bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the binding of deoxyribonuclease I to G‐actin by capillary electrophoresis
Authors: Carter Laura K. , Christopherson Richard I. , dos Remedios Cristobal G.
Published: May 2005

Deoxyribonuclease I (DNase I) is an actin monomer‐sequestering actin binding protein (ABP) that inhibits the rate and extent of actin polymerisation in vitro by forming a high affinity, stoichiometric 1:1 complex. Using capillary zone electrophoresis (CZE), we have studied the interaction between G‐actin and DNase I to evaluate the capability of CZE to determine the dissociation constant (Kd value) for this interaction. We used (i) an uncoated fused‐silica capillary and ultraviolet (UV) detection at 214 nm; (ii) a hydrophilic‐coated capillary with UV detection at 214 nm; and (iii) a hydrophilic‐coated capillary with laser‐induced fluorescence (LIF) detection. Using procedure (ii), a Kd value of approximately 0.03 μM was obtained by simulation of binding data. We conclude that CZE combined with a LIF detector has the capacity to extend the determination of Kd values from the micromolar range to the nanomolar range. Subsequent determination of Kd values for other actin‐binding proteins should provide information on interactions between the binding sites on actin for these proteins and their spatial relationship.
Escherichia coliproteome analysis using the gene‐protein database
Authors: VanBogelen Ruth A. , Abshire Kelly Z. , Moldover Brian , Olson Eric R. , Neidhardt Frederick C.
Published: May 2005

The gene‐protein database ofEscherichia coliis a collection of data, largely generated from the separation of complex mixtures of cellular proteins on two‐dimensional (2‐D) polyacrylamide gel electrophoresis. The database currently contains about 1600 protein spots. The data are comprised of both identification information for many of these proteins and data on how the level or synthesis rates of proteins vary under different growth conditions. Three projects are underway to further elucidate theE. coliproteome including a project to localize on 2‐D gels all of the open reading framed encoded by theE. colichromosome, a project to determine the condition(s) under which each open reading frame is expressed and a project to determine the abundance and location of each protein in the cell. Applications for proteome databases for cell modeling are discussed and examples of applications in therapeutic drug discovery are given.
Towards a proteome project of cyanobacterium Synechocystis sp. strain PCC6803: Linking 130 protein spots with their respective genes
Authors: Sazuka Takashi , Ohara Osamu
Published: May 2005

Following the complete sequencing of the genome of the univellular cyanobacterium, Synechocystis sp. strain PCC6803, within our institute, a protein‐gene linkage map of this photosynthetic microorganism was successfully constructed for 130 high abundance proteins present on two‐dimensional gels. An additional six proteins were analyzed, but were probably encoded extrachromosomally. In order to demonstrate the usefulness of this protein‐gene linkage map, we analyzed the changes that occur in cellular proteins after illumination of PCC6803 cells. The results indicate that this protein‐gene linkage map greatly simplifies the identification process of such modulated genes. After illumination, at least three distinctive spots with reduced intensity were detected on two‐dimensional gels and the corresponding genes of two of these were successfully identified as chaperonin 2 and a Tortula ruralis rehydrin‐related gene. Thus, the combination of the protein‐gene linkage map and two‐dimensional gel electrophoresis should permit a comprehensive analyses of the proteins encoded by the genome (i.e., “proteome”) of this photosynthetic autotroph. This post‐genome project represents a productive way of exploiting the information obtained from the sequencing of the cyanobacterium genome.
Characterisation of basic proteins from Spiroplasma melliferum using novel immobilised pH gradients
Authors: Cordwell Stuart J. , Basseal David J. , Bjellqvist Bengt , Shaw Denis C. , Humphery‐Smith Ian
Published: May 2005

Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) has become the method of choice for efficient separation of complex protein mixtures. Previously, analysis of the Spiroplasma melliferum proteome (protein complement of a genome) has been performed with pH 3–10 and narrow range pH 4–7 IPG gel strips. We report here on the use of novel 18 cm basic (pH 6–11) immobilised pH gradients (IPG) to increase the resolution of protein spots visible within 2‐D gels. These gradients were synthesised to emulate the gradient of commercially available IPG gel strips in a 5 cm region of overlap so as to attempt construction of a more complete map of cellular protein expression. Approximately 50 additional gene products were detected from S. melliferum that were not previously well‐resolved or visible using wide‐range pH 3–10 IPG gel strips. Twenty‐seven of these were electrotransferred to polyvinylidene difluoride (PVDF) membrane and analysed by N‐terminal protein microsequencing. Protein spots with an initial peak yield of as little as 100 femtomoles (fm) were sequenced to 5–10 amino acid residues, demonstrating the importance of improved sample handling procedures and analytical technologies. Many essential metabolic enzymes were shown to have basic pI, including: glyceraldehyde‐3‐phosphate dehydrogenase, pyruvate kinase, carbamate kinase and lactate dehydrogenase. A very basic protein (pI ≈︁ 11.0) was identified as uridylate kinase, an enzyme indirectly associated with pyrimidine biosynthesis and thought be absent in some members of the bacterial class Mollicutes. The advent of novel basic (pH 6–11) IPGs has allowed the visualisation of a significantly greater percentage of the ‘functional proteome’, that portion of the total protein complement of a genome actively translated within a specific time frame, on 2‐D electrophoresis gels. This will aid in the characterisation of translated gene products in conjunction with genome sequencing initiatives.
Global analysis of physiological responses in marine bacteria
Authors: Östling Jörgen , McDougald Diane , Marouga Rita , Kjelleberg Staffan
Published: May 2005

In this paper, we present results from studies on marine Vibrio species, in which complex adaptive responses have been investigated. The results of two‐dimensional polyacrylamide gel electrophoresis serve to illustrate the usefulness of a global approach, and how it can be combined with other methodologies in order to achieve an improved understanding of the means by which bacteria adapt to alterations in environmental conditions. The overall strategies described in this paper are particularly useful for studies of bacteria for which efficient genetic tools, background genotypes and in depth physiological data are not yet available.
Development of a Haemophilus two‐dimensional protein database
Authors: Cash Phillip , Argo Evelyn , Langford Paul R. , Kroll John S.
Published: May 2005

Members of the Haemophilus genus are responsible for various human infections including respiratory infections and meningitis. The complete nucleotide sequence of the Rd strain of Haemophilus influenzae has been reported and represents a valuable resource to investigate gene expression within this bacterial group. We described previously the application of two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) to characterise the proteins of Haemophilus influenzae (Cash et al., Electrophoresis 1995, 16, 135–148). We have extended these data with comparative studies of the proteins from other members of the Haemophilus genus (specifically H. parainfluenzae, H. haemolyticus and H. parahaemolyticus) to identify homologous proteins and, by extension, the genes encoding them, among these bacteria. The proteins extracted from each of these bacterial isolates were compared by coelectrophoresis to the 2‐D protein profile of the reference nontypable strain of H. influenzae (HI‐64443) used as the basis for the 2‐D protein database. A composite reference 2‐D protein profile of HI‐64443 was derived from three independent analyses of the soluble bacterial proteins. Between 21% and 37% of the HI‐64443 proteins from the reference 2‐D protein profile comigrated with proteins in the other isolates from the Haemophilus genus. This compared with 62% and 64% comigration when HI‐64443 was compared with the Eagan and Rd strains of H. influenzae, respectively. The 2‐D protein profile of the Rd strains of H. influenzae was compared to that of HI‐64443 by coelectrophoresis; 64% of the proteins detected for the Rd strain comigrated with proteins found for HI‐64443 when analysed in parallel. The capacity of 2‐D PAGE to investigate global interactions of gene expression was applied to the analysis of superoxide dismutase (SOD) expression in H. influenzae strain Eagan. A “knock‐out” mutant in the sodA gene which encodes [Mn]‐SOD was characterised with respect to proteins synthesis compared to the parental isolate. From these analyses, the primary product of sodA was provisionally identified as a protein with a molecular mass of 25 500 Da and an estimated pI of 6.55. Quantitative changes in the expression of two other proteins in the SOD mutant were detected by comparison with the parental isolate. These data are discussed in relation to the development of a 2‐D protein database for H. influenzae and related bacteria to investigate genome homologies and gene expression.
Cloning of minisatellite‐containing sequences from two‐dimensional DNA fingerprinting gels reveals the identity of genomic alterations in low‐grade gliomas of different patients
Authors: Marczinek Karola , Sugiyama Akio , Hampe Jochen , Thiel Gundula , Lehmann Karin , Neumann Regina , de Leeuw Wilio J. F. , Nürnberg Peter
Published: May 2005

Two‐dimensional (2‐D) DNA fingerprinting was used to investigate genomic changes in human low‐grade gliomas of different subtypes. DNA variations were identified in the 2‐D hybridization patterns as spot losses or gains. Computer‐aided matching of spot patterns from different patients revealed a clustering of spot changes at particular areas in the gel. Representative spots of each cluster were cloned using a spot cloning protocol which includes the preparation of a duplicate and a master gel. The DNA fragments of the 2‐D gels were transferred to DEAE and nylon membrane, respectively. After hybridization of the master blot with a minisatellite core probe, the position of a particular spot was determined with reference to the lambda DNA fragments used as external markers in both gels. The gel spot DNA was recovered from the DEAE membrane by high salt elution and was polymerase chain reaction (PCR)‐amplified after ligation of adaptor oligo cassettes. The PCR products were cloned and used as locus‐specific probe for the rehybridization of the 2‐D blots. One of these probes detected a spot loss in 7 of 28 low‐grade gliomas of different subtypes analyzed. Another probe revealed a characteristic intensity shift in 8 of 9 pilocytic astrocytomas between two neighboring spots. The target sequence of this highly specific effect was assigned to chromosome 11q14 by in situ hybridization of a P1 clone harboring the affected genomic region. Thus, we successfully established a spot cloning procedure for the generation of locus‐specific probes that may be instrumental in the discovery of the ciritical early events of glioma pathogenesis.
Optimization of a hexaplex DNA amplification from short tandem repeat and amelogenin loci
Authors: Miścicka‐Śliwka Danuta , Grzybowski Tomasz , Woźniak Marcin
Published: May 2005

An automated DNA profiling system based on the multiplex amplification of highly polymorphic short tandem repeat (STR) markers and the amelogenin locus was developed. Five STR loci with nonoverlapping allele size ranges have been utilized in the multiplex amplifications, including HUMD1S103, HUMTH01, HUMD21S11, HUMD18S51, and HUMFIBRA. One primer for each locus was labeled with a fluorescent dye (fluorescein) which allows detection on the single wavelength ALF DNA Sequencer (Pharmacia Biotech). As part of the detailed evaluation of the suitability of the hexaplex system for routine forensic use, the effect of variation in amplification parameters on the efficiency of the system was examined. Polymerase chain reaction amplification conditions were optimized to provide specific, robust amplification of forensic samples.
Molecular sequences of two minisatellites in blacklip abalone, Haliotis rubra
Authors: Huang Bixing , Chai Zhonglin , Hanna Peter J. , Gough Keith H.
Published: May 2005

In the cloning and sequencing of growth‐promoting genes of the blacklip abalone, Huliotis rubra (Leach, 1814), two DNA variable number of tandem repeats (VNTRs) were identified in abalone cDNA libraries. One contained a 33 bp repeat unit (5′‐CCCAAGGTCCCCAAGGTCAGGGAGGCGAAGGCT‐3′) located in the 3′ untranslated region of a putative growth hormone (GH) gene, and the repeat was designated as GHR. The other contained an 18 bp repeat unit (5′‐ACCCGGCGCTTATTAGAG‐3′) located in the 3′ untranslated region of a putative molluscan insulin‐related peptides (MIP) gene, and was designated as MIPR. Primers flanking the two VNTR repeat regions were derived from sequence information. One hundred blacklip abalones were collected along the Victorian coastline and used in a preliminary population study. The range of GHR alleles containing the 33 bp basic unit repeat motif included 7 to 20 repeats, with allele GHR 8 not being identified. The most frequent alleles contained GHR 16 and 17 repeats (56.0% and 16.5%, respectively). Four types of alleles were identified in MIPR, viz 4, 5, 6 and 7 repeats. The alleles containing 6 and 5 repeats were the most frequent (50.0% and 41.5%, respectively). Overall, the results indicate that these two DNA minisatellites have use in abalone studies, including paternity resting, triploid testing, population genetic structure, and gene flow.
Capillary electrophoretic detection of metabolites in the urine of patients receiving hypoglycemic drug therapy
Authors: Roche Matthew E. , Oda Robert P. , Lawson George M. , Landers James P.
Published: May 2005

Micellar electrokinetic chromatography (MEKC) in tandem with diode array detection (DAD) has been exploited as an analytical method for the separation and detection of sulfonylurea drugs. The ultimate goal is the development of an assay to detect these drugs or their metabolites in urine as a means of diagnosing sulfonylurea drug abuse. Using a spearation buffer consisting of 5 mM borate/5 mM phosphate/75 mM sodium cholate, separation of both the second and third generation sulfonylurea drugs can be achieved. The characteristic absorbance spectra associated with each of the third generation drugs, glipizide and glyburide, allow for their identification in mixtures. Coinjection of glyburide, its primary metabolite, hydroxy glyburide, and glipizide demonstrated that the metabolite was resolved from the parent drug but shared its absorbance spectral properties. MEKC analysis of a series of solid phase‐extracted urine samples from patients prescribed glipizide or glyburide, as well as from control patients not ingesting the drug, showed that the parent compounds were difficult to detect in the urine. However, the use of DAD allowed for detection of metabolites in the urine of these patients. With glyburide patients, only primary metabolites were detected, while urine from patients on glipizide showed a series of peaks whose absorbance spectra was consistent with the presence of both primary and secondary metabolites. In addition, the intensity of the metabolite peaks corresponded reasonably well with the respective dose and in vivo time interval associated with the urine collection. This study shows that MEKC with DAD has potential for further exploration as a clinical assay for detecting surreptitious abuse of sulfonylurea drugs.
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

Pharmaceuticals 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

The 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

The 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

A 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

Several 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

Almonds 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

We 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

Nuclear 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

The 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

This 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

The 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

The 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

Previous 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

This 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

Rab7 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

After 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

The 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

We 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.
Redefining the separation factor: A potential pathway to a unified separation science
Authors: Bowser Michael T. , Bebault Gwendolyn M. , Peng Xuejun , Chen David D. Y.
Published: May 2005

Understanding the separation process in capillary electrophoresis (CE) leads to the unification of the theories for separation science. While the separation of analytes is governed by equilibria in chromatography, and by (centrifugal) field in ultracentrifugation, the separation in CE is governed by both equilibria and (electric) field. Therefore, a comprehensive separation theory that describes the separation process of analytes in CE should be able to describe the separation processes in both chromatography and ultracentrifugation. In this paper, we propose that individual capacity factors for each analyte species be used to describe the migration behavior of an analyte. The effect of field on each analyte species, as well as the effect of equilibria are considered in deriving a generalized equation that is applicable for all separation techniques. The separation factor defined at present does not directly relate to the migration rates of the analytes, and therefore can not be used in a generalized theory. We propose that the ratio of the migration rates of a pair of analytes (γ) should be used as the separation factor, instead of the ratio of the two capacity factors. When γ is used to describe the separation of two closely migrating analytes, all separation techniques have the same resolution equation.
Simultaneous chiral separation of 3,4‐methylenedioxymethamphetamine (MDMA), 3‐4‐methylenedioxyamphetamine (MDA), 3,4‐methylenedioxyethylamphetamine (MDE), ephedrine, amphetamine and methamphetamine by capillary electrophoresis in uncoated and coated capillaries with native β‐cyclodextrin as the chiral selector: Preliminary application to the analysis of urine and hair
Authors: Tagliaro Franco , Manetto Giulia , Bellini Silvana , Scarcella Daniele , Smith Frederick P. , Marigo Mario
Published: May 2005

The importance of the chiral analysis of amphetamine‐related substances in both clandestine preparations and biological samples is widely recognized. For this purpose, capillary electrophoresis was successfully applied by several authors, but only few reports concerned ring‐substituted amphetamines, which represent the main components of “ecstasy”, a widely abused “recreational” substance. In the present work, the simultaneous chiral analysis of ephedrine, amphetamine, methamphetamine, 3,4‐methylenedioxymethamphetamine (MDMA), 3‐4‐methylenedioxyamphetamine (MDA) and 3,4‐methalenedioxyethylamphetamine (MDE) is reported, by using capillary electrophoresis with native β‐cyclodextrin (15 mM) as the chiral selector. After preliminary tests at different pH values (phosphate buffer 100 mM, pH 2.5–9.0) and with bare or coated fused‐silica capillaries, the optimized conditions were: pH 2.5 phosphate, uncoated capillary (45 cm × 50 μm inner diameter), potential 10 kV. Detection was either by fixed wavelength (200 nm) or multiwavelength (190–400 nm) UV absorbance. Under these conditions, good resolution was obtained for all the analytes, with excellent chiral selectivity and efficiency. The sensitivity for the individual enantiomers was better than 0.2 μg/mL, analytical precision was characterized by relative standard deviation values < 0.8% (⩽ 0.15% with internal standardization) for migration times intra‐day and < 2.0% (⩽ 0.54% with internal standardization) day‐to‐day; linearity, in the range 0.156–40 μg/mL, and accuracy were also satisfactory. After a simple liquid‐liquid extraction, urine samples could be analyzed with a sensitivity well below the recommended NIDA cut‐off of 500 ng/mL. For hair samples, it was necessary to increase the sensitivity by applying a field‐amplified sample stacking procedure, which allowed the chiral determination of MDA, MDMA and MDE at concentrations occurring in real samples from ecstasy users, with the possibility of recording UV spectra of the peaks.
Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a capillary electrophoresis instrument
Authors: Lazaruk Katherine , Walsh P. Sean , Oaks Frank , Gilbert Dennis , Rosenblum Barnett B. , Menchen Steve , Scheibler Don , Wenz H. Michael , Holt Cydne , Wallin Jeanette
Published: May 2005

Automated fluorescence analysis of polymerase chain reaction (PCR)‐amplified short tandem repeat (STR) systems by capillary electrophoresis (CE) is becoming an established tool both in forensic casework and in the implementation of both state and national convicted offender DNA databases. A new capillary electrophoresis instrument, the ABI Prism 310 Genetic Analyzer, along with the Performance Optimized Polymer 4 (POP‐4) provides an automated and precise method for simultaneously analyzing ten flourescently labeled STR loci from a single PCR amplification kit, which provides a power of discrimination of approximately one in five billion from a single PCR amplification. Data are presented on sizing precision, sizing accuracy, and resolution for the STR loci in the AmpFlSTR Profiler™ kit. Sizing accuracy is highly dependent on the electrophoresis system, and therefore the reporting of alleles based on the nucleotide size obtained from an electrophoresis system is not recommended for forensic work. The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelie ladder, allows for accurate genotyping of STR loci. Sizing precision of ⩽ 0.16 nucleotide standard deviation was obtained with this system, thus allowing for accurate genotyping of length variants that differ in length by a single nucleotide.
Amperometric pH regulation – a flexible tool for rapid and precise temporal control over the pH of an electrolyte solution
Authors: Hagedorn Rolf , Korlach Jonas , Fuhr Günter
Published: May 2005

Temporal control over both pH and ionic strength of an electrolyte solution with high accuracy was achieved with a dynamic, computer feedback‐controlled amperometric pH‐stat device consisting of four pH‐regulating electrodes placed in electrolyte reservoirs that are separated by dialysis membranes from a central compartment. Theoretical predictions of the behavior of this arrangement, obtained by computer simulation, were validated by running temporal pH programs such as step functions, oscillations, and linear pH gradients. Deviations from nominal values given by the computer program are within the limits of accuracy of the pH‐measuring electrodes. No volume changes accompany a change of pH or conductivity since ions are forced to leave or enter the central compartment through the membranes by the electrical force applied between the pH‐regulating electrodes. The device is flexible, easy to use and easily miniaturized. We discuss a wide range of possible applications in biochemistry and cell science. These include automated pH adjustment, isoelectric protein separation, amperometric measurement of enzyme kinetics and the response of cell cultures to well‐defined pH changes.
Methods for determination of electrophoretic mobility and stability of complexes originating in solutions during the chiral discrimination process
Authors: Vespalec Radim , Boček Petr
Published: May 2005

An equation for the calculation of electrophoretic mobility of kinetically labile complexes originating in solutions during the chiral discrimination process is derived. The mobility of the complex is calculated from that of a fully ionized racemic compound, measured in absence of the chiral selector, and from the effective mobilities of its enantiomers, corresponding to the concentration of the chiral selector causing their maximum difference. Correct values of stoichiometric stability constants of both enantiomers may be calculated from the mobility of the complex obtained in this way. Both the mobility and the stability values hold only for the experimental conditions used and the selected background electrolyte. The proposed method is demonstrated for the separation of the fully ionized N‐t‐BOC‐DL‐tryptophan with β‐cyclodextrin in 20 mM aqueous solution of α‐hydroxyisobutyric acid, adjusted with NaOH to pH 4.5. Mobility of the complex is 8.4 × 10−9 m2V−1s−1 at 25°C. The stability constants of D‐ and L‐enantiomers of N‐t‐BOC‐DL‐tryptophan with β‐cyclodextrin, KD and KL, obtained from migration data using this mobility of the complex, are 374 ± 37 M−1 and 336 ± 31 M−1, respectively. The geometrical mean value of calculated stability constants, 355 M−1, agrees perfectly with the value of 350 M−1, calculated from the same experimental data by another procedure recently.