high cysteine (HC) proteins, low cysteine (LC) proteins and very low cysteine (VLC) proteins. The method described is reproducible, rapid and inexpensive enough to be suitable for mass screening. Overall the results were more informative than previously reported one‐dimensional separations and indeed this technique may well be more suited to forensic and/or population investigations than the much more laborious and time‐consuming two‐dimensional techniques.
Characterizing allelic proteins for genome mapping in maize
Authors: Touzet Pascal , Morin Catherine , Damerval Catherine , Guilloux Martine Le , Zivy Michel , De Vienne Dominique 
Published: May 2005

AbstractMapping cDNA probes in order to construct genetic linkage maps is becoming a widespred strategy for genome analysis and gene isolation, particularly in cultivated plant species. Nevertheless, almost all cDNAs reveal two or more unlinked loci, making it difficult to identifiy the gene(s) actually expressed. In a highly polymorphic species, such as maize, two‐dimnsional polyacrylamide gel electrophoresis (2‐D PAGE) of proteins may circumvent this limitation. With the analysis of various segregating populations, we previously showed that the apparent position shifts of proteins on the 2‐D gels are monogenic and codominant. In this paper we compared allelic proteins on the basis of their high performance liquid chromatography (HPLC) profile and partial amino acid sequences. In a sample of 20 position shifts, the allelic proteins appeared to be similar in all but one case, strongly suggesting that polymorphism of structural genes is involved. Thus 2‐D PAGE could prove to be a useful tool for genome mapping: when a cDNA probe detects several loci, a position shift of the encoded protein will allow the identification of the gene translated in the organ considered.
Polymorphic human specific Alu insertions as markers for human identification
Authors: Novick Gabriel E. , Novick Corina C. , Yunis Juan , Yunis Emilio , Martinez Kianfa , Duncan George G. , Troup Gary M. , Deininger Prescott L. , Stoneking Mark , Batzer Mark A. , Herrera Rene J. 
Published: May 2005

AbstractAlu sequences represent the largest family of short interspersed repetitive elements (SINEs) in humans with 500 000 copies per genome. Recently, one Alu subfamily was found to be human specific (HS). We originally described the use of polymorphic HS Alu insertions as a tool in population studies and recently as tools in DNA fingerprinting and forensic analysis. In this report, we will use this simple polymerase chain reaction (PCR) base technique for the detection of HS Alu insertion polymorphisms. We will test the resolving power of this DNA profiling approach in both population genetics and paternity assessment. At the population level, we will describe the genotypic distribution of five polymorphic Alu insertions among 3 populations from the American continent, one of African origin, the other two Amerindians. Insight into their relationships will be provided. At the family level, we will examine one European American family of seven individuals and the same pedigree will also be characterized by way of the two systems currently and widely used to ascertain paternity: PCR‐sequence specific oligonucleotide probe hybridization (PCR‐SSO) and PCR‐restriction fragment length polymorphism (PCR‐RFLP) of human leucocyte antigen (HLA) class II molecules, and a standard RFLP protocol used in forensic casework and paternity studies. The importance and strengths of the method as well as its perspectives for future use in filiation studies will be evaluated.
Genotyping of human deoxyribonuclease I polymorphism by the polymerase chain reaction
Authors: Yasuda Toshihiro , Nadano Daita , Tenjo Etsuko , Takeshita Haruo , Sawazaki Kazumi , Nakanaga Masao , Kishi Koichiro 
Published: May 2005

AbstractWe recently completely elucidated the molecular basis of genetic polymorphism in human deoxyribonuclease I and found it to be controlled by four codominant alleles, DNASE1*1, *2, *3 and *4. In this paper we describe a novel DNase I‐genotyping system that could be used directly on DNA samples using the polymerase chain reaction (PCR) based on the three nucleotide substitutions underlying the protein polymorphism. The system consists of three independent reactions. Since the substitutions neither suppress nor create any known enzyme recognition site in the DNase I gene, two separate mismatched PCR followed by XhoI digestion methods were introduced to discriminate between the DNASE1*1 (or *3) and the DNASE1*2 (or *4) alleles, and to detect the DNASE1*4 allele. An amplification refractory mutation system was employed to detect DNASE1*3. A 100% correlation was found between the results of this genotyping method and those obtained by phenotyping using conventional isoelectric focusing. The high sensitivity and specificity of this genotyping method allows us to survey DNase I‐polymorphism in small DNA samples.
Lung tumor cells: A multivariate approach to cell classification using two‐dimensional protein pattern
Authors: Schmid Hans‐Rudolf , Schmitter Doris , Blum Philippe , Miller Mark , Vonderschmitt Dieter 
Published: May 2005

AbstractHigh resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) is a powerful research tool for the analytical separation of cellular proteins. The qualitative and quantitative pattern of polypeptides synthesized by a cell represents its phenotype and thus defines characteristics such as the morphology and the biological behavior of the cell. By analyzing and comparing the protein patterns of different cells it is possible to recognize the cell type and also to identify the most typical features of these cells. In applied pathology it is often difficult to identify the tissue of origin and the stage or grade of a neoplasia by cellular morphology analyzed by classical or immunostaining procedures. The protein pattern itself is the most characteristic feature of a cell and should therefore contribute to the identification of the cell type. For this reason we separated protein fractions originating from different lung tumor cell lines using 2‐D PAGE and we compared the resulting patterns on a multivariate statistical level using correspondence analysis (CA) and ascendant hierarchical clustering (AHC). The results indicate that (i) protein patterns are highly typical for cells and that (ii) the comparison of the protein patterns of a set of interesting cell types allows the identification of potentially new marker proteins. 2‐D PAGE is thus a unique and powerful tool for molecular cytology or histopathology, unveiling the protein expression level of tissues or cells.
A spot cloning method for restriction landmark genomic scanning
Authors: Ohsumi Tomoya , Okazaki Yasushi , Hirotsune Shinji , Shibata Hideo , Muramatsu Masami , Suzuki Harukazu , Taga Chiharu , Watanabe Sachihiko , Hayashizaki Yoshihide 
Published: May 2005

AbstractWe introduce two new methods for target cloning of DNA fragments corresponding to spots on the two‐dimensional profile of restriction landmark genomic scanning (RLGS). One is a restriction trapper‐based method and the other is a polymerase chain reaction (PCR) mediated method. Both are designed to select the target DNA fragments from a large amount of unlabeled background DNA fragments in the RLGS gel which produce background clones. The restriction trapper method is simple, with a cloning efficiency that is not biased by the length of the target DNA nor by its GC content. On the other hand, the PCR‐mediated method is efficient for cloning DNA fragments from a small amount of starting materials. These methods provide us with powerful tools for isolating DNA clones identified by the RLGS system as interesting spots. This paper reports the precise protocols of these methods and discusses their application and usefulness.
The use of restriction landmark genomic scanning to scan the mouse genome for endogenous loci with imprinted patterns of methylation
Authors: Shibata Hideo , Yoshino Kiyoshi , Muramatsu Masami , Plass Christoph , Chapman Verne M. , Hayashizaki Yoshihide 
Published: May 2005

AbstractRestriction landmark genomic scanning (RLGS) has been used to screen endogenous loci for imprinted patterns of methylation. The screening method is based upon the identification of genetic variation in RLGS profiles between different strains and determining whether specific variant landmarks are transmitted equally to the progeny of reciprocal F1 matings. The RLGS profiles of C57BL/6 (B6) and DBA/2 (D2) and their reciprocal hybrids were produced with two enzyme combinations that used NotI as the landmark enzyme and two combinations that used BssHII. An estimated 13% of the spots are either B5‐ or D2‐specific in these tests, giving a total of nearly 1000 variant loci that were examined for imprinted methylation. Three candidate loci for imprinted regulation were identified in these analyses. We also used crosses of more genetically diverse parents to increase the number of variant loci screened. Interspecific crosses of B6 with the M. musculus strain PWK and intrasubspecific crosses between B6 and the M. molossinus strain MSM expanded the levels of variation between the parental strains in the cross to an estimated 31% and 26%, respectively. The RLGS patterns for one NotI combination and one BssHII profile were examined for each of these crosses, giving approximately 2000 additional loci that were screened for imprinted patterns of methylation. Eight loci with imprinted patterns of transmission were observed out of 3040 loci tested. The chromosomal locations for the three B6 and D2 specific loci, Irlgs 1–3, were identified using BXD recombinant inbred strain analysis. Irlgs 1 and 3 are B6‐ and D2‐specific loci that had the same strain distribution pattern which mapped to the central region of chromosome 9. Irlgs 2 (U2afbp‐rs) was mapped to the proximal region of chromosome 11, which was reported as an imprinted region identified with uniparental disomy mice. An imprinted gene, U2 auxiliary factor binding protein‐related sequence (U2afbp‐rs), was identified for Irlgs2 locus, which encoded 51 kDa protein that had significant homology to the human U2af 35 kDa small subunit.
Enhancing protein solubilization with nondetergent sulfobetaines
Authors: Vuillard Laurent , Marret Nathalie , Rabilloud Thierry 
Published: May 2005

AbstractA significant increase in the yield of protein extraction by the addition of sulfobetaine‐type mild solubilization agents is shown for microsomal membrane proteins and proteins from lyophilized platelets, with a maximum increase in yield of up to 100%. These agents are also applied to native isoelectric focusing in immobilized pH gradients. The absence of significant denaturation induced by these solubilizers is shown for creatine kinase.
A highly discriminating octoplex short tandem repeat polymerase chain reaction system suitable for human individual identification
Authors: Oldroyd Nicola J. , Urquhart Andrew J. , Kimpton Colin P. , Millican Emma S. , Watson Stephanie K. , Downes Teresa , Gill Peter D. 
Published: May 2005

AbstractThrough the use of fluorescence‐based polymerase chain reaction systems, a highly discriminating multiplex with the potential for individual identification has been developed. The use of multiple dye technology enabling loci with overlapping size ranges to be co‐amplified has enabled us to successfully amplify seven tetranucleotide short tandem repeat loci within a single reaction in a discriminating power in the region of 1 × 109. Three out of the seven loci employed exhibit alleles differing in size by only 2 bp as opposed to the conventional 4 bp, which results in such loci being more powerful in terms of distinguishing between samples, particularly when co‐amplified in this manner. The size ranges of the loci contained within the system are such that windows still exist for the inclusion of additional loci at a later stage, which could increase the discriminating power of the system still further. In addition, further weight and utility is lent to the system through the incorporation of a simple and reliable sex test involving the amplification of a segment of the X–Y homologous gene Amelogenin.
Analysis of underivatized amino acids by capillary electrophoresis using constant potential amperometric detection
Authors: Guo Yong , Colón Luis A. , Dadoo Rajeev , Zare Richard N. 
Published: May 2005

AbstractA mixture of native (underivatized) amino acids is separated by capillary electrophoresis under alkaline conditions (pH ≈︁ 12) and amperometrically detected with a copper‐disk microelectrode. A simple design facilitates capillary‐electrode alignment without the need for micropositioning equipment. The limits of detection for the amino acids are in the low μm concentration range, and the signal response is linear over 2–3 orders of magnitude. This procedure is applied to analyze the amino acid hydrolysis products from cytochrome c.
Sensitivity enhancement and second‐dimensional information from solid phase extraction‐capillary electrophoresis of entire high‐performance liquid chromatography fractions
Authors: Strausbauch Michael A. , Madden Benjamin J. , Wettstein Peter J. , Landers James P. 
Published: May 2005

AbstractA novel separation technique is demonstrated for peptide analysis of entire 100 μL high‐performance liquid chromatography (HPLC) fractions in a single, low pH, capillary zone electrophoresis (CZE) separation. The method employs a hybrid capillary consisting of a removable inlet section containing a reverse‐phase packing and a standard CZE bare‐fused silica separation capillary. The packed tip allows sample to be concentrated at flow rates greater than 20 μL/min and released in a controlled manner for CZE separation. Separations are comparable to standard CZE with minor modifications in selectivity. Using a mixture of model peptides, the hybrid capillary is shown to effect enhancement in sensitivity of ≥ 100‐fold. Five fractions from the HPLC separation of peptides obtained from the digestion of bovine serum albumin (BSA) were analyzed by the solid phase extraction‐capillary electrophoresis (SPE‐CE) method which was shown to provide rapid second‐dimensional information. Practical concentration limits of detection for peptides are calculated to be 1‐‐10 ng/mL with this technology.
Extended theory of the electrophoretic mobility‐shift analysis of nonspecific protein‐DNA complexes, featuring cooperativity
Authors: Coombs Robert O. , Cann John R. 
Published: May 2005

AbstractThe simulated electrophoretic mobility‐shift behavior of a model system, in which the nonspecific binding of a protein to a DNA fragment is cooperative, was compared with the experimental behavior of the DNA: histone‐like bacterial protein (HU) system. It was concluded that the binding of HU to an 88 bp DNA fragment is, at least, not highly cooperative. The theory of mobility‐shift analysis was extended even further to encompass high affinity sequence‐specific binding of protein to a DNA fragment followed by weak nonspecific binding, the latter governed by conditional probabilities. In addition to featuring a ladder of incremental protein‐DNA complexes, the computed mobility‐shift patterns placed emphasis upon stabilization of weak, nonspecific complexes in gel cages.
Variation of the pH of the background electrolyte due to electrode reactions in capillary electrophoresis: Theoretical approach and in situ measurement
Authors: Corstjens Hugo , Billiet Hugo A. H. , Frank Johannes , Luyben Karel C. A. M. 
Published: May 2005

AbstractElectrode reactions during the electrophoretic process may change the pH of the buffer and subsequently the migration behavior of solutes with resultant loss of reproducibility. A theoretical treatment of pH variations due to electrolytic processes is presented. The choice of buffer appears to have a dramatic influence on the pH variations observed, even if substantial buffer action is expected at the pH chosen. The experimental evaluation of the separation of 4‐hydroxy‐3‐methoxycinnamic acid and 3‐hydroxybenzoic acid reveals that the quality of the separation decreases continuously from a baseline separation observed in the first experiment to a comigration of the two solutes (resolution = 0) in the ninth experiment. A pH decrease of about 0.05 pH units accounts for the observed changes in mobility.A novel in situ pH measurement approach is presented, in which the mobility, peak area, and peak height of an indicator dye are related to the pH in the capillary. This enables the identification and quantification of pH variations during electrophoretic runs: the pH decreases at the anodic side already after the first experiment and pH variations as small as 0.02 pH units can be measured. The variations in peak height appear to be less suited. The calculated pH variations are in close agreement with the ones obtained experimentally.
Toxicant‐induced alterations in two‐dimensional electrophoretic patterns of hepatic and renal stress proteins
Authors: Witzmann Frank A. , Fultz Carla D. , Lipscomb John C. 
Published: May 2005

AbstractRecent studies in this laboratory and by others suggest that two‐dimensional polyacrylamide gel electrophoresis of proteins (2‐DE) possesses significant utility in the detection of chemical toxicity and in providing information regarding toxic mechanism. After having identified a set of specific heat‐shock and glucose‐regulated proteins whose expression in rodent liver and kidney is highly conserved and constitutive, we compared the effect of in vivo exposure to perfluoro‐n‐octanoic acid and perfluoro‐n‐decanoic acid on their expression. The following stress proteins were identified, their x, y coordinate positions mapped, and abundance statistically analyzed and compared: hsp32, hsp60, hsc70, hsp70, hsp90, grp75, grp94, protein disulfide isomerase (PDI), and ER60. We report here that the stress response to perfluorocarboxylic acids is tissue‐, toxicant‐, and stress protein class‐specific and dose‐related. Furthermore, because nearly all of the proteins studied were constitutively expressed at detectable levels in both liver and kidney, the 2‐DE stress protein pattern may be suitable to future toxicologic screening applications.