bluuebunny/crossref_metadata_2025_split huggingface dataset:
Screening of a highly polymorphic microsatellite for microheterogeneity in human identification
Authors: Miścicka‐Śliwka Danuta , Grzybowski Tomasz , Czarny Jakub , Woźniak Marcin 
Published: May 2005

AbstractSingle‐strand conformation polymorphism (SSCP) analysis combined with automated laser fluorescence detection is proposed as a comprehensive, rapid and sensitive method for screening sequence variation of the human beta‐actin‐related pseudogene (HUMACTBP2). Eleven sequenced alleles representing each type of known sequence variant of HUMACTBP2 locus were studied. Allelic variants of the same size but different sequence structures are easily resolved on the basis of their secondary conformation. Fifty ACTBP2 amplification products previously typed on a denaturing gel were repeatedly examined to determine the utility of SSCP analysis in terms of ease of interpretation and reproduction capabilities of the conformational patterns. Eleven sequenced ACTBP2 allelic variants were used as external conformation standards in polymerase chain reaction (PCR)‐SSCP subtyping. This enabled identification of polymorphism in a particular length variant and therefore consistent discrimination between heterozygous samples appeared identical on denaturing gels. Of five “homozygous” samples, one was shown to be heterozygous for two distinct alleles of the same size but different sequences. Thus, the method provides a unique possibility for detecting false homozygotes. The technique complements both denaturing gel electrophoresis and DNA sequencing in studies on the overall variability of the ACTBP2 locus.
Detection of sequence variation in parasite ribosomal DNA by electrophoresis in agarose gels supplemented with a DNA‐intercalating agent
Authors: Zhu Xing Quan , Chilton Neil B. , Gasser Robin B. 
Published: May 2005

AbstractThis study evaluated the use of a commercially available DNA intercalating agent (Resolver Gold™) in agarose gels for the direct detection of sequence variation in ribosomal DNA (rDNA). This agent binds preferentially to AT sequence motifs in DNA. Regions of nuclear rDNA, known to provide genetic markers for the identification of species of parasitic ascarid nematodes (order Ascaridida), were amplified by polymerase chain reaction (PCR) and subjected to electrophoresis in standard agarose gels versus gels supplemented with Resolver Gold. Individual taxa examined could not be distinguished reliably based on the size of their amplicons in standard agarose gels, whereas they could be readily delineated based on mobility using Resolver Gold‐supplemented gels. The latter was achieved because of differences (approximately 0.1–8.2%) in the AT content of the fragments among different taxa, which were associated with significant interspecific differences (approximately 11–39%) in the rDNA sequences employed. There was a tendency for fragments with higher AT content to migrate slower in supplemented agarose gels compared with those of lower AT content. The results indicate the usefulness of this electrophoretic approach to rapidly screen for sequence variability within or among PCR‐amplified rDNA fragments of similar sizes but differing AT contents. Although evaluated on rDNA of parasites, the approach has potential to be applied to a range of genes of different groups of infectious organisms.
Affinity isoelectric focusing of human serum apolipoprotein B‐containing lipoproteins on agarose gels with cyclodextrins
Authors: Ichimura Mitsuko , Okamoto Motoi , Mori Shuji , Tohge Hiroko , Ochi Koji , Harada Hideo 
Published: May 2005

AbstractIn an attempt to subfractionate apolipoprotein B‐containing lipoproteins (apoB‐Lp), agarose isoelectric focusing (IEF) of apoB‐Lp in human sera was carried out in the presence of two kinds of cyclodextrins (CDs), which have different affinities for cholesterol. ApoB‐Lp in the agarose gels was detected by immunofixation, followed by protein staining. In the presence of both 11.4 mg/mL α‐CD and 3.51 mg/mL β‐CD, but not either one alone, IEF separated a maximum of six apoB‐Lp bands. Electrophoresis failed to separate the apoB‐Lp subfractions under similar conditions with CDs. The patterns of apoB‐Lp in IEF with the CDs appeared to differ in some sera. Since the subfractions of apoB‐Lp can be separated without ultracentrifugation of serum, the present method serves for the screening of apoB‐Lp heterogeneity on native serum lipoproteins.
Mixed‐mode separation of polycyclic aromatic hydrocarbons (PAHs) in electrokinetic chromatography
Authors: Luong John H. T. , Guo Yong 
Published: May 2005

AbstractA mixed‐mode separation technique has been developed and optimized for the separation of the 16 Environmental Protection Agency (EPA) priority polycyclic aromatic hydrocarbons (PAHs). The procedure utilized two different buffer additives as pseudo‐stationary phases with different selectivities towards the analytes. Sodium dioctyl sulfosuccinate (DOSS) displayed selectivities for PAHs which were somewhat similar to the C18 phase in reversed‐phase high performance liquid chromatography (HPLC). High acetonitrile content required for an effective separation prevented the formation of micelles as confirmed by fluorescence spectroscopy. Consequently, the separation could be attributed to the solvophobic association of the PAH molecules with hydrophobic chains of the DOSS surfactant. In another mode of separation, sulfobutylether‐β‐cyclodextrin (SB‐β‐CD) separated the 16 PAHs on the formation of inclusion complexes with the PAHs, and exhibited different selectivities for the PAHs compared to DOSS. SB‐β‐CD and DOSS were then combined in the running buffer to form a mixed pseudo‐stationary phase for the separation of the 16 PAHs. Due to the different selectivities of SB‐β‐CD and DOSS for the PAHs, the separation of the 16 PAHs was appreciably improved compared to that using DOSS or SB‐β‐CD alone. All the 16 PAHs were baseline‐resolved using an optimized running buffer containing 22.5 mM DOSS, 15 mM SB‐β‐CD, 15% acetonitrile and 5 mM hydroxypropyl‐β‐cyclodextrin in 6 mM borate at pH 9.
Molecular size and net charge of pathogenesis‐related enzymes from barley (Hordeum vulgare L., v. Karat) infected with Drechslera teres f. teres (Sacch.) Shoem
Authors: Rothe Gunter M. , Welschbillig Nora , Reiss Ernst 
Published: May 2005

AbstractMolecular size and net charge of isoforms of pathogenesis‐related (PR) chitinase, β‐1,3‐glucanase and peroxidase were studied in uninfected barley (Hordeum vulgare L., ν. Karat) leaves and in barley leaves infected with the pathogenic fungus Drechslera teres f. teres (Sacch.) Shoem. Molecular characteristics were determined by time‐dependent polyacrylamide gradient gel electrophoresis under native conditions and by applying an extended version of the computer program MOL‐MASS (Rothe, G. M., Weidmann, H., Electrophoresis 1991, 12, 703–709). Uninfected barley leaves contained predominantly one peroxidase isozyme but also three very weak peroxidases. Activities of all of these three peroxidases increased considerably after infection with Drechslera teres. The molecular masses of peroxidases 1 and 3 were estimated to be 38 ± 5 and 42 ± 7 kDa and their apparent valences at pH 8.4 were Z = 3.13 and 3.20, respectively. Amongst the chitinase isoforms, chitinase 1 and chitinase 2 appeared after infection, while chitinase 3 was also observed in uninfected leaves of barley. The molecular mass of chitinase 3 (31 ± 6 kDa; f/fo = 1.20) was larger than that of chitinase 1 (20 ± 2 kDa; f/fo = 1.04) and chitinase 2 (23 ± 3 kDa; f/fo = 1.06). The valence of constitutive chitinase 3 (Z = 1.44 ± 0.81) at pH 8.4 was lower than that of adaptive chitinase 1 (Z = 3.27 ± 1.02) and chitinase 2 (Z = 2.96 ± 1.38). Infection of barley leaves with Drechslera teres also induced the hydrolytic enzyme β‐1,3‐glucanase 1; β‐1,3‐glucanase 2 appeared in uninfected and in infected leaves. Constitutive β‐1,3‐glucanase 2 was smaller (molecular mass 19 ± kDa; f/fo = 1.05) than adaptive β‐1,3‐glucanase 1 (molecular mass 26 ± 4 kDa; f/fo = 1.07). The valence of adaptive β‐1,3‐glucanase 1 (Z = 9.58 ± 4.17) was approximately threefold that of β‐1,3‐glucanase 2 (Z = 2.80 ± 0.93).
Alterations in the expression of P2X1 receptors in failing and nondiseased human atria
Authors: Berry Desiree , Yao Mu , Barden Julian A. , Balcar Vladimir J. , Hansen Mitchell A. , Bennett Maxwell R. , Keogh Anne , dos Remedios Cristobal G. 
Published: May 2005

AbstractThis is the first report of the analysis of the ATP‐specific P2X1 receptor subunit in human hearts. We have examined homogenate samples of human left atria for the presence of P2X1 receptors using Western blots. Anti‐P2X1 immunoreactivity was detected in populations of nondiseased atria as well as in atria from explanted hearts from patients with terminally failing heart conditions such as dilated cardiomyopathy. At least three groups of P2X1 immunoreactive proteins were detected in the Western blots with approximate molecular mass values of 50, 70, and 160 kDa. We report changes in expression of their 50 and 70 kDa components. These changes may be related to the type of deficit in these hearts since the changes have been observed in hearts with decreased ejection fractions characteristic of dilated cardiomyopathy.
Peptide mass fingerprint sequence coverage from differently stained proteins on two‐dimensional electrophoresis patterns by matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS)
Authors: Scheler Christian , Lamer Stephanie , Pan Zaoming , Li Xin‐Ping , Salnikow Johannes , Jungblut Peter 
Published: May 2005

AbstractIdentification of proteins separated by two‐dimensional electrophoresis (2‐DE) is a necessary task to overcome the purely descriptive character of 2‐DE and a prerequisite to the construction of 2‐DE databases in proteome projects. Matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) has a sensitivity for peptide detection in the lower fmol range, which should be sufficient for an analysis of even weakly silver‐stained protein spots by peptide mass fingerprinting. Unfortunately, proteins are modified by the silver staining procedure, leading to low sequence coverage. Omission of glutaraldehyde increased the sequence coverage, but this improved sequence coverage is still clearly below the sequence coverage starting with Coomassie Brilliant Blue (CBB) R‐250‐stained spots. Other factors additionally seem to modify proteins during silver staining. By decreasing the protein amount, the advantage of very sensitive detection on the gel is lost during identification, because the resulting low sequence coverage is not sufficient for secure identification. Low‐quantity proteins can be identified better starting with CBB G‐250 or Zn‐imidazol‐stained proteins. In contrast, for high‐quantity CBB R‐250‐stained spots, a sequence coverage of up to 90% can be obtained by using only one cleaving enzyme, and up to 80% was reached for medium‐quantity spots after combination of tryptic digest with Asp‐N‐ and Glu‐C digest.
Rapid identification of comigrating gel‐isolated proteins by ion trap‐mass spectrometry
Authors: Arnott David , Henzel William J. , Stults John T. 
Published: May 2005

AbstractIn the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gel were analyzed and each was found to contain a number of proteins that subseqently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin in situ on a polyvinylidence difluoride (PVDF) membrane following electroblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) followed by an intial database search with the peptide mases failed to identify the proteins. The peptides were separated by reversed‐phase capillary high performance liquid chromatography (HPLC) in anticipation of subsequent Edman degradation, but mass analysis of the chromatographic fractions by MALDI‐MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by capillary HPLC‐electropspray ionization‐ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partialsequence was deduced for a number of peptides. Two stages of fragmentation (MS3) were used in one case to determine additional dequence. Database searches, each using a single peptide mass plus partial sequence, identified four proteins from a single electrophoretic band at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifications were corroborated by the presence ofmultiple matching peptide masses in the MALDI‐MS spectra. In addition, a novel sequence, not found in protein or DNA databases, wes determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and for de novo determination of peptide sequence. Reanalysis of the fragmentation data with a modified database searching algorithm showed that the same sets of proteins were identified from a limited number of fragment ion masses, in the absence of mass spectral interpretation or amino acid sequence. The implications for protein identification solely from fragment ion masses are discussed, including advantages for low signal levels, for a reduction of the necessary interpretation expertise, and for increased speed.
Routine identification of proteins from sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels or polyvinyl difluoride membranes using matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS)
Authors: Fernandez Joseph , Gharahdaghi Farzin , Mische Sheenah M. 
Published: May 2005

AbstractAs the resource laboratory for Rockefeller University our emphasis continues to be on methodology development for the routine analysis of low abundance proteins isolated from native sources. In the past ten years, gel electrophoresis of proteins has become the method of choice for the preparation of microgram and submicrogram quantities of protein for primary structural characterization, and over 95% of the samples submitted for protein identification are either in a gel or bound to polyvinyl difluoride membranes (PVDF). As such, we employ multiple microanalytical approaches encompassing Edman sequence degradation, amino acid and matrix assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometric analysis to provide an intergrated protein characterization of such samples. Here we describe the two major services we employ when providing protein identification from in‐gel or PVDF‐bound proteins.
Recycling isoelectric focusing and isotachophoresis
Authors: Bier Milan 
Published: May 2005

AbstractOf all electrophoretic methods, isoelectric focusing offers the highest resolution and is best suited for preparative applications. Over the years, several instruments were developed for this purpose, all operating in free fluids, in the absence of gels or other supporting matrices. In such systems, the avoidance of gravity or electrically driven convections is essential. Successful stratagems for fluid control included rapid recycling or rotation, in combination with either fine porosity screens or narrow gaps between parallel plates. The most successful apparatus so far is the Rotofor, in which fluid is stabilized by combining horizontal rotation with fine porosity screen partitioning. Recycling isotachophoresis offers the potential of separating proteins at high concentration. A new concept of tangential electrophoresis is described. To optimize the use of these devices for protein separation, low molecular weight, biologically acceptable buffers of known composition are essential. The buffering system developed for this purpose comprises a series of binary buffers that cover the pH range in steps of 1 pH unit or less. The pH gradient can be custom‐designed and is of remarkable stability in operation.
pH‐Regulated electroretention chromatography: Towards a new method for the separation of proteins according to their isoelectric points
Authors: Korlach Jonas , Hagedorn Rolf , Fuhr Günter 
Published: May 2005

AbstractThe pH‐dependent electroretention behavior of model proteins cytochrome c and ribonuclease A was studied in a hollow fiber arrangement, similar to that used in electrical field‐flow fractionation. Field‐induced immobilization of the proteins at the inner wall of the fiber was a function of the pH adjusted in the solution surrounding it, indicating that the pH inside the fiber lumen, relevant for protein migration, quickly equilibrates to the regulated value outside. A complete separation of the model proteins was achieved. Advantages of the principle as well as prospects for the development of a technique separating more than two protein species according to their isoelectric points are discussed.
Separation of viable from radiation‐induced apoptotic lymphocytes by free‐flow electrophoresis
Authors: Chestkov Valery , Baibakov Boris , Radko Sergey P. , Chrambach Andreas 
Published: May 2005

AbstractA human lymphocyte population undergoing apoptosis in vitro due to γ‐irradiation was fractionated by free‐flow electrophoresis in triethanolamine – Na‐acetate buffers, containing up to 50 mM NaCl, with pH 6.0, 7.2 and 8.5, made isotonic by addition of sucrose. As shown by a flow cytometric analysis of the eluate, the distribution of apoptotic lymphocytes is shifted to the range of higher electrophoretic mobilities relative to that of viable ones at pH 8.5, yielding cell fractions enriched in apoptotic cells by a factor of 3 to 5. The difference in rates of electrophoretic migration observed at a mildly alkaline pH but not at a neutral or mildly acidic one suggests that the surface of apoptotic lymphocytes is more acidic than that of viable ones.
Lectin‐gradient gel affinity electrophoresis of glycoproteins
Authors: Taketa Kazuhisa , Kamakura Kozue , Fukazawa Masako , Ikeda Satoru , Taga Hiroko 
Published: May 2005

AbstractLectin‐gradient agarose gel affinity electrophoresis was developed for identification of glycoforms of glycoproteins in lectin affinity electrophoresis. Gradation of lectin was done by stacking agarose gel blocks with increasing concentrations of lectin (discontinuous system) and by keeping the plate in a moist chamber at 4°C overnight (continuous system) before electrophoresis. On the visualization of separated glycoform lines, the antibody‐affinity blotting was superior for low concentrations of α‐fetoprotein. Fluoresceine isothiocyanate (FITC) labeling of whole serum proteins, enzyme activity staining for alkaline phosphatase, and prestaining for lipoproteins were also applicable for visualization of proteins at higher concentrations. The conventional Western blotting can not be recommended because of the competition between lectin and glycoproteins in binding to nitrocellulose membrane. Lectin‐gradient affinity electrophoresis also had a wide application for optimization of the condition of lectin affinity electrophoresis.
Continuous flow isoelectric focusing for purification of proteins
Authors: Soulet Nathalie , Roux‐de Balmann Hélène , Sanchez Victor 
Published: May 2005

AbstractTo estimate the feasibility of isoelectric focusing in a continuous mode, the conditions for obtaining stable and linear pH gradients were first investigated, using a mixture of carrier ampholytes. Then the operating parameters allowing the isoelectric focusing of bovine serum albumin as model protein were determined. A theoretical approach was used to find conditions minimizing the electrohydrodynamic effect, mainly responsible for protein dispersion, for the separation of bovine serum albumin and α‐lactalbumin from bovine milk.
The effects of electroendosmosis in agarose electrophoresis
Authors: Guo Yaojun , Li Xinhui , Fang Yong 
Published: May 2005

AbstractThe effects of agarose gel strips without and with 0.03 mr electroendosmosis (EEO) on isoelectric focusing (IEF) were studied. It is shown that only agarose without EEO can be used for IEF. The effects of electrode buffer strips using agarose with different EEO of 0, 0.03, 0.08, 0.20 mr on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and native PAGE were also studied. It apparently did not affect SDS‐PAGE, but affected native PAGE to a certain extent. The higher the EEO value was, the more water was present on the agarose gel strip during a separation. Agarose gel strips with an EEO of 0.2 mr are not suitable as electrode buffer strips for native PAGE.
Comparison of different methods to produce single‐strand DNA for identification of canned tuna by single‐strand conformation polymorphism analysis
Authors: Rehbein Hartmut , Mackie Ian M. , Pryde Susan , Gonzales‐Sotelo Carmen , Perez‐Martin Ricardo , Quinteiro Javier , Rey‐Mendez Manuel 
Published: May 2005

AbstractBy using single‐strand conformation polymorphism (SSCP) analysis of three amplicons of the cytochrome b gene obtained by the polymerase chain reaction (PCR) it was possible to differentiate between various species of tunas and bonitos processed as canned fish. Four different techniques were used to produce single‐strand DNA (ssDNA): (i) Denaturation of double‐strand DNA (dsDNA) by formamide and alkali, (ii) two‐step asymmetrical PCR, (iii) one‐step asymmetrical PCR, and (iv) exonuclease digestion of the phosphorylated strand of dsDNA. The technique rendering optimal results depended on the type of amplicon (i.e. the sequence).
The combination of Indian ink staining with immunochemiluminescence detection allows precise identification of antigens on blots: Application to the study of glycosylated barley storage proteins
Authors: Eynard Lucia , Laurière Michel 
Published: May 2005

AbstractThe localization after blotting of specific spots in two‐dimensional electrophoretic protein pattern was achieved using, in that order, Indian ink protein staining and immunodetection with chemiluminescence on the same membrane. Indian ink did not inhibit significantly the antibody reactions even after overnight staining. It produces permanent staining that did not quench the chemiluminescent signal, recorded on a film. This allowed perfect matching between the specific and the total protein patterns. The procedure was applied to the identification of glycoproteins present in barley storage protein preparations.
Proteins of rat serum: II. Influence of some biological parameters of the two‐dimensional electrophoresis pattern
Authors: Miller Ingrid , Haynes Paul , Gemeiner Manfred , Aebersold Ruedi , Manzoni Cristina , Lovati Maria Rosa , Vignati Mara , Eberini Ivano , Gianazza Elisabetta 
Published: May 2005

AbstractThis report complements the database already detailed for serum proteins of healthy adult male rats (P. Haynes et al., Electrophoresis 1998, 19, 1484–1492). The influence on the two‐dimensional electrophoresis (2‐DE) pattern of certain physiological conditions (sex, age) was studied as well as of changes in thyroid metabolism. We have extended the information about the major components of rat serum by identifying the proteins typical for the response to acute inflammation. Analyzing 27 spots, six proteins not found in control sera could be recognized; migration at overlapping or close positions with five already characterized proteins was observed for some. A compilation of all our rat data can be accessed through: http://weber.u.washington.edu/∼ruedilab/aebersold.html.