bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of α‐fetoprotein‐P4 sugar chain as α 2→6 monosialylated biantennary complex‐type oligosaccharides with an exposed galactose on the mannose α 1→6 arm by two‐dimensional extended agarose gel‐lectin affinity electrophoresis
Authors: Taketa Kazuhisa , Fujii Yasuto , Aoi Toshie , Taga Hiroko , Nishi Shinzo 
Published: May 2005

AbstractErythroagglutinating phytohemagglutinin (E‐PHA)‐reactive α‐fetoprotein (AFP)‐P4 and E‐PHA weakly‐reactive AFP‐P3 of sera from cord blood at term and from patients with hepatocellular carcinomas showed a mobility of monosialo‐AFP in two‐dimensional extended agarose gel‐E‐PHA affinity electrophoresis. Asialo‐AFP had as high an affinity for E‐PHA as that of monosialo‐AFP‐P4, while disialo‐AFP, AFP‐P2, revealed a negligible affinity for E‐PHA. For Allomyrina dichotoma lectin (allo A), asialo‐AFP had no affinity with a mobility of AFP‐A1s, monosialo‐AFP‐P4 had the highest affinity with a mobility of AFP‐A3, and monosialo‐AFP‐P3 had an intermediate affinity with a mobility of AFP‐A2s. The affinity of AFP‐P4 for E‐PHA was reduced by galactosidase digestion, indicating the presence of galactose residues, one sialylated and the other exposed. From the known high affinity of E‐PHA for biantennary complex‐type oligosaccharides with a terminal β 1→4 galactose of the Man α 1→6 antenna and from that of allo A for an α 2→6 sialylated β 1→4 galactose of the Man α1→3 antenna, together with the nonbisected biantennary structure of AFP‐P4 as deduced from its high affinity for concanavalin A, the sugar chain structure of AFP‐P4, which increases frequently in hepatocellular carcinomas, was identified as α 2→6 monosialylated nonbisected biantennary complex‐type oligosaccharides with an exposed β 1→4 galactose of the Man α 1→6 arm and with or without fucosylated core N‐acetylglucosamine, and AFP‐P3 as similar oligosaccharides with an exposed β 1→4 galactose of the Man α 2→3 arm.
Electrophoretic characterization of wheat grain allergens from different cultivars involved in bakers' asthma
Authors: Weiss Walter , Vogelmeier Claus , Görg Angelika 
Published: May 2005

AbstractIn order to identify and characterize the wheat grain allergens involved in bakers' asthma, proteins were sequentially extracted from whole‐meal flour. The polypeptide composition of the individual solubility fractions (albumin/globulin, gliadin and glutenin) was analyzed by sodium dodecyl sulfate‐gel electrophoresis (SDS‐PAGE), and high‐resolution two‐dimensional gel electrophoresis with immobilized pH gradient 4–9 in the first dimension (IPG‐Dalt). The resolved polypeptides were transferred onto an immobilizing polyvinylidene difluoride membrane and incubated with a pooled serum from four asthmatic bakers. Bound IgE was demonstrated by autoradiography using125I‐labeled anti‐human IgE. Our study demonstrated that the serum of the bakers allergic to flour contained IgE antibodies which bound to numerous polypeptides of all three solubility fractions. The highest percentage of IgE binding was observed with certain albumin and/or globulin polypeptides, whereas the gliadins and glutenins exhibited considerably less allergenicity. SDS‐PAGE revealed that the protein which bound the highest percentage of IgE from the sera of the allergic bakers is a 27 kDa albumin. More detailed investigations using IPG‐Dalt demonstrated that this allergen is not a single polypeptide but consists of several polypeptide spots that differ in their isoelectric points. Quantitative studies using computer‐assisted laser densitometry revealed that the amount of patients' IgE bound by these particular polypeptides differed considerably between the seven wheat cultivars examined, ranging from 13% to 53% of the total radioactive uptake.
Semi‐dry PhastTransfer detection of biotinylated cell surface molecules
Authors: Abdi Kaveh , Li Xiaopu , Mentzer Steven J. 
Published: May 2005

AbstractThe PhastTransfer system is a semi‐dry electrophoretic unit designed to optimize the transfer of small amounts of protein. Because of its efficiency, we adapted the PhastTransfer system for the detection of labeled membrane molecules. Biotin was used as the membrane molecule label because it permitted the long‐term storage of labeled lysates as well as the flexibility of derivatizing several different functional groups. After immunoaffinity separation using magnetic microspheres, the protein was electrophoretically separated with the PhastSystem and transferred with the PhastTransfer unit. Using an avidin‐linked enzyme amplification system, less than 10 ng of loaded protein could be detected on the transfer membrane. Based on these findings, the PhastTransfer system is a fast, reproducible, and convenient method for the transfer of small quantities of labeled cell surface protein.
Electroblotting proteolytic products from native gel for direct N‐terminal sequence analysis: An approach for studying protein‐protein interaction
Authors: Wang Feng , Su Chang , Hollfelder Kurt , Waddington Doreen , Pan Yu‐Ching E. 
Published: May 2005

AbstractProteins which are electroblotted from native gels onto polyvinylidene difluoride (PVDF) membranes are suitable for detailed structural analysis. This method, in conjunction with limited proteolysis and N‐terminal sequencing, has been used to study the molecular interactions between native protein molecules. The interaction between recombinant interleukin‐2 (rIL‐2) and its receptor (rIL‐2Rα) was examined as a model system. The working strategy consists of (i) proteolysis of rIL‐2Rα and rIL‐2Rα/rIL‐2 complex, (ii) separation of the major proteolytic products by native polyacrylamide gel electrophoresis followed by electroblotting onto PVDF membrane, and (iii) sequence analysis of the blotted protein bands for the identification of peptide regions sensitive to proteolysis. Results have indicated that the exon 3 encoded region in rIL‐2Rα is sensitive to proteolysis regardless whether it is complexed with rIL‐2 or not. This suggests that no major conformational changes occur in rIL‐2Rα during interaction with rIL‐2. This electroblotting approach is, therefore, useful for studying protein‐protein interaction in solution.
Skimmed‐milk blocking improves silver post‐intensification of peroxidase‐diaminobenzidine staining on nitrocellulose membrane in immunoblotting
Authors: Ludány Andrea , Gallyas Ferenc , Gaszner Balázs , Andrásfalvy Bertalan , Szücs György , Kellermayer Miklós 
Published: May 2005

AbstractA silver‐staining procedure for enhancing the sensitivity of protein detection on nitrocellulose membranes in immunoblotting is described. After completing any peroxidase‐Ni‐diaminobenzidine immunostaining, nitrocellulose sheets are placed in a physical developer, containing sodium tungstate and ascorbic acid, until the desired grade of silver‐intensification has been reached. In this way a 16‐ to 64‐fold amplification of intensity of the initial immunostaining can be achieved. False positive silver staining of protein bands and of background are suppressed by replacing bovine serum albumin, the blocking agent most frequently used in immunoblotting, with skimmed milk.
Antibodies bound to nitrocellulose in acidic buffers retain biological activity
Authors: Hoffman Wayne L. , Jump Adrien A. , Ruggles Ann O. , Kelly Patrick J. 
Published: May 2005

AbstractThis report compares the binding of proteins to nitrocellulose membranes in acidic buffers (pH 2 and 3) with binding in neutral buffer (pH 7). Initially, similar amounts of antibodies and other proteins bound to the nitrocellulose membrane in both acidic and neutral buffers. However, the susceptibility of individual proteins to displacement (stripping) from the membrane by the milk blocking agent depended on the pH of the buffer used to bind the proteins to the membrane. Most proteins that bound to nitrocellulose in acidic buffers were relatively resistant to milk‐stripping compared to proteins bound in pH 7 buffer. Acid‐binding of proteins to nitrocellulose also decreased the amount of protein that was stripped from the nitrocellulose membrane when Tween 20 was included in the washing buffer. After correcting for the amount of antibody remaining on the membrane after the milk block, it was found that acid‐bound antibodies were unchanged in biological activity when compared with the same antibodies bound at neutral pH. These results suggest that acid‐binding of proteins could increase the sensitivity of nitrocellulose membrane assays that use milk and/or Tween 20.
Intra‐species cross‐reactivity of house dust mite allergens separated by protein blotting and detected by selective elution of mite components and IgE antibodies
Authors: O'Neill Geraldine M. , Baldo Brian A. 
Published: May 2005

AbstractA complex and varied antibody response to allergens, in particular Dermatophagoides pteronyssinus, is observed in the serum of allergic patients. To examine the role of cross‐reacting antigenic determinants in allergy, a strategy has been devised for isolating antibody populations that bind to individual polypeptides of an allergen source separated by sodium dodecyl sulfate‐polyacrylamide electrophoresis and electroblotted to nitrocellulose. Polypeptides are identified on the blots, desorbed and coupled to CNBr‐activated paper discs. The coupled discs are then used as immunoadsorbent surfaces for the binding of antibodies from allergic sera. Antibodies eluted from the polypeptide immunoadsorbents are then used to reprobe protein blots of the allergen. Results are presented which demonstrate strong cross‐reaction between two major mite allergens, Der p I and Der p III, and some weaker intra‐species cross‐reactivities.
Immune response to a hapten of fluorescein isothiocyanate in a single mouse analyzed by two‐dimensional affinity electrophoresis
Authors: Nakamura Kazuyuki , Mimura Yusuke , Takeo Kazusuke 
Published: May 2005

AbstractImmune response to a hapten of fluorescein isothiocyanate (FITC) in a single BALB/c strain mouse was analyzed by two‐dimensional affinity electrophoresis (2D‐AEP). Anti‐FITC antibodies were induced by immunization with FITC‐conjugated bovine serum albumin. The antibodies were separated into a large number of spots of IgG due to differences in their isoelectric points(pI) and binding affinities to the FITC ligand. These spots consisted of IgG families which were composed of several spots having an identical affinity to the ligand but a different pI. The spots were not clearly detected in the antiserum taken on day 7 after the primary immunization, but on day 21 the spots of IgG were clearly detected, with a high diversity and specificity for the ligand. The size and number of IgG spots were markedly increased by the secondary immunization; however, the third immunization did not increase the size and number of IgG spots. The IgG spots of each family were specifically stained with an antimouse IgG subclass antibody. Furthermore, a monoclonal antibody (FL‐D6) was separated by 2D‐AEP into a single family which consisted of seven IgG1 spots having an identical affinity to FITC but different pIs. Therefore, each of the IgG families of anti‐FITC antibodies in the antiserum can be generated by a single clone of anti‐FITC antibody‐producing cells. The substitution of dextran T 2000 or lipopolysaccharide for bovine serum albumin as a carrier for FITC induced much smaller amounts of anti‐FITC antibodies with a low diversity but high specificity to FITC.
Humoral immune response against Helicobacter pylori as determined by immunoblot
Authors: Faulde Michael , Cremer Josef , Zöller Lothar 
Published: May 2005

AbstractAn immunoblot method has been evaluated to diagnose Helicobacter pylori infection serologically by comparing 69 serum specimens from patients with a positive Gram stain and/or culture result and a positive urease test on biopsy material, as well as 51 serum specimens from patients with at least 4 negative urease tests, and negative microscopy and culture results. Sensitivity and specificity was found to be 100%. Recognition of the cross‐reacting flagellin (66 kDa), flagellar sheath protein (51 kDa), and a 14 kDa protein are not a criterion for a current H. pylori infection. On the other hand, any combination of at least two of the 180, 120, 90, 75, 67, 29.5 and 19 kDa bands were diagnostic of infection. Three H. pylori strains, which were compared with both gel electrophoretic analyses and immunoblot reactivity, exhibited in part strong qualitative and quantitative differences that particularly affect the 120 kDa pathogenic factor, the large urease subunit and other proteins especially in the molecular mass range from 50 to 67 kDa. IgG immunoblot patterns showed that the choice of H. pylori strain, as well as a reproducible and standardizable antigen preparation, is of great importance for the reliability of serodiagnostic tests. The immunoblot method was found to be a valuable tool for the semiquantitative confirmation of results achieved with other serological methods as well as optimization and quality control of the antigens used for serodiagnostic purposes.
On the limits of near‐equilibrium DNA gel electrophoretic sequencing
Authors: Slater Gary W. , Mayer Pascal , Drouin Guy 
Published: May 2005

AbstractHigh field intensities deeply affect the migration mechanisms of DNA in gels. The corresponding large electric forces lead to far‐from‐equilibrium transport properties such as the limiting mobility, increased diffusion and strong pulsed field responses. We present a theoretical study of the conditions required to retain a near‐equilibrium reptation transport behaviour. We then examine the possibility of increasing both the field intensity and the gel concentration to sequence large single‐stranded DNA molecules rapidly and efficiently. Finally, we discuss possible experimental approaches based on our theoretical predictions.
Simple repetitive (GAA)n loci in the human genome
Authors: Siedlaczek Ina , Epplen Cornelia , Rieß Olaf , Epplen Jörg T. 
Published: May 2005

AbstractIn order to investigate the organization (and inheritance) of simple tendem (GAA)n repeats in the human genome, different restriction enzymes were employed for DNA digestion followed by separation of the resulting fregments by agarose gel electrophoresis. Frequently cutting enzymes (4 bp recognition sites) revealed highly complex multilocus banding patterns after conventional horizontal submarine gels. Fragments larger than 25 kb, resulting from digestions with rarely cutting enzymes (6 bp recognition sites), were separated by pulsed‐field gel electrophoresis (PFGE). Hybridizations were carried out directly in the gel matrix. Nearly all of the enzymes produce at least one predominant signal band after hybridization with (GAA)6. The frequency of (GAA)n stretches was estimated on human chromosome 4 by probing a respective cosmid library. In comparison with other simple di‐, tri‐ and tetranucleotide repeats, (GAA)n stretches appear underrepresented. Hybridization of a fetal human brain cDNA library indicated very few expressed (GAA)n repeats. These data are discussed with particular reference to genomic organization and other simple repetitive trinucleotide stretches.
The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences
Authors: Bjellqvist Bengt , Hughes Graham J. , Pasquali Christian , Paquet Nicole , Ravier Florence , Sanchez Jean‐Charles , Frutiger Séverine , Hochstrasser Denis 
Published: May 2005

AbstractThe focusing positions in narrow range immobilized pH gradients of 29 polypeptides of known amino acid sequence were determined under denaturing conditions. The isoelectric points of the proteins calculated from their amino acid sequences matched with good accuracy the experimentally determined pI values. We show the advantages of being able to predict the position of a protein of known structure within a two‐dimensional gel.
Optimizing a method of protein extraction for two–dimensional electrophoretic separation of proteins from planarians (Platyhelminthes, Turbellaria)
Authors: Collet Joan , Baguñà Jaume 
Published: May 2005

AbstractDifferent procedures for microscale extraction of proteins from small amounts of tissue of planarians (Platyhelminthes, Turbellaria) to be analyzed by two‐dimensional gel electrophoresis (2‐D PAGE) are compared. Three extraction methods were assessed: (i) extraction of soluble proteins with nondenaturing Tris buffers, (ii) extraction with Tris buffer containing the anionic detergent sodium dodecyl sulfate (SDS), and (iii) denaturing extraction under reducing conditions in the presence of urea and Nonidet P‐40 (NP‐40) with or without SDS. Buffers combining minute concentrations of SDS (0.01%), denaturing concentrations of urea (8M) and alkaline pH solubilized the greatest number of proteins without detectable proteolysis. Neither the presence of protease inhibitors nor higher concentrations of SDS improved protein extraction. We have applied this method to planarians to detect proteins specific to the pharynx. The resulting two‐dimensional pattern shows a larger number of specific spots than in previous extraction methods.
Electrophoretic analysis of plant cysteine and serine proteinases using gelatin‐containing polyacrylamide gels and class‐specific proteinase inhibitors
Authors: Michaud Dominique , Faye Loïc , Yelle Serge 
Published: May 2005

AbstractInclusion of gelatin in polyacrylamide gels provides a sensitive way of detecting multiple proteolytic activities in crude extracts from any source. The present study describes a method allowing discrimination between cysteine and serine proteinases in plant extracts, using gelatin‐containing gels in combination with class‐specific proteinase inhibitors. Preincubation of extracts with 4 mM phenylmethylsulfonyl fluoride, a serine proteinase inhibitor, or with 25 μM L‐trans‐epoxysuccinyl‐L‐leucylamido(4‐guanidino) butane, a cysteine proteinase inhibitor, allowed the identification of enzymes from both classes in extracts of tomato fruit and papaya latex. The efficiency of the two low molecular weight inhibitors used was very high, and the irreversibility of the inhibiting effect was maintained during electrophoresis conducted in the presence of sodium dodecyl sulfate. The analytic procedure described here, with a detection threshold of less than 100 pg enzyme, is the first that allows quick and accurate discrimination of plant cysteine and serine proteinases separated in electrophoretic gels. This simple and rapid technique could be of interest for studying the evolution of class‐specific proteinases in plant extracts during various developmental, physiological, and pathogenic processes. It is also potentially applicable to the majority of eucaryotic and procaryotic systems.
Precise quantitation of chloramphenicol acetyl transferase reporter mRNA by competitive polymerase chain reaction
Authors: Greco Anna , Massé Thierry , Madjar Jean‐Jacques 
Published: May 2005

AbstractA strategy, based on competitive polymerase chain reaction (PCR) after reverse transcription, was developed to quantitate mRNA of the E. coli chloramphenicol acetyl transferase (CAT) gene. Precise quantitation of this rare mRNA is achieved by co‐amplification of a constant amount of the complementary DNA (cDNA) with successive dilutions of a competitive DNA template of known concentration and with the use of the same primers. The unique EcoRI site located in the CAT coding sequence has been abolished in the competitive DNA. The two oligonucleotide primers used are both located in the CAT coding sequence at equal distance from the EcoRI site. After amplification, the PCR products from the cDNA to be quantified and from the competitor DNA are discriminated by EcoRI digestion, followed by separation of the resulting fragments by agarose gel electrophoresis. DNA amplified from the target cDNA is the only DNA digested by EcoRI into two fragments of identical size co‐migrating in an agarose gel. After ethidium bromide staining, comparison of the intensity of fluorescence of the resulting bands in a competition series permits us to estimate precisely the amount of CAT cDNA and therefore of the mRNA to be quantified.
Characterization of E‐PHA‐reactive α‐fetoprotein isoforms by two‐dimensional lectin affinity electrophoresis
Authors: Taketa Kazuhisa , Fujii Yasuto , Taga Hiroko 
Published: May 2005

AbstractErythroagglutinating phytohemagglutinin (E‐PHA)‐dependent isoforms of human α‐fetoprotein (AFP) from cord blood were analyzed for their carbohydrate structures by two‐dimensional electrophoresis with E‐PHA combined with extended agarose gel electrophoresis or with affinity electrophoresis with concanavalin A or Allomyrina dichtoma lectin. By means of neuraminidase and/or β‐galactosidase treatment, AFP‐P2 was identified as α2→6 disialo‐AFP, AFP‐P3 as having biantennary structures with α2→6 monosialylated galactose of the Mannose (Man) α1→6 arm, AFP‐P4 as having α2→6 monosialylated galactose of the Man α1→3 arm, and AFP‐P5 as disialo‐AFP with α2→3 sialylated galactose of the Man α1→6 antenna and with α2→6 sialylated galactose of the other antenna. Desialylated AFP with the terminal galactose of the Man α1→6 antenna with or without the galactose of the other arm also had a migration of AFP‐P4, and other hydrolytic intermediates without the terminal galactose of the Man α1→6 arm with and without the galactose of the other antenna had mobilities of AFP‐P3s and AFP‐P3, respectively. Thus, the present system of two‐dimensional lectin affinity electrophoreses would provide a model for the determination of the sugar chain structure of glycoproteins.
Affinity maturation of anti‐hapten antibodies in a single mouse analyzed by two‐dimensional affinity electrophoresis
Authors: Nakamura Kazuyuki , Mimura Yusuke , Tanaka Tatehiko , Fujikura Yoshihisa , Takeo Kazusuke 
Published: May 2005

AbstractAffinity maturation of anti‐hapten antibodies in an inbred mouse (BALB/c) was analyzed by two‐dimensional affinity electrophoresis (2D‐AEP) in which the antibodies specific to a hapten of dinitrophenyl (DNP) group were separated into a large number of IgG families according to the differences in their isoelectric points (pI) and in their affinities for the ligand of DNP immobilized in the gel matrices. Each of the IgG families consisted of several spots showing an identical affinity for the ligand but different pI, and the spots were specifically stained by immunostaining with an anti‐murine IgG subclass antibody. These results lead us to the conclusion that each of the IgG families is derived from a single clone of antibody‐producing cells specific to DNP. The mass and affinity of the IgG families varied in the course of immunization with DNP‐conjugated chicken serum albumin (DNP‐CSA). The first injection of DNP‐CSA induced a small amount of IgG families showing a wide variety in their affinity for DNP. The second injection induced a large amount of IgG families, especially of families having a medium affinity for DNP. Additional injections, however, did not change the mass or affinity of the IgG families significantly. This suggests that an antigen‐induced somatic mutation of the immunoglobulin gene does not occur frequently to mature the affinity of antibodies by the additional injections after the second injection. 2D‐AEP enables us to analyze the affinity maturation of antibodies in vivo in a single mouse and to document the subclass switch of the antibody in the course of immunization.
Characterization and quantification of mink serum amyloid A protein using two‐dimensional electrophoresis with immobilized pH gradients
Authors: Bruun Cathrine Foyn , Sletten Knut , Husby Gunnar , Marhaug Gudmund 
Published: May 2005

AbstractUsing hydrophobic interaction chromatography, two‐dimensional electrophoresis with an immobilized pH gradient in the first dimension and semidry blotting, three isoforms of mink serum amyloid A protein (SAA) were characterized and studied during chronic inflammation. Compared to conventional methods that have been applied to SAA, the major advantages of the present combination of methods are: (i) use of small serum volumes, (ii) rapid extraction, (iii) high resolution, and (iv) high yield of proteins.