bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chiral o‐phthaldialdehyde reagents for fluorogenic on‐column labeling of D‐ and L‐amino acids in micellar electrokinetic chromatography
Authors: Tivesten Anna , Folestad Staffan 
Published: May 2005

AbstractFollowing a recent communication from this laboratory (A. Tivesten et al., J. High Resol. Chromatogr. 1996, 19, 229–233) where on‐column chiral derivatization of D‐ and L‐amino acids in micellar electrokinetic chromatography (MEKC) was demonstrated for the first time, we now present further details of the labeling procedure. The basis of the method is the consecutive injection of a sample and the reagent onto the capillary as two discrete plugs. By utilizing their difference in mobility, the zones are mixed by the electrophoretic process in a controllable way. In this way the amino acids are both derivatized within a few seconds and subsequently separated in a single step. Compared with precolumn derivatization, dilution of the original sample is minimized, which is why the method is highly useful for microchemical analytical work, i.e., labeling of nano‐ to picoliter samples. Four different chiral thiols were compared in this study, 2,3,4,6‐tetra‐O‐acetyl‐l‐thio‐β‐D‐glucopyranose (TATG), N‐acetyl‐L‐cysteine (AC), N‐acetyl‐D‐penicillamine (AP), and N‐acetyl‐L‐cysteine (IBC). Together with o‐phthaldialdehyde (OPA) these constitute the chiral reagent. The reaction rate as well as the spectroscopic and chromatographic properties of the formed derivatives were examined. It was found that the fastest reaction is obtained with OPA/TATG, as was the case with L‐alanine (L‐ala), and that the rate is greatly affected by the presence and concentration of acetonitrile or methanol. Moreover, OPA/TATG yields superior resolution of D‐ and L‐amino acids over the other OPA/thiol combinations in a sodium dodecyl sulfate (SDS) micellar buffer, whereas the OPA/AC and OPA/IBC‐amino acid derivatives have a higher fluorescence quantum yield. With laser‐induced fluorescence detection (He‐Cd, 325 nm) the mass limit of detection is at the low amol level.
Identification of wallaby milk whey proteins separated by two‐dimensional electrophoresis, using amino acid analysis and sequence tagging
Authors: Molloy Mark P. , Herbert Ben R. , Yan Jun X. , Williams Keith L. , Gooley Andrew A. 
Published: May 2005

AbstractMicropreparative two‐dimensional polyacrylamide gel electrophoresis has been used to separate milk whey proteins from the Tammar wallaby (Macropus eugenii). We have used a combination of amino acid analysis and N‐terminal sequence tagging as a rapid and sensitive method to identify the major whey proteins. Using these techniques, we confidently identified α‐lactalbumin and late lactation protein. While these are the only two M. eugenii whey proteins with a corresponding SWISS‐PROT entry, we demonstrate that by using amino acid analysis and matching across species boundaries, we can identify previously unsequenced conserved wallaby whey proteins including β‐lactoglobulin and serum albumin.
Strategies for whole microbial genome sequencing and analysis
Authors: Fraser Claire M. , Fleischmann Robert D. 
Published: May 2005

AbstractThe introduction of methods for automated DNA sequence analysis nearly a decade ago, together with more recent advances in the field of bioinformatics, have revolutionized biology and medicine and have ushered in a new era of genomic science, the study of genes and genomes. These new technologies have had an impact on many areas of research, including the association between genes and disease, in DNA‐based diagnostics, and in the sequencing of genomes from human and other model organisms. The demonstration in 1995, that automated DNA sequencing methods could be used to decipher the entire genome sequence of a free‐living organism, Haemophilus influenzae, was a milestone in both the genomics and microbial fields [1]. Since the first report of the complete sequence of H. influenzae, these methodologies have been adopted by laboratories around the world. The complete genomic sequence of five eubacterial species [1–5], one archaea [6], and the eukaryote, Saccharomyces cerevisiae [7], have been reported in the last 18 months. At the beginning of 1997 more than a dozen microbial genome projects are at or near completion, with many others in progress. It is likely that in the next few years we will see the complete sequence of perhaps as many as 30–40 microbial genomes. In this article, we will review methods for whole genome sequencing and analysis and examine how this information can be exploited to better understand microbial physiology and evolution.
Proteome analysis of Saccharomyces cerevisiae: A methodological outline
Authors: Fey Stephen J. , Nawrocki Arkadiusz , Larsen Martin R. , Görg Angelika , Roepstorff Peter , Skews Gerard N. , Williams Richard , Larsen Peter Mose 
Published: May 2005

AbstractProteome analysis offers a unique means of identifying important proteins, characterizing their modifications and beginning to describe their function. This is achieved through the combination of two technologies: protein separation and selection by two‐dimensional gel electrophoresis, and protein identification and characterization by mass spectrometry. This methodological outline sketches the strengths and weaknesses of the two central technologies used, and provides both practical tips and the theoretical background for their utilization. One application of these technologies is illustrated by the characterization of genes, revealed by sequencing, but which have no — or only weak homology — to any other known genes. Other applications, for example the identification of protein markers for particular human diseases, are only referred to. The aim of the article is thus to provide the basis for a sound understanding of the full potential and limitations of proteome analysis.
Stress proteins in Listeria monocytogenes
Authors: Phan‐Thanh Luu , Gormon Thierry 
Published: May 2005

AbstractThe proteins induced by the different stress conditions in Listeria monocytogenes were analyzed by two‐dimensional (2‐D) electrophoresis with the aid of a computerized 2‐D gel analysis system. The stress conditions imposed were pH 4, pH 10, 0.015% sodium dodecyl sulfate (SDS), 0.03% sodium deoxycholate and 4% ethanol. As previously seen for heat shock and cold shock, more than half of the proteins normally synthesized by Listeria cells were repressed under these stress conditions. Conversely, the synthesis of a great number of proteins was increased and novel proteins appeared upon stress. Each stress factor induced a specific set of proteins. These stress proteins were characterized by their apparent molecular mass and isoelectric point. No universal stress proteins were found to be common to all the stresses studied, while some proteins were commonly induced by two or three stress conditions. The degree of dissimilarity in stress responses was best illustrated by the induction of only two proteins common to exposure to the two detergents SDS and sodium deoxycholate. This work, together with that on heat and cold shock, constitutes the basic step for the identification of stress proteins in Listeria.
Identification and characterization of Brucella ovis immunogenic proteins using two‐dimensional electrophoresis and immunoblotting
Authors: Teixeira‐Gomes Ana P. , Cloeckaert Axel , Bézard Guy , Bowden Raúl A. , Dubray Gérard , Zygmunt Michel S. 
Published: May 2005

AbstractIn a previous report, proteins from Brucella melitensis were characterized by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) and N‐terminal microsequencing. In the present report, we have extended this study to the second etiologic agent in ovine brucellosis, B. ovis, responsible for ram epididymitis and infertility. The combination of 2‐D gel electrophoresis and protein microsequencing facilitated the location and identification of the major proteins of B. ovis on the 2‐D pattern. These proteins comprised cytoplasmic, periplasmic and some membrane proteins except the major outer membrane proteins. By comparing 2‐D gel profiles of B. ovis with that of B. melitensis described previously, a few proteins with different expression levels were readily identified. Serum from a ram naturally infected with B. ovis was used in immunoblotting studies to identify immunogenic proteins recognized during the course of infection. This serum showed antibody reactivity against approximately 82 protein spots. Twenty‐one of these proteins were identified either by use of monoclonal antibodies or by N‐terminal microsequencing. Several proteins previously described in earlier Brucella works were identified: the 89 kDa outer membrane protein, DnaK, GroEL, BP26, and Cu‐Zn superoxide dismutase. Eight proteins had amino acid sequences homologous to those of various proteins from other bacteria found in protein databases: NikA, dihydrolipoamide succinyltransferase, a hypothetical 31 kDa protein, malate dehydrogenase, succinyl‐CoA synthetase alpha subunit, an amino acid ABC type transporter, Leu/ Ile/ Val‐binding protein precursor, and ClpP. The remaining eight proteins had N‐terminal sequences lacking similarity to existing database entries. Thus, the 2‐D PAGE analysis provided a convenient first approach in the characterization of immunogenic proteins.
Ancient DNA from polynesian rats: Extraction, amplification and sequence from single small bones
Authors: Matisoo‐Smith Elizabeth , Allen John S. , Ladefoged Thegn N. , Roberts R. Mere , Lambert David M. 
Published: May 2005

AbstractThe human colonisation of Polynesia was a major event in world prehistory. It represents one of the last human population migrations, and one which required crossing major water barriers. Though the subject of Pacific population origins has been approached by scholars from numerous fields for nearly a century, recent years have seen the problem addressed by human geneticists [1–5]. Since the initial report describing the recovery of DNA from skeletal remains [6], ancient DNA studies have also focused on the Pacific region [7, 8]. In this paper we present the results of ancient DNA analyses of Rattus exulans, an animal that was transported by ancestral Polynesians through the Pacific to the far reaches of the Polynesian triangle. Analysis of DNA of R. exulans skeletal remains has many advantages over studies of ancient human remains, yet the one drawback has been the recovery of ancient DNA from single bones of these very small rodents. We have successfully modified standard extraction protocols for ancient DNA [9] and have consistently extracted, amplified and sequenced mitochondrial DNA (mtDNA) from less than 0.1 g of R. exulans bone and tooth samples recovered from archaeological sites throughout the Pacific, ranging from 400 to 2000 years old.
A study of the genetic diversity of Mycobacterium tuberculosis isolated from patients in the eastern province of South Africa using random amplified polymorphic DNA profiling
Authors: Richner Sharon M. , Meiring Jill , Kirby Ralph 
Published: May 2005

AbstractThis population genetics study was done to determine the degree of genetic diversity amongst clinical isolates of Mycobacterium tuberculosis in one of the largest provinces of South Africa. Three hundred and fifty‐nine individual cultures were obtained from single patients over a nine‐month period. Bacterial DNA was extracted and amplified with two arbitrary ten‐mer primers, using the random amplified polymorphic DNA‐polymerase chain reaction (RAPD‐PCR) technique. RAPD markers were separated by agarose gel electrophoresis, visualised with ethidium bromide, and then analysed for similarity using the GelCompar programme. The isolates were seen to fall into two major population groups. A high degree of genetic diversity was seen, with a total of 350 unique strains occurring amongst the 359 isolates. The majority of drug‐resistant isolates grouped together in the smaller population group. The genetic data were also correlated with geographical locality. It was found that a slight preponderance of isolates that grouped together closely, vis. that were either genetically identical or very similar (similarity index of approximately 97%), were from areas that were considered to be geographically distant. Antibiotic resistance patterns were also examined, revealing a total of 16 different resistance profiles among the 68 drug‐resistant isolates. RAPD‐PCR has proved to be a cost‐ and time‐effective technique, suitable for such a large‐scale population genetics study. It has provided useful information on the genetic diversity of an organism which is responsible for an increasing amount of morbidity and mortality in South Africa's second‐largest province.
Tetra‐and pentanucleotide short tandem repeat instability in gastric cancer
Authors: Silva Filipe , Gusmão Leonor , Alves Cíntia , Seruca Raquel , David Leonor , Amorim António 
Published: May 2005

AbstractThe association between genetic instability in repetitive DNA domains and cancer has been reported in different types of malignancies. In this work we perform a comparative study of 29 gastric tumors with paired normal tissue using seven tetra‐(FES/FPS, VWA31/A, HTPO, TH01, MBPB) and pentanucleotide (CD4, TP53) STR polymorphic markers regarding loss of heterozygosity and replication error status. Furthermore, we compare the gene frequencies obtained in normal tissue from patients with those of a normal control population from the same area, looking for allele associations between any of these polymorphic loci and gastric cancer risk. The results have shown that FES/FPS and TP53 present the higher rates of somatic instability. The observed results for TP53 are in accordance with those previously reported in gastric carcinogenesis, while instability of FES/FPS is for the first time reported in this tumor type. Our data suggest that different loci show different rates of instability and/or loss of heterozygosity and do not seem to consist of a result of an RER+ phenotype affecting several genomic repetitive domains. Furthermore, the instability in markers TH01, MBPB, TP53, and FES was generally detected in genotypes involving alleles with a high number of repeats. Comparing gene frequencies in patients and normal controls, no significant differences were found, although longer alleles are consistently more frequent in patients for the markers MBPB, TH01, and CD4.
United Arab Emirates population data on three DNA tetrameric short tandem repeat loci: HUMTHO1, TPOX and CSF1PO — derived using multiplex polymerase chain reaction and manual typing
Authors: Bayoumi Riad A. , Al‐Gazali Lihadh I. , Jaffer Uzma , Nur‐E‐kamal Mohamed S. A. , Dawodu Adekunle , Bener Abdulbari , Eapen Valsamma , Budowle Bruce 
Published: May 2005

AbstractAllele and genotype frequencies for three tetrameric short tandem repeat (STR) loci: HUMTHO1, TPOX, and CSF1PO, were determined in a United Arab Emirates (UAE) national population sample (n= 119). The loci were amplified simultaneously and the PCR products were separated in polyacrylamide DNA sequencing gels. Detection of the DNA fragments was accomplished using silver staining. Allele designations were determined by comparison to an allelic ladder. One allele at each locus was sequenced to confirm the nature of the repeats and their number. Alleles at the HUMTHO1 and TPOX loci were distributed bimodally, while CSF1PO showed unimodal distribution. The observed heterozygosities were 76% for HUMTHO1, 64% for TPOX, and 71% for CSF1PO. No deviation from the Hardy‐Weinberg expectations was observed in the genotype distribution for the loci TPOX and CSF1PO, but the HUMTHO1 locus did depart from HWE based on the likelihood ratio and the exact test. No correlation was detected between the alleles at any of the three pairs of loci. The allelic frequency data of these three loci in the UAE population sample can thus be used in human identity testing.
Genetic variation in Asiatic lions and Indian tigers
Authors: Shankaranarayanan Pattabhiraman , Banerjee Moinak , Kacker Raj Kumar , Aggarwal Ramesh Kumar , Singh Lalji 
Published: May 2005

AbstractPrevious reports have suggested that Asiatic lions and tigers are highly inbred and exhibit very low levels of genetic variation. Our analyses on these species have shown much higher degrees of polymorphism than reported. Randomly amplified polymorphic DNA (RAPD) analysis of 38 Asiatic lions, which exist as a single population in the Gir Forest Sanctuary in India, shows an average heterozygosity of 25.82% with four primers. Sperm motility studies by our colleagues corroborate this data. In Indian tigers, microsatellite analysis of five CA repeat loci and multilocus fingerprinting using Bkm 2(8) probe on a population of 22 individuals revealed a heterozygosity of 22.65%. Microsatellite analysis at loci Fca 77 and Fca 126 revealed polymorphism amongst the Asiatic X African lion hybrids, which has enabled us to use these as markers to discriminate the pure Asiatic lions from the hybrids. A similar analysis was used to identify hybrids of Indian and Siberian tigers through polymerase chain reaction (PCR) amplification of hair samples. To ascertain the variation which existed before the population bottleneck at the turn of the present century, microsatellite analysis was performed on 50‐ to 125‐year‐old skin samples from museum specimens. Our results show similar levels of genetic variability as in the present population (21.01%). This suggests that low genetic variability may be the characteristic feature of these species and not the result of intensive inbreeding. DNA fingerprinting studies of Asiatic lions and tigers have helped in identifying individuals with high genetic variability which can be used for conservation breeding programs.
Measurements of catecholamine‐mediated apoptosis of immunocompetent cells by capillary electrophoresis
Authors: Bergquist Jonas , Josefsson Elisabet , Tarkowski Andrej , Ekman Rolf , Ewing Andrew 
Published: May 2005

AbstractSingle cell analysis with capillary electrophoresis, a technique capable of detecting zeptomole quantities (10−21 mole) of neurochemical species, has been used to demonstrate that lymphocytes are capable of active synthesis of dopamine and norepinephrine. Exposure of lymphocytes to catecholamines at concentrations as low as 10 nM leads to decreased proliferation and differentiation, e.g. interferon‐γ (IFN‐γ), interleukin‐4 (IL‐4) and immunoglobulin (Ig). In addition, both inhibition of dopamine uptake with nomifensine and inhibition of packing of catecholamines into vesicles with tetrabenazine, results in sinificantly lower levels of dopamine and norepinephrine (p < 0.01 and p < 0.05, respectively). The catecholamine‐dependent inhibition of T‐ and B‐lymphocyte activity is mediated via an induction of a Bcl‐2/Bax and Fas/FasL involved apoptosis. These findings indicate a novel mechanism for regulation of lymphocyte activity in the central nervous system, whereby elevated regional levels of catecholamines might lead to the immunoprivilege of the brain.
Hydrophobic peptide mapping of clinically relevant heptathelical membrane proteins by capillary electrophoresis
Authors: Dong Maoqing , Oda Robert P. , Strausbauch Michael A. , Wettstein Peter J. , Landers James P. , Miller Laurence J. 
Published: May 2005

AbstractThe structural investigation of G protein‐coupled receptors has been hindered by the lack of techniques to effectively resolve the hydrophobic peptides obtained by chemical or proteolytic cleavage, as well as the minute amounts of protein typically isolated. We have developed a capillary electrophoresis method for efficient separation of hydrophobic peptides using a cyanogen bromide digest of bacteriorhodopsin as a model for these clinically important membrane proteins. This procedure includes (i) solubilization of the protein digest in acetic acid; and (ii) electrophoresis using an acetic acid‐based buffer system augmented by acetonitrile and hexane sulfonic acid, in a Polybrenecoated fused silica capillary. The potential for detection sensitivity to be increased at least 100‐fold by use of on‐line solid‐phase extraction on C18‐silica is shown. This approach is potentially useful for peptide fingerprinting of sparse and extremely hydrophobic membrane receptors.
Identification of monoclonal proteins in serum: A quantitative comparison of acetate, agarose gel, and capillary electrophoresis
Authors: Katzmann Jerry A. , Clark Raynell , Wiegert Elaine , Sanders Elisabeth , Oda Robert P. , Kyle Robert A. , Namyst‐Goldberg Colette , Landers James P. 
Published: May 2005

AbstractA selected group of 308 sera were analyzed by capillary electrophoresis (CE), agarose gel electrophoresis (AGE), and cellulose acetate electrophoresis (CAE) and evaluated for abnormalities that would suggest the presence of a monoclonal protein. The sensitivity (an electrophoretic abnormality in sera that contained a monoclonal protein) and specificity (a normal electrophoretic pattern in sera that did not contain a monoclonal protein) was determined for each electrophoretic procedure. CAE was the most specific procedure and CE was the most sensitive. The increase in sensitivity of CE was primarily due to increased detection of cryoglobulins and free light chains. The quantitation of the gamma region and/or monoclonal antibody peaks by CE was similar to results obtained by AGE. Quantitation of very large monoclonal protein peaks (> 3.0 g/dL) by on‐line absorption detection (CE) yielded higher results than quantitation by dye‐binding (AGE).
Development of a lipoprotein profile using capillary electrophoresis and mass spectrometry
Authors: Macfarlane Ronald D. , Bondarenko Pavel V. , Cockrill Steven L. , Cruzado Ingrid D. , Koss William , McNeal Catherine J. , Spiekerman A. Michael , Watkins Layle K. 
Published: May 2005

AbstractA new program for lipoprotein characterization is outlined where capillary electrophoresis (CE) plays a central role in the analysis of intact lipoprotein serum components and the apoprotein domains. The first characterization step involves separation and particle density analysis of very low‐, low‐, and high‐density lipoprotein fractions (VLDL, LDL, HDL) by ultracentrifugation and image analysis. VLDL, HDL, and LDL fractions are analyzed by capillary electrophoresis. Sodium dodecyl sulfate (SDS) at low concentrations in the background electrolyte used in the CE analysis is incorporated into the lipoprotein particle without appreciable delipidation, as determined by ultracentrifuge particle density analysis. Increasing the concentration of SDS results in extensive delipidation, resulting in the release of apoproteins (apo) which are detected as components of the electropherogram. Apo B‐100 is detected in the delipidated VLDL and LDL fractions along with micelles of the lipids. Micelles from LDL delipidation have uniform charge densities. Apo A‐I and A‐II are detected in the HDL fraction. A new method for lipoprotein delipidation is introduced where the lipoprotein fraction is adsorbed on a reversed‐phase hydrophobic cartridge. Delipidation and recovery of the apoprotein fractions is made by serial clutions with acetonitrile. CE of the lipid‐free apoprotein mixture shows the presence of apoC‐I,II,III and apoE in the VLDL fraction, and apoA‐I,II apoC‐I and apoE in the HDL fraction. Electrospray ionization mass spectrometry analysis gives the isoform distribution for each apoprotein. The identification of the apoproteins in the electropherograms is the first step in developing a CE‐based quantitation method for measuring serum levels of these apoproteins and their distribution between the lipoprotein fractions. The assay described in this paper is being used as a level 2 and 3 cardiac risk profile analysis for individuals with normal lipid profiles who have a documented or family history of cardiovascular disease.