bluuebunny/crossref_metadata_2025_split huggingface dataset:
The serine proteinase inhibitory proteins of the chondrodystrophoid (beagle) and non‐chondrodystrophoid (greyhound) canine intervertebral disc
Authors: Melrose James , Taylor Thomas K. F. , Ghosh Peter 
Published: May 2005

AbstractTrypsin inhibitory proteins of low buoyant density (ρ ≤ 1.35 g/mL) fractions were prepared by CsCl density gradient ultracentrifugation of 4 M guanidinium hydrochloride extracts of lumbar beagle and greyhound annulus fibrosus and nucleus pulposus from animals aged 1 to 6 years. Affinity blotting with biotinylated trypsin was used to identify active trypsin inhibitory protein species; these species were also identified immunologically by Western blotting using antibodies against bovine pancreatic trypsin inhibitor (BPTI), and human inter‐α‐trypsin inhibitor (ITI). None of the trypsin inhibitory species evident in Western blots were reactive with anti‐human α1‐proteinase inhibitor (α‐1‐PI), α2‐macroglobulin or secretory leucocyte proteinase inhibitor. The greyhound intervertebral disc samples generally had higher levels of active trypsin inhibitor species per unit weight of tissue extracted, and a more extensive range of inhibitor species. Inhibitor species of 30, 32, 34 kDa were identified in both beagle and greyhound intervertebral disc samples; these species were generally most prominent in the annulus fibrosus samples. In contrast, the nucleus pulposus samples contained relatively large trypsin inhibitor species; the anti‐BPTI detected an inhibitor species of ∼ 85–90 kDa; anti‐ITI detected species of 120–250 kDa; biotinylated trypsin detected species of 60–110 kDa. A small molecular mass trypsin inhibitor species of 6 kDa, which was of similar mobility to BPTI, was also detected in annulus fibrosus samples; however, this species did not react with anti‐BPTI.
Electrophoretic characterisation of fractions collected from gluten protein extracts subjected to size‐exclusion high‐performance liquid chromatography
Authors: Larroque Oscar R. , Gianibelli Maria C. , Batey Ian L. , Macritchie Fin 
Published: May 2005

AbstractThe electrophoretic analysis by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE; reduced and unreduced) of fractions, collected from a size exclusion‐high performance liquid chromatography (SE‐HPLC) separation of gluten proteins using a column with pore size of around 400Å, showed clear resolution for the seven elution ranges studied in two Australian bread wheat lines. Polymeric proteins — high molecular weight (HMW) glutenin subunits, low molecular weight (LMW) glutenin subunits, HMW albumins and some modified ω‐gliadins — appeared exclusively in the region within the first peak of the chromatogram (fractions 1 to 5), the limit being a region that resolved as a small peak before the large peak of gliadins and where some ω‐gliadins eluted. A larger proportion of HMW glutenin subunits and B subunits contributed to polymer formation of higher molecular weight. The polymer size decreased as the proportion of the other protein components increased.
Two‐dimensional gel electrophoresis of actin‐binding proteins isolated by affinity chromatography from human skeletal muscle
Authors: Coumans Joëlle V. F. , Humphery‐Smith Ian , dos Remedios Cristobal G. 
Published: May 2005

AbstractIn muscle cells actin exists as a mixture of monomeric (G‐actin) and filamentous actin (F‐actin) and ionic conditions strongly favor the formation of F‐actin. The existence of unpolymerized actin depends, among other factors, on proteins that bind to G‐actin, the so‐called G‐actin‐binding proteins (G‐ABPs). We have coupled monomeric actin to divinylsulphone‐activated agarose (Mini‐Leak) to isolate G‐ABPs in human skeletal muscle. Eluted proteins were analyzed by two‐dimensional gel electrophoresis (2‐DE), which shows that some proteins are selectively retained. Deoxyribonuclease I (DNase I) is known to bind residues at the “pointed end” of actin (subdomains 2 and 4) with a high affinity. When DNase I is bound to the actin Mini‐Leak before applying the skeletal muscle extract, the 2‐DE gels of the eluted proteins reveals differences when compared to gels of proteins eluted from actin‐Mini‐Leak and DNase I‐Mini‐Leak affinity columns. This strategy should detect ABPs which bind to sites other than the DNase I‐binding site and some may prove to be novel.
Applications of consensus polymerase chain reaction with subsequent electrophoretic distinction of amplificates
Authors: Krause Günter , Oduncu Fuat , Kaltstein Anja , Pusl Thomas , Pachmann Katharina , Gerzer Rupert 
Published: May 2005

AbstractConserved sequences within gene families permit the design of consensus primers that match several members of a given class of homologous genes. Polymerase chain reaction (PCR) products obtained with such consensus primers were characterized by restriction mapping or single‐strand conformation polymorphism (SSCP) analysis, using precast polyacrylamide minigels and automated silver staining. Examples for the electrophoretic distinction of consensus amplificates are presented in the fields of guanylyl cyclase expression studies and in the determination of B‐cell clonality in human blood samples. Guanylyl cyclase expression in inner ear tissues of guinea pigs was investigated by reverse transcription PCR using consensus primers with specificity for the subclass of particulate guanylyl cyclases. The resulting PCR products were assigned to three representatives of this group by restriction mapping. The consensus PCR approach enabled the detection of an unexpected receptor type, namely guanylyl cyclase C, in the inner ear. The distinction by SSCP analysis of denatured consensus amplificates was appropriate for the identification of clone‐specifically rearranged immunoglobulin heavy chain genes of B‐lymphocytes. Genomic DNA isolated from blood samples of leukemia patients served as the template for the consensus amplification of clone‐specific VDJ rearrangements. Rapid distinction and re‐identification of consensus PCR products was achieved by SSCP analysis for regular antigen receptor rearrangements and for t(14; 18) translocations. The potential of these procedures for detecting leukemia or lymphoma clones when monitoring minimal residual disease was assessed.Presented at the “Elektrophorese Forum '96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996.
Multistructure sequencing of N‐linked fetuin glycans by capillary gel electrophoresis and enzyme matrix digestion
Authors: Guttman András 
Published: May 2005

AbstractOligosaccharide sequencing by specific enzymatic digestion of N‐linked fetuin glycans using a carefully designed exoglycosidase matrix in conjunction with separation of the combined digests by high performance capillary gel electrophoresis is described. Due to the extremely high separation power and excellent reproducibility of capillary gel electrophoresis, in most instances no isolation of the individual oligosaccharides is necessary, enabling multistructure sequencing from a released glycan pool. By comparing the positions of the separated exoglycosidase digest fragments to maltooligosaccharides of known size, relative migration times and migration shifts are easily calculated. Hence, the particular sequence of each oligosaccharide in a released glycan pool can be proposed with high confidence. Additionally, with the use of high sensitivity laser‐induced fluorescence detection, complete sequence information can be attained from picomolar amounts of purified glycoproteins.
Separation of human plasma/serum proteins by capillary isoelectric focusing in the absence of denaturing agents
Authors: Manabe Takashi , Iwasaki Aiko , Miyamoto Hiromitsu 
Published: May 2005

AbstractHuman plasma/serum proteins were separated by capillary isoelectric focusing in the absence of denaturing agents. Proteins focused in a fused silica capillary were mobilized by replacing the catholyte sodium hydroxide to acetic acid. The performance of the separation of human plasma proteins has been examined by changing the duration of the step of isoelectric focusing, carrier ampholyte concentration, and plasma protein concentration. The separation patterns of plasma proteins were compared with those obtained by micro two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) run in the absence of denaturants in order to locate the major plasma proteins on the capillary electropherograms. Using the established electrophoretic conditions and the results of peak identification, proteins in the sera of IgG myeloma patients were analyzed by capillary isoelectric focusing. The advantages of capillary isoelectric focusing in plasma protein analysis compared with 2‐D PAGE are discussed.
Proteome analysis of Spiroplasma melliferum (A56) and protein characterisation across species boundaries
Authors: Cordwell Stuart J. , Basseal David J. , Humphery‐Smith Ian 
Published: May 2005

AbstractSpiroplasma melliferum (Class: Mollicutes) is a wall‐less, helical bacterium with a genome of approximately 1460 kbp encoding 800–1000 gene‐products. A two‐dimensional electrophoresis gel reference map of S. melliferum was produced by Phoretix 2‐D gel software analysis of eight high quality gels. The reference map showed 456 silver‐stained and replicated protein spots. 156 proteins (34% of visible protein spots) from S. melliferum were further characterised by one, or a combination, of the following: amino acid analysis, peptide‐mass fingerprinting via matrix assisted laser desorption ionisation‐time of light (MALDI‐TOF) mass spectrometry, and N‐terminal protein microsequencing. Proteins with close relationship to those previously determined from other species were identified across species barriers. Thus, this study represents the first larger‐scale analysis of a proteome based upon the attribution of predominantly ‘unique numerical parameters’ for protein characterisation across species boundaries, as opposed to a sequence‐based approach. This approach allowed all database entries to be screened for homology, as is currently the case for studies based on nucleic acid or protein sequence information. Several proteins studied from this organism were identified as hypothetical, or having no close homolog already present in the databases. Geneproducts from major families such as glycolysis, translation, transcription, cellular processes, energy metabolism and protein synthesis were identified. Several gene‐products characterised in S. melliferum were not previously found in studies of the entire Mycoplasma genitalium and Mycoplasma pneumoniae (both closely related Mollicutes) genomes. The presence of such geneproducts in S. melliferum is discussed in terms of genome size as compared with the smallest known free‐living organisms. Finally, the levels of expression of S. melliferum gene‐products were determined with respect to total optical itensity associated with all visible proteins expressed in exponentially grown cells.
Proteome studies of Saccharomyces cerevisiae: Identification and characterization of abundant proteins
Authors: Garrels James I. , McLaughlin Calvin S. , Warner Jonathan R. , Futcher Bruce , Latter Gerald I. , Kobayashi Ryuji , Schwender Brian , Volpe Tom , Anderson Damon S. , Mesquita‐Fuentes Rodrigo , Payne William E. 
Published: May 2005

AbstractTwo‐dimensional (2‐D) gel electrophoresis can now be coupled with protein identification techniques and genome sequence information for direct detection, identification, and characterization of large numbers of proteins from microbial organisms. 2‐D electrophoresis, and new protein identification techniques such as amino acid composition, are proteome research techniques in that they allow direct characterization of many proteins at the same time. Another new tool important for yeast proteome research is the Yeast Protein Database (YPD), which provides the sequence‐derived protein properties needed for spot identification and tabulations of the currently known properties of the yeast proteins. Studies presented here extend the yeast 2‐D protein map to 169 identified spots based upon the recent completion of the yeast genome sequence, and they show that methods of spot identification based on predicted isoelectric point, predicted molecular mass, and determination of partial amino acid composition from radiolabeled gels are powerful enough for the identification of at least 80% of the spots representing abundant proteins. Comparison of proteins predicted by YPD to be detectable on 2‐D gels based on calculated molecular mass, isoelectric point and codon bias (a predictor of abundance) with proteins identified in this study suggests that many glycoproteins and integral membrane proteins are missing from the 2‐D gel patterns. Using the 2‐D gel map and the information available in YDP, 2‐D gel experiments were analyzed to characterize the yeast proteins associated with: (i) an environmental change (heat shock), (ii) a temperature‐sensitive mutation (the prp2 mRNA splicing mutant), (iii) a mutation affecting post‐translational modification (N‐terminal acetylation), and (iv) a purified subcellular fraction (the ribosomal proteins). The methods used here should allow future extension of these studies to many more proteins of the yeast proteome.
Osmoresponsive proteins and functional assessment strategies in Saccharomyces cerevisiae
Authors: Blomberg Anders 
Published: May 2005

AbstractCells respond to increased external osmolarities by enhanced accumulation of compatible solutes. In yeast‐cells, mainly exemplified by Saccharomyces cerevisiae, the premier compatible solute is the polyhydroxy‐alcohol glycerol, the production of which is accompanied by overall metabolic changes. By applying two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) coupled to computerized image quantification, a large body of valuable physiological information relating to this stress‐adaptation has been gathered. One of the presumed key‐enzymes in the production of glycerol in the cell is glycerol 3‐phosphate dehydrogenase encoded by the GPD1 gene. The amount of this protein is enhanced during saline stress, and from 2‐D analysis linked to microsequencing it became apparent that the osmo‐regulated form contained a putative presequence. Sequence analysis of another salt‐induced spot in the 2‐D pattern revealed identity to a gene, YER062c, with previously unknown function. Biochemical characterization of this protein, including standard purification via chromatography and subsequent activity/specificity measurements, identified this salt‐regulated protein as the missing protein/gene in glycerol production, namely the glycerol 3‐phosphatase. The sequence of another salt regulated protein resolved in the 2‐D gel revealed identity to a bacterial dihydroxyacetone kinase, thus indicating salt induced glycerol dissimilation. Comparing Northern data to the 2‐D generated expression pattern revealed a strong correlation, indicating mainly regulation at the transcriptional level. In addition, altered expression during saline growth of some of the glycolytic enzymes was also apparent. Signalling mutants, either in the cAMP‐dependent protein kinase A pathway or in a protein kinase cascade, have been analyzed during osmotic stress via 2‐D PAGE, grouping proteins/genes apparently regulated via similar mechanismus. Proteome analysis has proven invaluable in the unravelling of the molecular physiology of yeast cells during adaptation and growth under osmotic stress, identifying vital components not selected by purely genetic approaches.
Amplification of anonymous DNA fragments using pairs of long primers generates reproducible DNA fingerprints that are sensitive to genetic variation
Authors: Gillings Michael , Holley Marita 
Published: May 2005

AbstractThe reproducibility and potential applications of anonymous amplification protocols can be improved by using pairs of primers, each of 18 to 24 bases, to replace the single 8 to 10 base primers normally used in randomly amplified polymorphic DNA (RAPD) or DNA amplification fingerprinting (DAF) methods. Amplification using large primer pairs (LP‐RAPD) generates 5 to 30 bands that can be resolved on standard agarose gels. Complex fingerprints can be readily generated from viruses, bacteria, fungi, plants, invertebrates and vertebrates. We also present evidence that a number of polymerase chain reaction (PCR) methods, including those based on the use of enterobacterial repetitive intergenic consensus (ERIC‐PCR) or microsatellite primed (MP‐PCR) sequence, may in essence operate by the same mechanism as LP‐RAPD. Using standard LP‐RAPD protocols, reproducible fingerprints can be generated from a single specimen using different thermocyclers, regardless of the mechanism used for thermocycling (air‐cooled, Peltier effect, or robotic arm). LP‐RAPD is sensitive to intraspecific and interspecific genetic variation, demonstrated here by analysis of mites and apple cultivars. Approximately 50% of LP‐RAPD products are expected to have different primers at either end. Polymorphic bands with this arrangement can be recovered from the gel and directly sequenced using the LP‐RAPD primers themselves. The efficiency of sequencing is improved by the length of the LP‐RAPD primers. This method has the potential to allow the production of allele‐specific species markers in less than two days.
Ancient and modern mitochondrial DNA sequences and the colonization of the pacific
Authors: Hagelberg Erika 
Published: May 2005

AbstractMitochondrial DNA (mtDNA) is a valuable tool for the study of recent human evolution because it is easy to analyse, is inherited uniparentally and has a relatively rapid rate of evolution. mtDNA analysis has been used extensively for the elucidation of the pattern of migrations of human populations. Several studies have focused on the Pacific because Polynesia was settled by humans for the first time relatively recently and there is a wealth of archaeological and linguistic data to complement genetic data on the region. Results of mtDNA analyses on modern‐day Pacific populations indicate reduced genetic variability, and suggest that the Polynesians descend from people who migrated relatively recently from island Southeast Asia and that a population bottleneck occurred during the settlement of the central Pacific. Several informative polymorphisms have been identified in the hypervariable control region of mtDNA in modern‐day Pacific populations that are helpful in tracing the ancestral affinities of these people. Studies of these mtDNA polymorphisms in ancient bones of prehistoric Pacific islanders indicate that the proto‐Polynesian colonizers may have descended from the early settlers of island Melanesia. Although fraught with technical difficulties, studies of ancient DNA can provide valuable evidence on the genetic affinities of past peoples.
Applications of microsatellite‐based Y chromosome haplotyping
Authors: Kayser Manfred , De Knijff Peter , Dieltjes Patrick , Krawczak Michael , Nagy Marion , Zerjal Tatiana , Pandya Arpita , Tyler‐Smith Chris , Roewer Lutz 
Published: May 2005

AbstractY‐chromosomal microsatellites have been investigated for the purposes of application to male identification, population genetics and population history. With nine markers, every male in a German population sample (n = 70) could be identified by an individual‐specific Y microsatellite haplotype. The analysis of 474 unrelated males of nine human populations with seven markers revealed 301 different Y haplotypes. The analysis of molecular variance (AMOVA) approach was used to detect male population characteristics of Y microsatellite haplotypes. With pairwise comparisons of inter‐population variance, most of the populations could be distinguished significantly. Sixty individuals from different male populations in Asia and Northern Europe carrying a novel Y‐chromosomal T→C transition show reduced microsatellite variability together with haplotype similarities. Microsatellite data suggest that the mutation occurred recently in Asia, supporting the hypothesis of Asian ancestry of some northern European populations.
Capillary electrophoresis of DNA for molecular diagnostics
Authors: Righetti Pier Giorgio , Gelfi Cecilia 
Published: May 2005

AbstractA number of applications of capillary zone electrophoresis (CZE) in sieving liquid polymers (notably linear polyacrylamides and cellulose) for the analysis of polymerase chain reaction (PCR) products of clinically relevant, diagnostic DNA, are reviewed. The fields covered are: human genetics, quantitative gene dosage, microbiology and virology, forensic medicine and therapeutic DNA (notably, antisense nucleotides). Some unique, novel developments are highlighted, such as: (i) nonisocratic CZE, i.e., temperature‐programmed CZE for detection of DNA point mutations; (ii) the synthesis of novel N‐substituted acrylamides, offering extreme resistance to alkaline hydrolysis coupled to high hydrophilicity. In the field of denaturing gradient gel electrophoresis (DGGE), as routinely performed in gel slabs, a novel methodology is described in CZE: double‐gradient DGGE. In this technique, two gradients are simultaneously applied along the migration direction: a chemical (or thermal) denaturing gradient, for partially unwinding homo‐ and hetero‐duplexes of DNA, and a porosity gradient, for recompacting diffuse bands melting over a broader range of denaturing conditions. It is thus demonstrated that chemical gradients, in addition to temperature gradients, can be easily implemented even in a capillary format.
Capillary electrophoresis as a clinical tool for the analysis of protein in serum and other body fluids
Authors: Oda Robert P. , Clark Raynell , Katzmann Jerry A. , Landers James P. 
Published: May 2005

AbstractMost electrophoretic analyses of proteins in the clinical laboratory are currently carried out by electrophoresis in acrylamide or agarose gels. This labor‐intensive method is now being challenged by capillary electrophoresis (CE) because of its potential for automated, rapid, high efficiency separations. The automatability of CE itself, combined with its amenability to interfacing with other robotized functions, positions this technology perfectly for the analysis of proteins in physiological matrices, such as serum, urine, and cerebrospinal fluid. The focus of this overview is to familiarize the clinical scientist with the research demonstrating the applicability of the technology to the clinical protein laboratory.
Analysis of small molecules for clinical diagnosis by capillary electrophoresis
Authors: Shihabi Zakariya K. , Friedberg Michael A. 
Published: May 2005

AbstractThe application of capillary electrophoresis (CE) for the analysis of small molecules in clinical research is growing steadily. Initial studies have dealt with separations of standards or compounds in clean matrices. However, later studies dealt with analysis of those compounds in serum, urine or tissues. Great progress has been accomplished in three areas of clinical interest: organic acids, amino acids and drug analysis. The analysis of these compounds by capillary electrophoresis has several distinct advantages: high resolution, simplicity, versatility and especially low operating costs. In many cases, the sample can be injected directly without complex pretreatment. Most of the described methods have been validated for their precision, linearity and accuracy. In forensic toxicology, the CE has been used for drug identification and as a complementary analytical method.