bluuebunny/crossref_metadata_2025 huggingface dataset:
The resolution between two native proteins and between their sodium dodecyl sulfate‐complexes in agarose and polyacrylamide gel electrophoresis
Authors: Chen Nong , Chrambach Andreas
Published: May 2005

Commercial gel electrophoresis apparatus with intermittent fluorescence scanning of the migration path (HPGE‐1000 apparatus, LabIntelligence) makes it possible to measure band width and migration distance as a function of the duration of electrophoresis. As a result, resolution can be evaluated quantitatively and therefore different gel media can be compared objectively. The resolution of fluorescein carboxylate labeled conalbumin (molecular mass 86 kDa) and soybean trypsin inhibitor (22.7 kDa) in gel electrophoresis was found to increase as a function of the gel type in the order SeaKem GTG‐, SeaKem Gold‐agarose, 2% N,N′‐methylenebisacrylamide cross‐linked polyacrylamide, MetaPhor‐XR‐, and SeaPrep‐agarose. The advantage in resolving capacity of SeaPrep agarose over the polyacrylamide gel was by a factor of up to five. The resolving capacity of the agaroses was in indirect relation to the degree of electroendosmosis. In all media, resolution increased with migration distance (time). The same proteins when reacted with sodium dodecyl sulfate (SDS) resolve (i) better at up to 6% SeaPrep agarose concentration than in polyacrylamide, as in the gel electrophoresis of the native proteins; (ii) less effectively, by contrast, at SeaPrep agarose concentrations > 6%, than in polyacrylamide gel; and (iii) significantly better in 4–6% SeaPrep agarose than in 4–6% SeaKem GTG agarose. Since Ferguson plot analysis in both agarose and polyacrylamide gels shows that the two SDS‐proteins are larger than the native proteins with which they are complexed, the superiority of polyacrylamide gels above 7% appears to be correlated with the fact that its mean pore radius, estimated for both media using identical assumptions and identical rigid spherical standards — proteins, is approximately seven times larger than that of SeaPrep agarose in the concentration range of 3–8%, and that therefore the molecular “fit” in polyacrylamide is closer than that in SeaPrep agarose of the concentration range used. The dependence of resolution on the ratio of particle radius to mean pore radius (“fit”) is also suggested by the fact that the two SDS‐proteins resolve in a biphasic dependence on gel concentration in both agarose and polyacrylamide, with a maximum at 6% agarose and 10% polyacrylamide.
Capillary electrophoresis of pesticides: V. analysis of pyrethroid insecticides via their hydrolysis products labeled with a fluorescing and UV absorbing tag for laser‐induced fluorescence and UV detection
Authors: Karcher Arron , El Rassi Ziad
Published: May 2005

Some representative standard pyrethroid insecticides, namely permethrin, phenotrin, cypermethrin, sanmarton and fenpropathrin, were subjected to base hydrolysis with the aim of facilitating the indirect determination of these neutral species of low water solubilities by aqueous capillary electrophoresis. This first involved the base fragmentation of the pyrethroids in alcohol buffer (pH 12.0), and then the selective tagging of the carboxylated hydrolytic products with 7‐aminonaphthalene‐1,3‐disulfonic acid (ANDSA) via a condensation reaction in the presence of organic soluble carbodiimide. The tagging of the hydrolytic products with ANDSA imparted each of the derivatives with two strong sulfonic acid groups whose permanent charges were necessary for achieving aqueous capillary electrophoresis. In addition, the labeling with ANDSA allowed the detection of the derivatives at low levels by capillary electrophoresis laser‐induced fluorescence. The geometric and optical isomers of the ANDSA derivatives of the pyrethroid hydrolytic products were best separated when using electrolyte systems composed of sodium phosphate buffer, pH 6.5, containing n‐octylglucoside chiral surfactant in the presence of small amounts of acetonitrile (e.g., 10% v/v).
From genome to proteome: Protein map of Haemophilus influenzae
Authors: Langen Hanno , Gray Chris , Röder Daniel , Juranville Jean‐François , Takacs Béla , Fountoulakis Michael
Published: May 2005

High‐resolution two‐dimensional (2‐D) polyacrylamide gel electrophoresis allows the separation of complex biological mixtures (i.e., several hundred proteins from a bacterial cell lysate) in a single experiment. In this report proteins from Haemophilus influenzae were separated by 2‐D gels and analyzed by peptide mass fingerprinting and/or amino acid analysis. By comparing the peptide mass profiles and the amino acid composition with the Haemophilus influenzae database, 119 protein spots were identified. The combination of amino acid analysis and peptide mass fingerprinting is a powerful tool for a rapid and economical identification of a large number of proteins resolved by 2‐D gels. Studies on gene regulation and changes of protein expression upon drug treatment require quick and serial analysis techniques to efficiently identify potential new drug targets.
Two‐dimensional map of Haemophilus influenzae following protein enrichment by heparin chromatography
Authors: Fountoulakis Michael , Langen Hanno , Evers Stefan , Gray Chris , Takács Béla
Published: May 2005

Two‐dimensional gel electrophoresis separates several hundred protein molecules in one single experiment and is efficiently used to study the products expressed by different genomes. Low‐copy‐number gene products are invisible on a stained two‐dimensional map and must be enriched such that sufficient amounts are present for visualization and identification. We investigated the enrichment of proteins of the bacterium Haemophilus influenzae by chromatography on immobilized heparin which has affinity for growth and protein biosynthesis factors. Total soluble proteins of the microorganism were fractionated on Heparin‐Actigel which resulted in enrichment of approximately 160 proteins. The eluates, representing about 40% of the applied proteins, were analyzed by two‐dimensional gel electrophoresis and the protein spots were characterized by amino acid composition analysis and matrix‐assisted laser desorption ionization mass spectrometry. The proteins enriched by chromatography on the heparin gel were not exclusively low‐copy‐number gene products and they did not exclusively belong to one single class of proteins. The proteins that bound to the heparin gel are indicated in a two‐dimensional protein map which includes more than 110 newly identified proteins.
Identifying the major proteome components of Haemophilus influenzae type‐strain NCTC 8143
Authors: Link Andrew J. , Hays Lara G. , Carmack Edwin B. , Yates John R.
Published: May 2005

With the completion of the Haemophilus influenzae Rd genomic sequence, we know the identity of most of the theoretical proteins in the proteome of this bacterium. However, the most abundant components of the actual proteome are unknown. Using mass spectrometry and two‐dimensional gel electrophoresis (2‐DE), we sequenced and analyzed the most abundant proteins observed in the ATCC reference strain of H. influenzae, NCTC 8143 (303 of ≈︁ 400 Coomassie‐stained 2‐DE spots). To automate the identification of 2‐DE spots, we coupled a liquid autosampler to a microcolumn liquid chromatography electrospray ionization tandem mass spectrometer capable of identifying 22 spots per day. From the 303 sequenced spots, we identified 263 unique proteins. Most of the abundant proteins lie in an isoelectric point range of pH 4–7 and a molecular mass range of 10–100 kDa. Of the observed proteins, the most abundant is the outer membrane protein P2. Based on variety and abundance, proteins involved in energy metabolism and macromolecular synthesis are the dominant classes of proteins. Unexpectedly, tryptophanase was identified as a highly abundant protein in the strain NCTC 8143 whose sequence is rot present in the genome of the Rd strain. By searching the tandem mass spectra against the translated genomic sequence, we identified several proteins which were not annotated in the genomic sequence. Surprisingly, 22% of the identified 2‐DE spots represent isoforms in which gene products with the same primary sequence have different observed pI and Mr, indicating that these proteins are post‐translationally processed. Although most proteins' predicted and observed isoelectric points and molecular masses show reasonable concordance, the observed values for several proteins deviate significantly from the predicted values. These anomalies may represent either highly processed proteins or misinterpretations of the genomic sequence. Using the technology developed in this project, the protein expression of other strains of H. influenzae grown under different environmental conditions can be compared to identify differences in their proteomes.
Peptide‐mass fingerprinting and the ideal covering set for protein characterisation
Authors: Wise Michael J. , Littlejohn Timothy G. , Humphery‐Smith Ian
Published: May 2005

The rules that govern the dynamics of protein characterisation by peptide‐mass fingerprinting (PMF) were investigated through multiple interrogations of a nonredundant protein database. This was achieved by analysing the efficiency of identifying each entry in the entire database via perfect in silico digestion with a series of 20 pseudo‐endoproteinases cutting at the carboxy terminal of each amino acid residue, and the multiple cutters: trypsin, chymotrypsin and Glu‐C. The distribution of peptide fragment masses generated by endoproteinase digestion was examined with a view to designing better approaches to protein characterisation by PMF. On average, and for both common and rare cutters, the combination of approximately two fragments was sufficient to identify most database entries. However, the rare cutters left more entries unidentified in the database. Total coverage of the entire database could not be achieved with one enzymatic cutter alone, nor when all 23 cutters were used together. Peptide fragments of > 5000 Da had little effect on the outcome of PMF to correctly characterise database entries, while those with low mass (near to 350 Da in the case of trypsin) were found to be of most utility. The most frequently occurring fragments were also found in this lower mass region. The maximum size of uncut database entries (those not containing a specific amino acid residue) ranged from 52 908 Da to 258 314 Da, while the failure rate for a single cutter in identifying database entries varied from 10 865 (8.4%) to 23 290 (18.1 %). PMF is likely to be a mainstay of any high‐throughput protein screening strategy for large‐scale proteome analysis. A better understanding of the merits and limitations of this technique will allow researchers to optimise their protein characterisation procedures.
Evaluation of algorithms used for cross‐species proteome characterisation
Authors: Cordwell Stuart J. , Humphery‐Smith Ian
Published: May 2005

The ability to effectively search databases for the identification of protein spots from two‐dimensional electrophoresis gels has become an essential step in the study of microbial proteomes. A variety of analytical techniques are currently being employed during protein characterisation. A number of algorithms used to search databases, accessible via the World Wide Web, depend upon information concerning N‐ and C‐terminal microsequence, amino acid composition, and peptide‐mass fingerprinting. The effectiveness of nine such algorithms, as well as COMBINED (software developed in this laboratory for identifying proteins across species boundaries) was examined. Fifty‐four ribosomal proteins from the Mycoplasma genitalium genome, and 72 amino acyl tRNA synthetases from the Haemophilus influenzae, M. genitalium and Methanococcus jannaschii genomes were chosen for study. These proteins were selected because they represent a wide range of sequence identities across species boundaries (22.7–100% identity), as detected by standard sequence alignment tools. Such sequence variation allowed for a statistical comparison of algorithm success measured against published sequence identity. The ability of analytical techniques used in protein characterisation and associated database query programs to detect identity at the functional group level was examined for proteins with low levels of homology at the gene/protein sequence level. The significance of these theoretical data manipulations provided the means to predict the utility of data acquired experimentally for non‐sequence‐dependent software in proteome analysis. The data obtained also predicted that ‘sequence tagging’ of peptide fingerprints would need to be accompanied by at least 11–20 residues of amino acid sequence for it to be widely used for protein characterisation across species boundaries.
Identification of procaryotic developmental stages by statistical analyses of two‐dimensional gel patterns
Authors: Vohradsky Jiří , Li Xin‐Ming , Thompson Charles J.
Published: May 2005

Multivariate statistical comparisons of two‐dimensional protein (2‐D) gel patterns were used for the first time to define stages of a biological developmental system. The differentiating procaryote, Streptomyces coelicolor, was radiolabeled in liquid cultures at 16 intervals during development, and radioactive proteins were separated and quantified on 2‐D gels. Cluster, principal component, and correlation analyses classified these gel patterns into four distinct groups, each reflecting a pattern of gene expression specific for a stage of development. These studies focused our attention on a phase of arrested growth as a key regulatory transition leading to secondary metabolism and a phase of renewed growth. Proteins whose synthesis was switched on or off during the “transitional” phase (some 21 and 18, respectively) were identified and will be the focus of future studies designed to identify their physiological or regulatory function.
First steps from a two‐dimensional protein index towards a response‐regulation map for Bacillus subtilis
Authors: Antelmann Haike , Bernhardt Jörg , Schmid Roland , Mach Hiltraut , Völker Uwe , Hecker Michael
Published: May 2005

Data on the identification of proteins of Bacillus subtilis on two‐dimensional (2‐D) gels as well as their regulation are summarized and the identification of 56 protein spots is included. The pattern of proteins synthesized in Bacillus subtilis during exponential growth, during starvation for glucose or phosphate, or after the imposition of stresses like heat shock, salt‐ and ethanol stress as well as oxidative stress was analyzed. N‐terminal sequencing of protein spots allowed the identification of 93 proteins on 2‐D gels, which are required for the synthesis of amino acids and nucleotides, the generation of ATP, for glycolyses, the pentose phosphate cycle, the citric acid cycle as well as for adaptation to a variety of stress conditions. A computer‐aided analysis of the 2‐D gels was used to monitor the synthesis profile of more than 130 protein spots. Proteins performing housekeeping functions during exponential growth displayed a reduced synthesis rate during stress and starvation, whereas spots induced during stress and starvation were classified as specific stress proteins induced by a single stimulus or a group of related stimuli, or as general stress proteins induced by a variety of entrely different stimuli. The analysis of mutants in global regulators was initiated in order to establish a response regulation map for B. subtilis. These investigations demonstrated that the alternative sigma factor σB is involved in the regulation of almost all of the general stress proteins and that the phoPR two‐component system is required for the induction of a large part but not all of the proteins induced by phosphate starvation.
Hybridization capture of microsatellites directly from genomic DNA
Authors: Refseth Unn Hilde , Fangan Bjørn Magne , Jakobsen Kjetill Sigurd
Published: May 2005

A rapid approach for isolation of microsatellites and other tandem repeated sequences in described. The method is based on hybridization capture of repetitive elements from digested genomic DNA using biotinylated oligonucleotide probes in solution and subsequent attachment to magnetic beads coated with streptavidin. Captured fragments are amplified by adapter polymerase chain reaction (PCR) and the PCR products enriched for microsatellites cloned directly into a T‐vector for sequencing. The results presented here show that this approach is highly effective, allowing di‐ and trinucleotide repeats to be isolated and sequenced directly from fish and mammalian genomic DNA within four to five days. Assuming a density and relative abundance of repeats with AC/GT motifs corresponding to that found in the human genome, the protocol presented gives at least a 35‐fold enrichment of AC/GT microsatellites using an (AC)10 oligo probe. In addition, four out of five sequences captured by a (CAG)9 oligo probe contained one or several CAG repeat arrays. The efficiency of this direct approach suggests that it can be used for extracting other types of tandem and interspersed repeated sequences (including transposons, rRNA and tRNA genes and proviruses) from vertebrate genomes.
Application of inter simple sequence repeat (ISSR) markers to plant genetics
Authors: Godwin Ian D. , Aitken Elisabeth A. B. , Smith Lawrence W.
Published: May 2005

Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Single‐locus SSR markers have been developed for a number of species, although there is a major bottleneck in developing SSR markers whereby flanking sequences must be known to design 5′‐anchors for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) fingerprinting was developed such that no sequence knowledge was required. Primers based on a repeat sequence, such as (CA)n, can be made with a degenerate 3′‐anchor, such as (CA)8RG or (AGC)6TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with 32P or 33P via end‐labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reaction yields 20–100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and report here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis usually detects a higher level of polymorphism than that detected with restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) analyses. Our data indicate that this is not a result of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.
Analysis of the internal transcribed spacer regions of ribosomal DNA in common airborne allergenic fungi
Authors: Gaskell Giles J. , Carter Dee A. , Britton Warwick J. , Tovey Euan R. , Benyon Fiona H. L. , Løvborg Uffe
Published: May 2005

Ribosomal DNA internal transcribed spacer (ITS) sequences were investigated in the genera Alternaria, Aspergillus, Cladosporium and Penicillium, to identify regions suitable for the design of genus‐ and species‐specific oligonucleotide probes. The ITS regions of all four genera were quite distinct. However, we discovered that the genera Aspergillus and Penicillium displayed high similarity in most of the ITS2 region. Whilst interspecies similarity within Alternaria and Cladosporium was high in both ITS regions 1 and 2, ITS1 similarity within Aspergillus and Penicillium was lower by comparison. We conclude that: (1) the ITS2 region is highly conserved between Aspergillus and Penicillium, and (2) the ITS1 region will allow for the development of specific probes to distinguish between Aspergillus and Penicillium.
On simple repetitive DNA sequences and complex diseases
Authors: Epplen Cornelia , Santos Eduardo J. M. , Mäueler Winfried , van Helden Paul , Epplen Jörg T.
Published: May 2005

Simple repetitive DNA sequences are abundantly interspersed in eukaryote genomes and therefore useful in genome research and genetic fingerprinting in plants, fungi and animals, including man. Recently, simple repeats were also identified in some prokaryotic genomes. Hence the same probes can be applied for multilocus DNA fingerprinting in medically relevant bacteria. Simple repeats including composite dinucleotide microsatellites are differentially represented in different compartments of eukaryote genomes. Expanded triplet blocks in and around certain genes may, for example, cause so‐called trinucleotide diseases in man. As a consequence, simple repetitive sequences should also be characterized with respect to their influences on the DNA structure, gene expression, genomic (in)stability and their development on an evolutionary time scale. Here three examples of microsatellites in the human major histocompatibility complex (HLA) are investigated, a (GT)n microsatellite situated 2 kb 5′ off the lymphotoxin α (LTA) gene, a (GAA)n block in the 5′ part of the HLA‐F gene and a composite (GT)n(GA)m stretch in the second intron of HLA‐DRBl genes. Grossly differing mutation rates are evident in these elements as well as varying linkage disequilibria. The unfolding of these simple repeats in distant human populations is covered including Caucasians, Bushmen and South American Indians. Furthermore, implications of simple repeat neighboring genes are discussed for the multifactorial diseases multiple sclerosis (MS), rheumatoid arthritis (RA) and early onset pauciarticular arthritis (EOPA). Polymorphisms of HLA‐DRBl and T cell receptor β variable (TCRBV) genes confer susceptibility for these autoimmune diseases as demonstrable by intronic simple repeat variability. Microsatellite polymorphisms within the TNF region reveal linkage disequilibria with HLA‐DRBl and different promotor alleles of the TNFA gene. Disease associations with TNFA microsatellite alleles are, on the one hand, secondary to associations with HLA‐DRBl genes (in MS) or they represent additional risk factors (in RA, EOPA) on the other hand. Evolutionary persistence, various structural conformations and the specific binding of nuclear proteins to several simple repeat sequences refute the preconceptions of biological insignificance for all of these ubiquitously interspersed elements.
Angiotensinogen gene variation in a population case‐control study of preeclampsia/eclampsia in Australians and Chinese
Authors: Guo Guanglan , Wilton Alan N. , Fu Yuliang , Qiu Hongyu , Brennecke Shaun P. , Cooper Desmond W.
Published: May 2005

Preeclampsia/eclampsia (PE/E) is a common disease of human pregnancy with a strong genetic component. The etiology of PE/E is unknown. Two recent reports indicated that the angiotensinogen gene (AGT) could be involved in susceptibility to PE/E. We performed a population‐based case‐control study in Australian and Chinese populations to investigate whether AGT is a good candidate gene for PE/E. A microsatellite polymorphism within AGT was typed as well as a molecular variant T235 (Met→Thr) of AGT using allele‐specific PCR and allele‐induced restriction site PCR. The allele distributions of the microsatellite and the variant T235 of AGT were significantly different between the two ethnic groups. However, no significant allele associations were found with disease when comparing PE/E patients and controls in Australian or Chinese populations, which is in contrast to the two earlier reports. The results suggest that the contribution of AGT to the occurrence of PE/E is small, if anything, and is not constant across populations.
Genetic determination of paternity and variation in male reproductive success in two populations of rhesus macaques
Authors: Bercovitch Fred B. , Nürnberg Peter
Published: May 2005

The fundamental framework for uncovering factors affecting the evolution of social behavior rests upon analyses of variation in reproductive success. In species where females mate with multiple males, paternity is invisible in the absence of genetic data. We determined paternity in two populations of rhesus macaques, Macaca mulatta, using both single locus and multilocus techniques. One troop, Group R, is one of four troops living on a 15 ha island (Cayo Santiago) off the coast of Puerto Rico, while the other troop, Group M, was translocated from Cayo Santiago to the Sabana Seca Field Station (Puerto Rico) in 1984. About a dozen human‐derived short tandem repeat (STR) markers have been found to be polymorphic in the study of populations and provide the initial paternity determination. Final evaluation of paternity is then confirmed by multilocus DNA fingerprinting using synthetic oligonucleotide probes. Body condition, age, and dominance rank have an impact on male progeny production, while canine size does not. We suggest that nonagonistic competition in the form of sperm competition and endurance rivalry will modulate male reproductive success. A large body size among males provides them with an advantage in both sperm competition and endurance rivalry. Comparison of the two populations indicated that demographic, social, ecological, and morphological factors interact to regulate variation in reproductive success among male nonhuman primates.
Use of capillary electrophoresis with laser‐induced fluorescence detection to assess messenger ribonucleic acid molecules amplified by the polymerase chain reaction: Applications in the cloning of cells
Authors: Personett David , Sugaya Kiminobu , Hammond David , Robbins Michael , McKinney Michael
Published: May 2005

Progressive and selective degeneration of specific classes of neurons occurs in the Alzheimer's disease (AD) brain. Differential vulnerability in this disease is evident even within supopulations that synthesize and release acetylcholine as a transmitter; i.e., basal forebrain cholinergic neurons degenerate but other classes of cholinergic neurons are relatively preserved. The basis for this selective vulnerability is unknown. Studies of differential neuronal vulnerability in AD would be facilitated if cell lines expressing neurotransmitter‐specific phenotypes could be cloned from the brain. Capillary electrophoresis (CE) with laser‐induced fluorescence (LIF) has been shown to be a sensitive method of detection and quantitation of the DNA products of the polymerase chain reaction (PCR). CE/LIF was combined with the PCR to detect phenotypic messenger RNA (mRNA) molecules, converted to cDNA using reverse transcriptase (RT), in cultures of virally immortalized brainstem progenitor cells produced during establishment of a cloning strategy. RT/PCR methods were developed for detection of the mRNAs for choline acetyltransferase (ChAT), the neuronal, constitutive isoform of nitric oxide synthase (c‐NOS), and the growth‐associated protein GAP‐43, three genes known to be expressed in central cholinergic neurons. A “nondestructive” method of screening cultured cells for their expression of c‐NOS was established using depolarization with medium containing 50 mM potassium ion. These approaches were first validated using cultured SN56 (cholinergic) and N1E‐115 (c‐NOS‐positive) neuroblastoma cells, and with primary brainstem cultures. For the cloning of novel cell lines, progenitor cells were isolated from the embryonic day 13 fetal brainstem and were immortalized by transfection with a retroviral vector that confers a temperature‐sensitive SV‐40 transforming activity and neomycin resistance. Cell colonies surviving in G418‐containing media were isolated and cloned by dilution. Clonal cultures were expanded by growth at 33°C, differentiated by switching to a low‐serum medium and growth at 39°C, and screened for depolarization‐induced accumulation of nitrite in the medium. The subset of putative c‐NOS‐positive clones (about 4%) were then screened for their expression of mRNAs using RT/PCR in combination with CE/LIF. This screening protocol proved to be powerful in the rapid isolation and phenotypic characterization of immortalized progenitor cells cloned from embryonic rat brainstem.
Development of a nonisotopic capillary electrophoresis‐based method for measuring glomerular filtration rate
Authors: Bergert Jan H. , Liedtke Robert R. , Oda Robert P. , Landers James P. , Wilson David M.
Published: May 2005

The conditions for quantitative measurement of nonisotopic iothalamate meglumine (Conray) in urine and plasma by capillary zone electrophoresis (CE) have been developed. The impetus for developing this methodology was to replace the traditional [125I]iothalamate glomerular filtration rate (GFR) marker assay, a routine tool in the measurement of kidney function. This new approach for measuring kidney function is attractive since it avoids the cost of administration of radioisotopic compounds to patients, as well as the cost associated with purchase and disposal of isotopic compounds and contaminated samples. The concentration of iothalamate in urine and plasma determined by CE can be used directly to calculate GFR. The GFR in patients injected with [125I]iothalamate and nonisotopic iothalamate simultaneously showed an excellent correlation (0.998) with between‐day coefficient of variation of 2.30% and a recovery of 102% and 98%, respectively, when added to urine and plasma. Interference from drugs and other urinary compounds is eliminated with this method. Collectively, this study has shown that CE is a cost‐effective alternative to the current methodology for measuring GFR.
Determination of serum corticosteroids by mixed micellar electrokinetic capillary chromatography with sodium dodecyl sulfate and sodium cholate
Authors: Wiedmer Susanne K. , Sirén Heli , Riekkola Marja‐Liisa
Published: May 2005

The application of mixed micellar electrokinetic capillary electrochromatography to the determination of the corticosteroid hormones cortisone, cortisol (hydrocortisone), and dexamethasone in serum samples is demonstrated. The serum samples were enzymatically hydrolyzed, precipitated, solid‐phase extracted, and analyzed by capillary electrophoresis. A micellar system of sodium dodecyl sulfate and sodium cholate buffered with an organic compound provided the electrolyte solution. Spiked blank serum samples were used for the linearity testing and limits of detection and quantitation were measured. Patient samples were analyzed and the concentrations of cortisol and cortisone were measured. The method, which was fast and easy to perform, was confirmed to be useful for the determination of corticosteroids in serum.
Characterization of stereoselectivity and genetic polymorphism of the debrisoquine hydroxylation in man via analysis of urinary debrisoquine and 4‐hydroxydebrisoquine by capillary electrophoresis
Authors: Lanz Matthias , Theurillat Regula , Thormann Wolfgang
Published: May 2005

Using capillary zone electrophoresis with a phosphate buffer at pH 2.5 containing 50 mM heptakis‐(2,3,6‐tri‐O‐methyl)‐β‐CD as chiral selector, the separation of the enantiomers of the main metabolite of debrisoquine (DEB), 4‐hydroxydebrisoquine (4‐OHDEB), is reported. For extraction of underivatized urinary DEB, S‐4‐OHDEB and R‐4‐OHDEB, a procedure using disposable cartridges containing a polystyrene‐based polymer was developed. A few nL of the extracts were analyzed in a 60 cm fused‐silica capillary of 50 μm ID and solute detection was effected at 195 nm. For all three compounds, a mean (n = 5) recovery of about 73% and a detection limit of about 150 ng/mL were noted. Data obtained with urines that were received for routine phenotyping with DEB and mephenytoin confirmed the almost exclusive formation of S‐4‐OHDEB. Under the described conditions, no R‐4‐OHDEB could be detected. With these data and those obtained employing no chiral selector in the buffer, differentiation between extensive metabolizer phenotypes (EM) and poor metabolizer phenotypes (PM) for DEB was unambiguously possible by the presence of a significant peak and no (or minor) peak for 4‐OHDEB, respectively. Data obtained for ten EM subjects and five PM subjects were found to agree with those generated by the routine assay based on gas chromatography. The capillary electrophoretic assays described are simple, reproducible (relative standard deviation of peak area ratios < 3%), require no sample derivatization, consume no halogenated organic solvents, and operate with inexpensive separation columns as well as small amounts of chemicals.
Determination of nitrite and nitrate reduction by capillary ion electrophoresis
Authors: Trushina Eugenia V. , Oda Robert P. , Landers James P. , McMurray Cynthia T.
Published: May 2005

Production of nitrates and nitrites is a common step in many methodologies used to measure nitric oxide (NO) and NO‐derived products in biological fluids. We report conditions that allow the rapid separation and quantification of nitrite from nitrate ions in biological fluids by capillary ion electrophoresis (CIE). CIE can be used to directly quantify nitrites and nitrates near the millimolar range. To detect lower levels, we have used CIE to monitor the reduction of nitrites and nitrates to NO for chemiluminescence detection. For reduction reactions, we directly compared the ability of three commonly used agents — potassium iodide (KI), mercuric chloride (HgCl2) and vanadium chloride (VCl3) — to reduce nitrite and nitrate ions to NO. Nitrites/nitrates can be efficiently reduced to NO at 37°C using vanadium chloride (100%) or HgCl2 (80%). However, these CE‐derived conditions cannot simply be extrapolated to chemiluminescence measurements. Vanadium (III) yields high background in the photomultiplier that diminishes the sensitivity of chemiluminescence measurement to that outside of physiological ranges. We find that reactions carried out at 37°C in 2 M HCl using HgCl2 is efficient using both techniques.
Analysis of endotoxins by capillary electrophoresis
Authors: Freitag Ruth , Fix Marina , Brüggemann Oliver
Published: May 2005

Endotoxins are part of the outer membrane of gram‐negative bacteria such as E. coli. Upon entering the blood stream, they cause a violent, sometimes life‐threatening, response of the immune system. Endotoxins are lipopolysaccharides (LPS), lacking optically active groups, and their detection in the underivatized state can be difficult. In this paper the potential of capillary electrophoresis (CE) for LPS analysis is investigated. By using a standard phosphate buffer method, concentrations down to 100 μg/mL can be detected within 6 min. The detection limit can be lowered by one order of magnitude by using a sodium dodecyl sulfate (SDS)/borate buffer, pH 9.2. In this buffer, the SDS serves to homogenize the size of the LPS aggregates, while the borate forms complexes with the diol groups of the molecule, thereby enhancing its optical activity. The formation of LPS‐affinity complexes with the UV‐active polymyxin B or labeling of the LPS with a fluorophore (fluorescein isothiocyanate) was unsuccessful. Best results, in terms of detection limit and speed, were obtained with an indirect UV‐detection CE method. By using a strongly UV‐active electrophoresis buffer, endotoxins could be detected as “negative” peaks. In this case, a detection limit of 3 μg/mL (35 pM) was determined. Proteins and other UV‐active substances did not disturb the assay, since they generated no detectable signals. The indirect UV detection was used to quantify the residual LPS content of a DNA preparation from E. coli.
Detection and quantitative characterization of artificial extra peaks following polymerase chain reaction amplification of 14 short tandem repeat systems used in forensic investigations
Authors: Meldgaard Michael , Morling Niels
Published: May 2005

Detection on automated DNA sequencers of polymerase chain reaction (PCR) products of tetra‐ and penta‐nucleotide short tandem repeat (STR) loci frequently reveals one or more extra peaks along with the true, major allele peak. The most frequent extra peak pattern is a single smaller peak which is one repeat unit shorter than the true allele peak. The existence of such artificial peaks is of special importance when the methods are used for forensic investigations because the artificial extra peaks may simulate true alleles when samples containing mixtures of DNA from different individuals are analyzed. We have investigated the relative levels of formation of extra peaks in 14 STR marker systems. We found that not only the parameters of the PCR but also factors determining the stringency during the post‐PCR and pre‐electrophoresis handling of samples were of importance for the formation of extra peaks. In our hands, the amounts of extra peaks were reduced (i) if the samples were effectively denatured immediately before loading, (ii) if they contained substantial amounts of formamide (i.e. > 50%), and (iii) if the temperature of the electrophoresis gel was above a certain level (i.e. > 43°C). The results suggest that extra peaks may in part be due to re‐annealing of the PCR product under suboptimal conditions. When efforts had been made to reduce the post‐PCR formation of extra peaks, the relative peak areas of the extra peaks ranged from 1% to 17% of those of the true alleles. Similar results were obtained when the PCR products were analyzed under native conditions. Low‐copy genome analysis excluded that somatic heterogeneity of the STR regions caused the extra peaks. The systems HumVWA31A, HumFibra/FGA, and D21S11 were especially affected by low‐stringency conditions, while Hum‐TH01, HumCD4, and D12S391 were virtually unaffected by low‐stringency conditions. Replacement of the Taq DNA polymerase with DNA polymerases with lower processivity resulted in higher levels of extra peaks. Our results support the hypothesis that extra peaks are produced due to slipped‐strand mispairing.
Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing
Authors: Alba F. Javier , Daban Joan‐Ramon
Published: May 2005

We have studied the light emission efficiency of proteins labeled with different fluorescent dyes chemically excited by the bis(2,4,6‐trichlorophenyl)oxalate (TCPO)‐H2O2 reaction. Using this peroxyoxalate chemiluminescence system, the best results were obtained with proteins covalently labeled with 2‐methoxy‐2,4‐diphenyl‐3(2H)‐furanone (MDPF). Blotted proteins on polyvinylidene difluoride (PVDF) membranes can be labeled rapidly with MDPF. Our results demonstrate that energy from the excited intermediate produced in the TCPO‐H2O2 reaction can be efficiently transferred to MDPF‐labeled proteins in solution and on PVDF membranes. Although this nonenzymatic chemiluminescent system produces a background emission that reduces the sensitivity, the method developed in this work allows detection of 5 ng of protein in blots after 5 min exposure to X‐ray film. Chemiluminescence of MDPF‐labeled proteins on Western and slot blots may also be detected and quantified using a charge‐coupled device (CCD) camera or a storage phosphor imaging system. This chemiluminescent method allows the staining of the total electrophoretic pattern but does not preclude further N‐terminal sequencing and immunodetection of specific bands.
A sensitive electrophoretic method for the quantification of myosin heavy chain isoforms in horse skeletal muscle: Histochemical and immunocytochemical verifications
Authors: Rivero José‐Luis L. , Talmadge Robert J. , Edgerton V. Reggie
Published: May 2005

In adult horses, three myosin heavy chain (MyHC) isoforms can be identified by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunohistochemistry using specific anti‐MyHC monoclonal antibodies. This report studies the suitability of a consistent sections of equine gluteus medius muscle biopsies (n = 18). The method used (previously described by R. J. Talmadge and R. R. Roy; J. Appl. Physiol. 1993, 75, 2337–2340) resolved MyHCs in three bands: I, IIB or IIX, and IIA from the fastest to the slowest migration band. The success rate of the protocol for yielding three well‐differentiated MyHC bands was 100% and a subsequent quantification by densitometry for each MyHC isoform was obtained in all 18 muscle biopsies. The results obtained with this electrophoretic method were compared with routine myofibrillar adenosine triphosphatase histochemistry and immunohistochemistry using specific anti‐MyHC monoclonal antibodies. The percent composition of the three electrophoretically separated MyHC isoforms (I, IIA and IIB or IIX) showed strong positive correlation with percentage of the area occupied in the biopsies by the three major fiber types (I, IIA, and IIB) identified histochemically (r = 0.96, P < 0.001) and immunohistochemically (r = 0.94, P < 0.01). It can be concluded that the electrophoretic method used here for measuring MyHC content is a valid alternative for muscle fiber typing in horses. As it is less costly and time‐consuming than both qualitative histochemistry and immunohistochemistry, the method offers new prospects for application in equine experimental studies and veterinary medicine.
System peaks in capillary zone electrophoresis. 3. Practical rules for predicting the existence of system peaks in capillary zone electrophoresis of anions using indirect spectrophotometric detection
Authors: Macka Miroslav , Haddad Paul R. , Gebauer Petr , Boček Petr
Published: May 2005

A theoretical and experimental study of the existence and evolution of system peaks in capillary zone electrophoresis (CZE) with indirect spectrophotometric detection is presented with respect to the effect of the number of coions present in the background electrolyte (BGE). It is shown that in BGEs having only one coion (i.e., the UV‐absorbing probe anion), the sample produces only negative peaks due to each analyte anion and no system peaks, with the number of sample peaks corresponding to the number of analytes present in the sample injected. In BGEs containing two coions, a sample with one analyte anion produces one negative indirect detection peak and one system peak. The transition between BGEs having one coion and those with two coions has also been studied and it has been shown that an addition of ca. 5% of the second coion to a single coion BGE causes the resulting BGE to behave macroscopically as a regular two‐coion BGE. A descriptive model is proposed, based on transient isotachophoresis (transient stacking) of the sample species and of the coion from the BGE which has the closest mobility to the sample ion. This model explains qualitatively the formation and evolution of the sample peak (containing the sample species and being detected by indirect detection due to displacement of the UV‐absorbing probe in its zone) and the system peak (containing no sample species and being a vacancy in the continuum of coions of the BGE). It is shown that the system peak may be positive or negative as it corresponds to the situation where the vacancy of one component of the BGE results in an enhanced concentration of the other component. It has been demonstrated that the system peak is created by a vacancy of that component of the BGE which has the greatest difference in mobility relative to that of the sample species. On indirect detection in BGEs containing two coions the sample displaces predominantly the BGE coion which has a mobility closest to that of the analyte anion. In systems with BGEs containing two coions, a sample having n analytes produces n sample peaks and one system peak, the sign and magnitude of which are dependent on the sum of the UV absorbances of the analytes involved. The effect of bicarbonate from atmospheric CO2 has also been studied and it has been shown that weakly alkaline BGEs with a single anionic UV‐absorbing coion, such as those currently used for anionic analyses with indirect detection, may suffer from the presence of system peaks due to bicarbonate.
Elution concentration of proteins cut from two‐dimensional polyacrylamide gels using Pasteur pipettes
Authors: Kristensen Dan Bach , Inamatsu Mutsumi , Yoshizato Katsutoshi
Published: May 2005

The present study devised a new procedure for concentrating proteins cut from primary two‐dimensional polyacrylamide gels prior to their structural characterization. Gel spots containing a protein were cut from several identical two‐dimensional gels and loaded on the top of a high tensile strength stacking gel in a Pasteur pipette. The protein was concentrated electrophoretically into a small volume in the narrow tip of the pipette. The concentrated protein was then blotted from the stacking gel onto a polyvinylidene diffluoride membrane, where the protein was subjected to tryptic on‐membrane digestion. Utilizing this system, we identified 22 proteins chosen randomly from two‐dimensional gels of proteins from rat dermal papilla cells by internal microsequencing. As much as 1.5 mL volume (cut gel spots in protein sample buffer) could be loaded onto the Pasteur pipettes, generally yielding a final on‐membrane area of approximately 2 mm2 after elution concentration and electroblotting onto polyvinylidene difluoride membranes. We concluded that this newly devised system is effective and useful for concentrating proteins prior to structural characterization, and that larger volumes than previously recommended can be effectively concentrated, with little or no effect on the final on‐membrane area occupied by the concentrated proteins.
Interleukin‐1β induced changes in the protein expression of rat islets: A computerized database
Authors: Andersen Henrik U. , Fey Stephen J. , Larsen Peter Mose , Nawrocki Arkadiusz , Hejnæs Kim R. , Mandrup‐Poulsen Thomas , Nerup Jørn
Published: May 2005

Insulin‐dependent diabetes mellitus is caused by an autoimmune destruction of the β‐cells in the islets of Langerhans. The cytokine interleukin 1 inhibits insulin release and is selectively cytotoxic to β‐cells in isolated pancreatic rat islets. The antigen(s) triggering the immune response as well as the intracellular mechanisms of action of interleukin 1‐mediated β‐cell cytotoxicity are unknown. However, previous studies have found an association of β‐cell destruction with alterations in protein synthesis. Thus, two‐dimensional (2‐D) gel electrophoresis of pancreatic islet proteins may be an important tool facilitating studies of the molecular pathogenesis of insulin‐dependent diabetes mellitus. 2‐D gel electrophoresis of islet proteins may lead to (i) the determination of qualitative and quantitative changes in specific islet proteins induced by cytokines, (ii) the determination of the effects of agents modulating cytokine action, and (iii) the identification of primary islet protein antigen(s) initiating the immune destruction of the β‐cells. Therefore, the aim of this study was to create databases (DB) of all reproducibly detectable protein spots on 10% and 15% acrylamide 2‐D gels of neonatal rat islets (10% and 15% DB), labeled under standardized culture conditions. 1235 and 557 spots were present in 5 of 5 gels in the 15% isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) DB, respectively, whereas 995 and 378 spots were present in 5 of 5 gels in the 10% IEF and NEPHGE DB, respectively, yielding a reproducibility of spot detection between 75.2% and 91.7%. In both DBs, the average coefficient of variation of the percentage of integrated optical density (CV% of %IOD) for spots present in all gels was between 42.4% and 45.7%. When the same sample was analyzed in consecutive sets of gels on different days (interassay analysis), the average CV% of %IOD was 35.5%–36.1%. When the same sample was analyzed repeatedly in one set of gels (intra‐assay analysis), the average CV% of %IOD was 30.2% in the IEF gels, while the average CV% of %IOD was 45.7% in the NEPHGE gels. Addition of interlenkin‐1β (IL‐1β) to the cultures resulted in statistically significant modulation or de novo synthesis of 105 proteins in the 10% gels. In conclusion, we present the first 10% and 15% acrylamide 2‐D gel protein databases of neonatal rat islets of Langerhans and demonstrate its usage to identify proteins altered in expression by IL‐1β

Part of this work was carried out at the Institute of Medical Microbiology, Århus University, Århus, Denmark

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