bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the gene for apolipoprotein E5‐Frankfurt (Gln81 ‐> Lys, Cys112 ‐> Arg) by polymerase chain reaction, restriction isotyping, and temperature gradient gel electrophoresis
Authors: Ruzicka Viktor , Mäz Winfried , Russ Andreas , Fisher Eva , Mondorf Werner , Groß Werner 
Published: May 2005

AbstractA new apolipoprotein (apo) E variant, apoE5‐Frankfurt, was identified in a 43‐year‐old male with moderate hypercholesterolemia. On isoelectric focusing in an immobilized pH gradient, apoE5‐Frankfurt migrated to a position more cathodic than apoE4 (Cys112 ‐> Arg). On sodium dodecyl sulfate‐gel electrophoresis, its apparent molecular weight could not be distinguished from that of the three common apoE isoforms (E2, E3 and E4). Restriction isotyping with CfoI (HhaI) showed that apoE5‐Frankfurt had arginine in positions 112 and 158 of the mature protein, suggesting that the mutation accounting for the additional positive charge had occurred in an ϵ4 allele. The third and the fourth exon of the apoE gene were amplified using the polymerase chain reaction and analyzed by temperature gradient gel electrophoresis. This suggested that there were two mutations in the fourth exon of the mutant allele. Cloning and sequencing disclosed that, apart from the exchange of arginine for cysteine in position 112, a C to A substitution replaced glutamine (CAA) in position 81 by lysine (AAA).
Differences in the isoelectric focusing patterns of serum and cerebrospinal fluid transthyretin
Authors: Szilágyi A. Katalin , Ha Nguyen Thu , Szetényi Judit , Puskás Éva 
Published: May 2005

AbstractTransthyretin isolated by polyacrylamide gel electrophoresis from human serum and cerebrospinal fluid, dissociated into its subunits, was subjected to isoelectric focusing in polyacrylamide gels containing 8 mole/L urea. The isoelectric focusing multi‐component patterns of serum and cerebrospinal fluid transthyretin differ in a characteristic way, having only one main protein zone in common. Double diffusion immunotest and immunoblotting revealed the immunological identity of serum and cerebrospinal fluid transthyretin and of the main components separated by isoelectric focusing. The different isoelectric focusing zones can be consistently explained when they are ascribed to structurally identical transthyretin subunits associated with different ligands specifically occurring in either serum or cerebrospinal fluid. Only the protein zone located at the same pI, in serum and cerebrospinal fluid transthyretin patterns may be assigned to ligand‐free subunits. Thus, the typical differences in isoelectric focusing patterns may point to different carrier functions of transthyretin in serum and cerebrospinal fluid.
A method for simultaneous determination of groupspecific component (GC) and alpha2HS‐glycoprotein (AHSG) by ultrathin‐layer isoelectric focusing and immunoblotting
Authors: Kuchheuser Werner , Krause Dieter 
Published: May 2005

AbstractA method for simultaneous determination of group‐specific component and alpha2‐HS‐glycoprotein by ultrathin‐layer isoelectric focusing and immunoblotting is described. The advantages of this combined technique are the saving of chemicals and time. The band patterns of both marker proteins were always reproducible.
Human erythrocyte pyrimidine 5′‐nucleotidase isozymes: Effect of sulfhydryl reagents and electrophoretic discrimination
Authors: Manco Licínio , Amorim António 
Published: May 2005

AbstractThe electrophoretic behavior of human red cell pyrimidine 5′ ‐nucleotidase iso zymes (UMPH1 and UMPH2) was studied on starch gels with and without treatment with thiol reagents. It was found that at least one reactive sulfhy‐dryl group occurs in the UMPH1 isozyme but not in the UMPH2 isozyme. An electrophoretic system is described that allows the discrimination of UMPH1 and UMPH2 isozymes.
Disulfide linked dimers of apolipoprotein D in urine
Authors: Blanco‐Vaca Francisco , Pownall Henry J. 
Published: May 2005

AbstractApolipoprotein (apo) D is a glycoprotein that contains at least one free cysteine. This allows the formation of disulfide linked dimers of apoD, a phenomenon that could interfere with the study of the isoforms of apoD. Consequently, it is important to consider the effects of hetero‐ and homodimer formation on the molecular heterogeneity of apoD as well as on the evaluation of the specificity of antibodies to this glycoprotein. The identification of apoD in urine has provided a potential new marker of tubular proteinuria [1]. Thus, we have studied the specificity of our polyclonal antibodies to apoD against the proteins present in normal urine, and at the same time, the existence of dimeric species of apoD linked by disulfide bonds in urine. The specimens were obtained from apparently healthy individuals and analyzed by Western blot. The results showed that apoD in urine exists as a mixture of monomers and dimers, the latter having apparent molecular weights different from those occurring in plasma. Only monomeric apoD was observed under reducing conditions, proving the monospecificity of the polyclonal apoD antibodies.
The rat liver epithelial (RLE) cell nuclear protein database
Authors: Wirth Peter J. , Luo Lin‐Di , Benjamin Timothy , Hoang Tanya N. , Olson Arthur D. , Parmelee David C. 
Published: May 2005

AbstractThe master two‐dimensional computer database of rat liver epithelial (RLE) cellular proteins (Wirth et al., Electrophoresis 1991, 12, 931–954) has been expanded to include detailed information concerning 1100 nucleoplasmic (cytosolic) and 850 particulate associated [35S]methionine labeled as well as 215 nucleoplasmic and 269 particulate associated [32P]orthophosphate labeled RLE nuclear polypeptides, respectively. The RLE nuclear protein database developed using the Elsie 5 gel analysis system contains both qualitative and quantitative annotations including polypeptide identification number, protein name (if known), molecular weight and pI information, quantitation and polypeptide spot shape, subcellular location, as well as specific information regarding transformation (chemical and spontaneous) and growth‐related characteristics. Microsequencing of polypeptides directly from two‐dimensional (2‐D) blotted membranes has recently been established in our laboratory and provides a highly efficient and rapid means of polypeptide identification in the absence of specific antibodies. At present the RLE protein database is still in the developmental stage and is continually being updated as additional information is obtained. Nonetheless, it is anticipated that knowledge obtained concerning the identification and characterization of specific transformation and/or growth regulatory proteins in the RLE in vitro cell system will not only have direct application to other rodent and human 2‐D protein databases currently under development but will also complement them.
Plasma and red blood cell protein maps: Update 1993
Authors: Golaz Olivier , Hughes Graham J. , Frutiger Séverine , Paquet Nicole , Bairoch Amos , Pasquali Christian , Sanchez Jean‐Charles , Tissot Jean‐D. , Appel Ron D. , Walzer Claude , Balant Luc , Hochstrasser Denis F. 
Published: May 2005

AbstractThis publication updates the reference plasma and red blood cell protein maps obtained with immobilized pH gradients. Seventeen polypeptide spots or chains were partially characterized by direct N‐terminal sequencing or by sequencing of peptides obtained from enzymatic digestion. Additional new polypeptides and previously known proteins are listed in a table and/or labeled on the protein maps, thus providing the 1993 update of the human plasma and red blood cell two‐dimensional gel SWISS‐2DPAGE database. SWISS‐2DPAGE and the SWISS‐PROT protein sequence databases are closely linked together through the use of common accession numbers.
Peculiarities in electrophoretic behavior of different serum albumins
Authors: Miller Ingrid , Gemeiner Manfred 
Published: May 2005

AbstractBehavior of serum albumins of different species was compared with regard to electrophoretic mobility under native conditions, molecular mass and isoelectric point in presence or absence of denaturing agents. In addition to serum samples from human, horse, cow, pig, cat, dog, sheep, goat, chicken, rabbit, mouse, and rat, commercially available standards or albumin preparations obtained by chromatographic procedures were analyzed. Feline and canine albumins showed marked differences in all investigated methods. Our experiments also revealed several factors influencing albumin patterns, especially during isoelectric focusing in immobilized pH gradients or in the presence of carrier ampholytes.
An efficient disk based data structure for rapid searching of quantitative two‐dimensional gel databases
Authors: Lemkin Peter F. , Wu Yecheng , Upton Kyle 
Published: May 2005

AbstractFast access of two‐dimensional (2‐D) gel quantitative databases is important for rapid searching for protein differences between sets of 2‐D gels from an experiment. The GELLAB‐II system organizes corresponding spots from the gels in the database into reference or “Rspot” sets. These Rspot numeric names index fixed regions in the paged composite gel database file. This is adequate for an existing database, but has several problems. (i) Building the initial database requires guessing how much disk space to pre‐allocate for each corresponding spot (i.e. spots from different gels). If it ever runs out of preallocated space during this process, it must expand the size of each corresponding set of spots copying the old database data into the new in‐place on the disk. (ii) When adding new gels or editing the database, if a new spot is created, the system may also go into this expansion mode. The time spent and wasted disk space can be appreciable – depending on the size of the database (order of 100 gel database). (iii) Because each set of corresponding spots is the same size, we waste space in most spot sets since they do not requuire the additional space a few spot sets require which contain additional fragmented spots. We present a new low‐level disk object‐based structure and algorithm paged indexed buckets (PIB), which optimizes disk space usage while having similar retrieval speed to the original method.
Consecutive silver staining and autoradiography of 35S and 32P‐labeled cellular proteins: Application for the analysis of signal transducing pathways
Authors: Luo Lin‐di , Wirth Peter J. 
Published: May 2005

AbstractThe methodology for the simultaneous analysis of protein synthesis concomitant with protein phosphorylation/dephosphorylation is described. The technique consists of metabolic labeling of rat liver epithelial (RLE) cells with [32P]orthophosphate and [35S]methionine, performing two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) of the mixed samples, followed by silver staining and subsequent autoradiography of the dried silver stained 2‐D PAGE electrophoretograms using two films placed back‐to‐back. The first film, which is positioned in direct contact with the dried silver‐stained gel, visualized both exposure to 35S and 32P while the second film recorded exposure to only 32P due to the differential energy levels of the two isotopes. The juxtapositioning of the silver‐stained images with the two autoradiographic film images permits the unambiguous mapping of the phosphorylated polypeptides back to their corresponding silver‐stained and methionine‐labeled counterparts. This strategy provides quantitative information utilizing both silver staining (measure of constitutive levels of protein expression) and metabolic labeling to measure rates of protein synthesis and/or degradation and phosphorylation and/or dephosphorylation using [35S]methionine and [32P]orthophosphate, respectively. We have utilized this methodology for the in vitro analysis of transforming growth factor type β1 (TGF‐β1)‐mediated signal transduction in RLE cells and have identified three nuclear polypeptides, 1 (pI4.95/Mr 97 kDa), 2 (5.00/85 kDa) and 3 (4.90/84 kDa) whose phosphorylation status is rapidly and transiently modulated by TGF‐β1. The methodology described should have wide applications in studies where it is desirous to measure protein synthesis and/or degradation concomitant with signal transduction pathways involving protein phosphorylation.
A comparative two‐dimensional gel protein database of the intact and regenerating newt limbs
Authors: Tsonis Panagiotis A. 
Published: May 2005

AbstractIn this paper we describe a two‐dimensional gel database of the regenerating newt limb. Protein synthesis was compared in the intact limb, in the 1‐week regenerating limb, representing the dedifferentiation stage, and in the 2‐week regenerating limb, representing the formation of the blastema. This comparative database provided data on differential expression of about 800 proteins during the process of limb regeneration. In addition, a map has been generated for these proteins for future guidance in characterizing further new, unknown proteins. The overall expression patterns of the proteins indicated that the dedifferentiation stage was marked by down‐regulation of most proteins, while the blastema formation was marked by the appearance of many new proteins. The potential use of such a database in isolating factors involved during limb regeneration is discussed.
Comparisons of protein changes in human and rodent hepatocytes induced by the rat‐specific carcinogen, methapyrilene
Authors: Richardson Frank C. , Strom Stephen C. , Copple Deborah M. , Bendele Raymond A. , Probst Gregory S. , Leigh Anderson N. 
Published: May 2005

AbstractThere is a growing concern that the rodent biossay may not always serve as an appropriate model to assess the carcinogenic risk for humans exposed to certain compounds. Mechanistic research that examines the effects of a compound in rodent and man could help in the interpretation of bioassay results. This paper reports a novel use of two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) technology to assess similarities and differences in the response of rodents and humans to the rat‐specific hepatocarcinogen, methapyrilene (MP). A sequential examination of rodent and human hepatic proteins was conducted following in vivo exposure of rats and mice and in vitro exposure of rat, mouse, and human hepatocytes to MP. Results showed that covalent modifications observed in rats and mice in vivo were duplicated both qualitatively and quantitatively in the corresponding in vitro systems and that these modifications correlated with carcinogenic susceptibility. Covalent modifications in human hepatocytes were minimal despite exposure to concentrations of MP that were 6‐fold higher than those used in rodent hepatocytes. These studies suggest that in the case of MP the rat is not the most appropriate model for assessing the human situation. Furthermore, these data show that in vitro‐in vivo comparisons based on 2‐D PAGE may provide adjunctive information for extrapolating rodent toxicity/bioassay results to human risk assessment.
Membrane electrophoresis of DNA
Authors: Heller Christoph , Beck Stephan 
Published: May 2005

AbstractWe have separated double stranded DNA fragments by electrophoresis using a membrane instead of a gel as separating matrix. Such a membrane, containing separated DNA fragments, is suitable for subsequent hybridization analysis. The method combines electrophoretic separation and transfer onto a solid support and therefore eliminates the need for a transfer or blotting step.
Typing of the 3′ hypervariable region of the apolipoprotein B gene: Approaches, pitfalls, and applications
Authors: März Winfried , Ruzicka Viktor , Fisher Eva , Russ Andreas P. , Schneider Wolfgang , Groß Werner 
Published: May 2005

AbstractApolipoprotein B‐100 is the principal protein component of lipoproteins with very low, intermediate, and low density. The interaction of apoB‐100 with low density lipoprotein (LDL) receptors is responsible for the uptake of LDL into cells. An AT‐rich hypervariable region is located adjacent to the 3′ end of the apoB gene. It consists of a variable number of tandemly repeated sequences (VNTR). Two approaches were used to analyze this polymorphism. In both, the region harboring the VNTR was amplified with the polymerase chain reaction (PCR). In the first method, fluorescently labeled primers were used in the PCR reactions and products were separated in agarose gels by means of an automated fluorescent fragment analyzer. In the second method, PCR products were analyzed in denaturing polyacrylamide gels and detected with silver staining. Even in the highly sophisticated automated system, agarose gel electrophoresis did not always enable unequivocal assignment of VNTR alleles. In contrast, denaturing polyacrylamide gel electrophoresis made it possible to distinguish the 15 bp differences between the VNTR alleles in a precise and simple manner. The VNTR polymorphism was typed in 234 individuals. Among these were 136 patients with coronary artery disease and 74 healthy controls. Thirteen alleles could be distinguished. The allele containing 49 repeats (VNTR‐49) was found in 9.2% of the coronary artery disease patients and in 4.7% of the controls. Thus, the VNTR‐49 allele increases relative coronary risk by about twofold. It is concluded that the apoB VNTR polymorphism is a potentially useful genetic marker. Since agarose gel electrophoresis may lead to ambiguous results, we prefer typing by denaturing polyacrylamide gel electrophoresis. This has to be accounted for, especially if the apoB VNTR polymorphism is applied to forensic studies.
Amplification of a Y‐chromosomal DNA sequence by the polymerase chain reaction for documentation of residual recipient cells in small samples from bone marrow, peripheral blood and cerebrospinal fluid after bone marrow transplantation
Authors: Suttorp Meinolf , Sprenger Christine , Dreger Peter , Sievers Erika , Claviez Alexander , Löffler Helmut , Schaub Jürgen , Schmitz Norbert 
Published: May 2005

AbstractThe long arm of the human Y‐chromosome contains about 800 to 5000 copies of the tandemly repeated DNA sequence DYZ1. A major part of the repeating unit (pHY10)has been cloned and sequenced [1]. Primers were designed to match a part of this repeat sequence for the amplification of a 154 bp fragment spanning the Eco RI restriction site of the unit [2]. Typical dilution experiments showed that this polymerase chain reaction (PCR) method allows the detection of 5 to 10 male cells among 100000 female cells [3], or in 500 μL of cerebrospinal fluid containing only one cell per μL. In addition, the quality of the DNA used for the amplification reaction is less critical, thus allowing analysis of long‐term stored samples such as bone marrow smears or dried blood stains spotted onto filter paper, which might contain partially degraded DNA [4]. We applied this technique to detect residual host cells in the clinical setting of human sex‐mismatched bone marrow transplantation (BMT). Fourteen patients, receiving transplantations because of leukemias could be supervised so far. Throughout the whole period of monitoring (days + 14 until +911 post BMT; median: 160 days), residual host cells were detected in all but three patients. Persistence of host cells in the early phase post‐BMT was mostly transient and probably due to long‐term surviving host T‐lym‐phocytes. Reappearance of host cells several months after BMT is highly suspicious of relapse from the underlying malignancy. Due to its high sensitivity, PCR is a valuable tool in monitoring the switch from recipient to donor cell population. In addition, the documentation of persisting or reappearing recipient cells could be associated with clinical relapse [5].
Agarose electrophoresis of DNA in discontinuous buffers, using a horizontal slab apparatus and a buffer system with improved properties
Authors: Zsolnai Attila , Orbán Lászlo , Chrambach Andreas 
Published: May 2005

AbstractUsing a horizontal slab apparatus with a buffer in the reservoirs at the level of the gel (“sea‐level electrophoresis”), the retrograde discontinuous buffer system reported by Wiltfang et al. for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of proteins was applied to DNA electrophoresis. This application yielded the advantages of an increased displacement rate of the moving boundary front and a decrease in the concentration of the counterion base in the resolving phase, which yielded reduced relative mobility values at equivalent gel concentrations and practicable low buffer concentrations. The change of relative mobilities (Rf) with a variation of field strength is decreased compared to that of the migration rate in the continuous Tris‐boric‐acid‐EDTA (TBE) buffer and thus the robustness of the system is improved, as well as the efficiency of separation. The system of Wiltfang et al. has in common with previously described discontinuous DNA systems, that it is able to stack DNA from dilute samples and is insensitive to sample components with lower net mobilities than DNA, such as acetate. However, the variance of Rf at constant current density in the discontinuous buffer system is not improved over that of the migration rate at constant field strength in the continuous TBE buffer.
On the use of dimensionless parameters in acid‐base theory. II. The molar buffer capacities of bivalent weak acids and bases
Authors: Rilbe Harry 
Published: May 2005

AbstractIt is shown that stoichiometric rather than thermodynamic dissociation constants can and should be used in the buffer capacity theory. The mean valence of bivalent weak acids and bases is deduced in a form with dimensionless parameters. The expressions for the molar buffer capacities of bivalent weak acids and bases are identical with that for a bivalent ampholyte, and the theory for the latter given earlier is thus valid in all details for bivalent weak acids and bases.
Pattern variations of polyclonal and monoclonal immunoglobulins of different isotypes analyzed by high‐resolution two‐dimensional electrophoresis
Authors: Tissot Jean‐Daniel , Hochstrasser Denis F. , Spertini François , Schifferli Jürg A. , Schneider Philippe 
Published: May 2005

AbstractHigh‐resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was used to analyze serum samples and purified immunoglobulins (Ig) obtained from “normal” individuals and from patients diagnosed with monoclonal gammopathies (MG) (n = 47; 5 IgA, 15 IgM, 15 IgG, 4 biclonal IgG, 1 IgD, 7 Bence Jones proteins). Polyclonal and monoclonal heavy (H) chains were located at different restricted gel positions according to their isotype. Monoclonal H chains appeared as sets of spots characterized by charge (pI) and size (Mr) microheterogeneity. Most of the monoclonal gamma chains were not seen on the gels (12/15). Supplementary polypeptides of 45–48 kDa were detected in serum samples containing monoclonal IgM, but were not seen in MG of other isotypes. However, these polypeptides were not specifically associated with monoclonal IgM because they were also found on protein maps of purified polyclonal IgM. Polyclonal light (L) chains appeared as cloudy bands containing several zones of higher density, whereas monoclonal L chains were usually resolved as single sharp spots. In 6 samples, monoclonal L chains were not seen, and in 9 samples, they appeared as two or more spots, characterized by different pI and/or Mr. In one sample obtained from a patient with a biclonal gammopathy, the L chains were resolved as 4 different spots. Our results confirm that 2‐D PAGE is an excellent tool to study Ig. Analysis of the L chain region of the gels was particularly informative. Several monoclonal L chains exhibited heterogeneous two‐dimensional spot patterns, suggesting that “subtile” clonal mutations of B‐cell lineage and/or posttranslational modifications were involved in their production.