bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractMicrosatellites 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

AbstractRibosomal 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

AbstractSimple 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

AbstractPreeclampsia/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

AbstractThe 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

AbstractProgressive 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

AbstractThe 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

AbstractThe 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

AbstractUsing 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

AbstractProduction 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

AbstractEndotoxins 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

AbstractDetection 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

AbstractWe 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

AbstractIn 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.