bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determination of pharmaceuticals in plasma by capillary electrophoresis without sample pretreatment reproducibility, limit of quantitation and limit of detection
Authors: Kunkel Annette , Günter Stefan , Wätzig Hermann 
Published: May 2005

AbstractPharmaceuticals in human plasma are determined on underivatized fused‐silica capillaries by micellar electrokinetic capillary chromatography (MEKC) without sample pretreatment. Our best method to date uses as running buffer a sodium dodecyl sulfate (SDS) containing borate buffer (60 mM with 200 mM SDS) at pH 10. Between runs, proteins adsorbed to the capillary wall are removed by an acetonitrile and SDS‐buffer rinsing regimen (50% v/v each). A day‐to‐day precision for relative peak areas of about 2% relative standard deviation (RSD; n >40) has been reached. Different rinsing approaches are discussed (salts, enzyme‐containing solutions, organic solvents, hydrofluoric acid). The separation system is tested in a concentration range between approximately 100 mg/L‐10 mg/L. Correlations between the limit of quantitation, the limit of detection and the signal/noise are discussed. The applicability of the system is demonstrated for the pharmaceuticals acetaminophen, salicylic acid, sulfamethoxazole, tolbutamide, and trimethoprim.
The fifth allele of the human deoxyribonuclease I (DNase I) polymorphism
Authors: Iida Reiko , Yasuda Toshihiro , Aoyama Masahiro , Tsubota Etsuko , Kobayashi Michiko , Yuasa Isao , Matsuki Takasumi , Kishi Koichiro 
Published: May 2005

AbstractThe fifth allele, DNASE1*5, of human deoxyribonuclease I (DNase I) has been discovered. Polymerase chain reaction fragments containing exon 5 of the DNase I gene were screened for DNA polymorphism using single‐strand conformation polymorphism (SSCP) analysis. DNAs from 114 unrelated Japanese and 81 German individuals were tested and a new variant was detected. By DNA sequencing analysis, this variant was found to be caused by a heterozygous G‐A transition at nucleotide position 1227 that results in a Val to Met substitution at amino acid position 92 of the mature enzyme. The nucleotide substitution was also confirmed by mismatched polymerase chain reaction (PCR)‐restriction fragment length polymorphism (RFLP) analysis. Genotyping of the variant could be carried out by three independent reactions based on PCR amplification, and phenotyping by isoelectric focusing followed by immunostaining. The results supported the presence of the fifth codominant allele, DNASE1*5, which generates a new isozyme.
Simple and rapid determination of the acetaldehyde dehydrogenase (ALDH2) genotypes by nonradioactive single‐strand conformation polymorphism analysis
Authors: Yuasa Isao , Umetsu Kazuo , Nakagawa Mayumi , Ikebuchi Jun , Inoue Terutaka , Irizawa Yoshito 
Published: May 2005

AbstractThe genotyping of mitochondrial acetaldehyde dehydrogenase (ALDH2) is very important in alcohol studies. We describe an ALDH2 genotyping method based on nonradioactive single‐strand conformation polymorphism (SSCP) analysis on mini‐gels following amplification with a mutated primer set. The three ALDH2 genotypes were clearly and unambiguously distinguished. This method was applied to the ALDH2 genotyping of 129 unrelated Japanese. The allele frequency of ALDH2*2 was calculated to be 0.271, which was consistent with the previous data. The method proved to be simple, rapid and reliable, and dispensed with isotopic reagent and expensive restriction enzymes and equipment. The SSCP method described here is valuable in routine ALDH2 genotyping.
Further genetic heterogeneity in acatalasemia
Authors: Góth László 
Published: May 2005

AbstractA T‐deletion at position 10 of exon 4 for catalase gene was reported as a novel mutation, causing a new genetic type of acatalasemia in Japan. This mutation, destroying a Hinf1 recognition site, was searched for in Hungarian acatalasemic (2) and hypocatalasemic (22) patients and in controls (27) by Hinf1 digestion and sequence analyses of a 203 bp polymerase chain reaction (PCR) product containing the entire exon 4. The Hinf1 polymorphism did not reveal any difference between controls and hypocatalasemic as well as acatalasemic patients. These results were confirmed by sequence analyses showing the T nucleotide for the two acatalasemic and for one unrelated hypocatalasemic patient, as well as for two controls. These findings represent further evidence that acatalasemia is heterogeneous at the DNA level.
The effect of sodium tetradecyl sulfate on mobility and antigen detectability of microtubule proteins in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Hodgkinson Julie L. , Steffen Walter 
Published: May 2005

AbstractSeveral factors been reported to influence the mobility of polypeptide in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) including the brand of SDS. Using microtubule proteins from axonemes of Lytechinus pictus and Spisula solidissima sperm and meiotic spindles of Spisula solidissima we demonstrate that the change in mobility was caused by sodium tetradecyl sulfate (STS), a major contaminant of many commercial SDS brands. We also examined the use of sodium tetradecyl sulfate and different SDS brands as a tool in extracting more information from immunoblot studies. Commercial SDS containing contaminants other than sodium tetradecyl sulfate reduced or eliminated the immunosignal from certain polypeptides and the loss of antigenicity could not even be recovered by immunoblot under “renaturing” conditions. It can thus be concluded that STS can be useful in separating and identifying comigrating polypeptides and in detecting additional immunobands in immunoblots.
Analysis of mandelonitrile lyase and β‐glucosidase from sweet almonds by combined electrophoretic techniques
Authors: Chiari Marcella , Gelain Arianna , Riva Serigo , Tura Debora 
Published: May 2005

AbstractAlmonds are a rich source of mandelonitrile lyase (oxynitrilase) and β‐glucosidase. The isolation of these two enzymes from sweet almonds reqires fractional ammonium sulfate precipitation followed by ion‐exchange chromatography on diethylaminoethyl‐(DEAE) and carboxymethylcellulose (CMC) columns. In the present investigation different electrophoretic techniques such as sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), isoelectric focusing in immobilized pH gradients (IEF‐IPG), and capillary electrophoresis were used to characterize these two enzymes. For the first time, β‐glucosidase and oxynitrilase were separated in an immobilized pH gradient of one pH unit. Capillary zone electrophoresis (CZE) was an excellent tool for analysis of the purity of enzyme preparations, achieving complete separation of various protein constituents in only 15 min. CZE showed a resolving capacity for the separation of enzyme forms comparable to that of isoelectric focusing in an immobilized pH gradient.
Difference gel electrophoresis. A single gel method for detecting changes in protein extracts
Authors: Ünlü Mustafa , Morgan Mary E. , Minden Jonathan S. 
Published: May 2005

AbstractWe describe a modification of two‐dimensional (2‐D) polyacrylamide gel electrophoresis that requires only a single gel to reproducibly detect differences between two protein samples. This was accomplished by fluorescently tagging the two samples with two different dyes, running them on the same 2‐D gel, post‐run fluorescence imaging of the gel into two images, and superimposing the images. The amine reactive dyes were designed to insure that proteins common to both samples have the same relative mobility regardless of the dye used to tag them. Thus, this technique, called difference gel electrophoresis (DIGE), circumvents the need to compare several 2‐D gels. DIGE is reproducible, sensitive, and can detect an exogenous difference between two Drosophila embryo extracts at nanogram levels. Moreover, an inducible protein from E. coli was detected after 15 min of induction and identified using DIGE preparatively.
Common nuclear matrix proteins in rat tissues
Authors: Korosec Thomas , Gerner Christopher , Sauermann Georg 
Published: May 2005

AbstractNuclear matrix proteins have been defined as insoluble residual proteins resulting from treatment of isolated nuclei with nucleases, detergents and high ionic strength buffers. They are considered as in part representing the proteins constituting the three‐dimensional framework of the interphase nucleus. Though cell‐specific nuclear matrix proteins have been differentiated from ubiquitously occurring (common) nuclear matrix proteins, the number and types of common nuclear matrix proteins have not yet been unequivocally established. In the present study nuclear matrix proteins were prepared from isolated nuclei of rat kidney, liver, lung, spleen and testes. The matrix proteins were separated by two‐dimensional (2‐D) electrophoresis and silver stained. Then the spot patterns were compared by computer‐assisted image analysis. Composite images were derived for nuclear matrix proteins of individual tissues. Finding between 396–483 spots per tissue, a total of 964 individual spots were registered. Of these, 102 were common nuclear matrix proteins, as appearing in each of the tissue‐characteristic images. The apparent molecular mass and pI data may serve for further identification of these nuclear proteins.
Capillary electrophoresis based immunoassays: A critical review
Authors: Schmalzing Dieter , Nashabeh Wassim 
Published: May 2005

AbstractThe purpose of this article is to review and evaluate the uses and potentials of capillary electrophoresis (CE) in immunoassay analysis (IA). This review will be divided into four sections. First, a brief summary of the fundamentals, applications and requirements of immunoassays in both research and clinical diagnostics will be given. This section will also cover the rationale behind the current research to combine CE and IA. Also, the specific needs to CE in this field will be addressed. Second, the modes of use of CE in IA will be discussed and typical applications for each will be given. Third, a separate section will be devoted to the investigation of performing immunoassays on micro fabricated devices, an interesting alternative to the conventional capillary‐based approach. Finally, a critical assessment of the current status and merits of this technology will be presented.
Pre‐, on‐ and post‐column derivatization in capillary electrophoresis
Authors: Bardelmeijer Heleen A. , Waterval Joop C. M. , Lingeman Henk , Hof Ron van 't , Bult Auke , Underberg Willy J. M. 
Published: May 2005

AbstractThis survey gives a short overview of the various reagents and procedures that can be used for pre‐, post‐ and on‐column derivatization in capillary electrophoresis. First there is an introduction about capillary electrophoresis as an analytical technique; this is followed by a discussion of the pros and cons of the various modes of derivatization and a comparison with liquid chromatography. In the following paragraphs the reagents for a number of functional groups are discussed. The emphasis is on derivatization of the amino group. Most of the information on the reagents and derivatization procedures is listed in tables together with information on the detection mode, analytes, sensitivity and samples. In addition to the amino group, information is given on labeling of aldehyde, keto, carboxyl, hydroxyl and sulfhydryl groups.
Capillary electrophoretic separations of enantiomers using cyclodextrin‐containing background electrolytes
Authors: Vigh Gyula , Sokolowski Alex D. 
Published: May 2005

AbstractThe 1996 primary literature papers which deal with the separation of enantiomers using cyclodextrins are reviewed here. Though the majority of the papers still use the neutral native cyclodextrins or the neutral derivatized cyclodextrins as resolving agents, there was a significant increase in number of separations which relied on charged cyclodextrins, both weak electrolytes and strong electrolytes, as resolving agents. Also, there was an increase in the number of papers which reported binding constants and correlated them with other physical or chemical characteristics of the analytes. Several successful minor enantiomer determinations were presented, pushing the reliable quantitation levels below 0.1%. Work continued on the simultaneous use of neutral and charged cyclodextrins to improve separation selectivity or peak resolution.
Free‐flow electrophoretic analysis of endosome subpopulations of rat hepatocytes
Authors: Stefaner Isabella , Klapper Herbert , Sztul Elizabeth , Fuchs Renate 
Published: May 2005

AbstractThe separation of functional early and late endosomes from other cellular compartments by free‐flow electrophoresis (FFE) has been previously demonstrated in nonpolarized cells [1, 2]. Here, using 125I‐labeled anti‐secretory component antibodies ([125I]SC Ab) and FITC‐labeled asialoorosomucoid (FITC‐ASOR) as markers of the transcytotic and lysosomal pathway, respectively, we demonstrate the separation of three distinct endosome subpopulations from polarized rat hepatocytes. Internalization of both markers at 16°C resulted in their accumulation in a common endosome compartment, indicating that both the transcytotic and the lysosomal pathways are arrested in the sorting early endosome at temperatures below 20°C. After chase of the markers from early endosomes into the transcytotic or the degradative route at 37°C, transcytotic endosomes carrying [125I]SC Ab migrated with an electrophoretic motility between early and late endosomes while late endosomes labeled with FITC‐ASOR were deflected more towards the anode than early endosomes. These data indicate that in rat hepatocytes, the transcytotic and lysosomal pathways utilize a common (i.e. early endosomes) and two distinct endosome subpopulations (i.e. transcytotic endosomes, late endosomes) prior to delivering proteins for biliary secretion or lysosomal degradation, respectively.
Acidic cytosolic proteins are preferentially imported into rat liver lysosomes
Authors: Aniento Fernando , Roche Enrique , Knecht Erwin 
Published: May 2005

AbstractPrevious studies have reported that lysosomes isolated from human diploid fibroblasts and from rat liver can selectively import and degrade specific proteins. We have now reinvestigated this selectivity using anin vitroassay with rat liver lysosomes and an extract of cytosolic proteins prepared from cultured cells labeled to equilibriums with [35S‐]methionine. Analysis by two‐dimensional gel electrophoresis and autoradiography of the cytosolic proteins bound to the lysosomal membrane and imported into the lysosomes shows that when all cytosolic proteins are simultaneously present in thein vitroassay the lysosomal uptake also occurs in a specific manner. These findings suggest that isolated lysosomes are able to discriminate among different proteins, selecting those with certain features for lysosomal degradation. Additional characterization of the cytosolic proteins which are selectively imported by lysosomes shows that a common structural feature of most, but not all, of these proteins is an acidic isoelectric point (pI<6.0) and a small or intermediate size. This observation is in agreement with earlier studies which established a relationship between thein vivohalf‐lives of cytosolic proteins in rat liver and their net charge, with acidic proteins, in general, being degraded more rapidly than neutral or basic proteins. The reasons for this preference are still uncertain, although a possible explanation is presented.
Natural resistance to infection with intracellular pathogens: Cross‐talk between Nramp1 and Lps genes
Authors: Kovářová Hana , Hajdúch Marián , Macela Alěs 
Published: May 2005

AbstractThis study was designed to analyze the association of Nramp1 and/or Lps genes with differential protein expression in macrophages in order to select candidate proteins that might be related to resistance/susceptibility to various microbial infections under the control of the Nramp1 and/or Lps genes. The macrophage cell lines derived from bone marrow of Nramp1 or Lps congenic mice were utilized and high‐resolution two‐dimensional electrophoreis (2‐DE), separating proteins according to their charge and size, was used as a window into alterations in gene expression and a means to compare the macrophages carrying a resistant allele of Nramp1 gene and/or normal allele of Lps gene, with their counterparts carrying either a susceptible allele of Nramp1 or defective allele of the Lps gene. We demonstrate that the changes of constitutive levels of two proteins named according to their isoelectric point/molecular weight (pI/Mr), p6.6/25 and p7.0/22, discriminate satisfactorily not only the macrophages congenic at the Nramp1 gene but also the macrophages congenic at the Lps gene, thus reflecting their common genotype (Nramp1r, Lpsn). Furthermore, the decreased constitutive levels of these proteins in macrophages carrying a defective allele of Lps but preserving a resistant allele of Nramp1 can be, at least in part, restored by stimulation with interferon gamma or lipopolysaccharide. 2‐DE immunoblot analysis identified the p7.0/22 protein as manganese superoxide dismutase. Bcl‐2 appears to be the best candidate for p6.6/25 as suggested by 2‐DE quantitative alterations and Western blot analysis. These proteins are important in the regulation of intracellular redox balance and the regulation of apoptosis in macrophages and their alterations might reflect closely the transport functions of ions or other charged substrates suggested for Nramp1 protein.
Flow cytometric sorting and biochemical characterization of the late endosomal rab7‐containing compartment
Authors: Méresse Stéphane , André Pascale , Mishal Zohair , Barad Marc , Brun Nicole , Desjardins Michel , Gorvel Jean‐Pierre 
Published: May 2005

AbstractRab7 is a small molecular weight GTPase that is known to be associated with late endocytic compartments. Studies in which wild‐type or mutant forms of this protein have been overexpressed in mammalian cells have indicated that rab7 plays a role in controlling membrane transport between late endocytic compartments. However, both the precise site(s) of action and localization of rab7 remain unclear. In the present study, we have used density‐gradient centrifugation in combination with a new epitope‐specific flow cytometric sorting method to isolate rab7‐containing vesicles from baby hamster kidney (BHK) cells. Electron‐micrographs of sorted elements showed a homogeneous population of vesicles that resembles late endosomes. The polypeptide composition of rab7‐containing vesicles was then analyzed by two‐dimensional (2‐D) gel electrophoresis. Rab7‐containing vesicles were enriched in the cation‐independent mannose 6‐phosphate receptor and especially in the precursor forms of cathepsin D. Taken together, these results show that the rab7‐containing vesicles are a component of the endocytic pathway that connects late endosomes and lysosomes and in which precursor forms of lysosomal hydrolases, segregated from their receptor, might be included.
Melanie II – a third‐generation software package for analysis of two‐dimensional electrophoresis images: II. Algorithms
Authors: Appel Ron D. , Vargas J. Reynaldo , Palagi Patricia M. , Walther Daniel , Hochstrasser Denis F. 
Published: May 2005

AbstractAfter two generations of software systems for the analysis of two‐dimensional electrophoresis (2‐DE) images, a third generation of such software packages has recently emerged that combines state‐of‐the‐art graphical user interfaces with comprehensive spot data analysis capabilities. A key characteristic common to most of these software packages is that many of their tools are implementations of algorithms that resulted from research areas such as image processing, vision, artifical intelligence or machine learning. This article presents the main algorithms implemented in the Melanie II 2‐D PAGE software package. The applications of these algorithms, embodied as the feature of the program, are explained in an accompanying article (R. D. Appel et al.; Electrophoresis 1997, 18, 2724–2734).
Typing of the short tandem repeat D8S347 locus with different fluorescence markers
Authors: Pöltl Roger , Luckenbach Christine , Fimmers Rolf , Ritter Horst 
Published: May 2005

AbstractThe short tandem repeat (STR) locus D8S347 was analyzed by capillary electrophoresis. Sequencing data and a population study of 203 individuals from a southwestern German population are presented. We detected 12 different alleles, 340–388 bp in length, and found 40 different genotypes. The heterozygosity index was 85.7%. Futhermore, we investigated the consequences of different fluorescent dyes, namely 6‐FAM, HEX, and ROX, on the ABI‐calculated fragment sizes of defined (i.e., sequenced) alleles (348–376 bp in length). 6‐FAM‐labeled fragments appear to be smaller than the corresponding HEX‐or ROX‐labeled fragments. On average, 6‐FAM‐labeled fragments differ by 3.52 bp from the sequencing data, HEX‐labeled ones by 2.04 bp, and ROX‐labeled ones by 1.42 bp. Generally, small alleles differ less from the expected sequencing data than larger ones.
Identification of bacteriophage T4 virion proteins by transverse pore‐gradient sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and dual amino acid labeling
Authors: Ferguson Peter L. , Coombs David H. 
Published: May 2005

AbstractWe have developed a horizontal N,N′‐methylenebisacrylamide (Bis) acrylamide gradient sodium dodecyl sulfate (SDS) gel system that permits the evaluation of the purity of individual protein bands in complex mixtures. A Bis gradient gel is poured vertically and, after polymerization, reoriented horizontally. A single large sample spanning the top of the gel is then run down at right angles to the gradient. The relative mobility of a few proteins varies considerably from the rest, causing them to merge with and cross other bands as the Bis concentration changes. Band splitting revealed that several bands previously thought to represent a single species are actually comprised of comigrating proteins. Once the Bis/monomer concentration offering the best separation was identified, we sought a simple method for identifying the genetic origin of bands, since many proteins now migrated in new positions on the gel. We reasoned that if infected cells were simultaneously labeled with [35S]methionine and [3H]leucine and the purified virion proteins analyzed to determine their 35S/3H ratio, we could identify most proteins by comparing this ratio with one calculated from the T4 DNA sequence. Our expectations were realized, and we here report the separation and identification of all T4 virion proteins. Finally, we comment on the incorporation of various changes to the original Laemmli SDS‐polyacrylamide gel formulations that have been reported in the literature.