bluuebunny/crossref_metadata_2025 huggingface dataset:
Two‐dimensional protein electrophoresis and multiple hypothesis testing to detect potential serum protein biomarkers in children with fetal alcohol syndrome
Authors: Robinson Mary K. , Myrick James E. , Henderson L. Omar , Coles Claire D. , Powell Mary K. , Orr Geoffrey A. , Lemkin Peter F.
Published: May 2005

Fetal alcohol syndrome (FAS) surveillance and intervention efforts are hampered by the lack of a specific biochemical test for diagnosis of the syndrome. Based on the hypothesis that abnormalities in growth and development (key features of FAS) involve altered protein metabolism, we analyzed serum proteins by two‐dimensional gel electrophoresis and image analysis to search for potential protein biomarkers of FAS. Serum samples from 12 participants in whom FAS had been diagnosed and 8 sex‐ and age‐matched participants whose mothers did not consume alcohol were analyzed in duplicate to determine whether the integrated intensities of matched proteins are significantly altered in children with FAS. Multiple hypothesis testing on 34 of the gels consisting of more than 1700 spots per gel revealed 21 proteins that we classified as potential protein biomarkers of FAS on the basis of significant t‐test differences at p < 0.02. We classified 8 of the proteins as candidate biomarkers on the basis of significant concentration differences between case and control subjects at p < 0.01. One of the proteins is clearly an isoform of retinol binding protein; two appear in the area of the gel where alcohol dehydrogenase is expected to appear; one appears to be an isoform of alpha‐1‐antitrypsin; three appear to be isoforms of the beta‐chain of haptoglobin; three may be forms of immunoglobulin light chains; and several others have not been associated with known proteins. No single protein differentiated all case subjects from control subjects, but stepwise canonical discriminant analyses revealed four groups of spots that distinguished between FAS case and control subjects with no misclassifications. Because of the small number of samples analyzed, we have not positively identified the proteins. It will be necessary to confirm our observations using additional samples before identifying each protein.
Effects of oxidative stress and Ca2+ agonists on molecular chaperones in human umbilical vein endothelial cells
Authors: Dreher Don , Vargas J. Reynaldo , Hochstrasser Denis F. , Junod Alain F.
Published: May 2005

Endothelial cell dysfunction is a key factor in oxidative stress‐related pathology. Disruption of Ca2+ homeostasis is thought to be responsible for much of the endothelial cell dysfunction in oxidative stress. The expression of molecular chaperones (MC), which stabilize protein structures in normal and in stress conditions, reflects the Ca2+ ‐dependent and ‐independent stress effects in the different cell compartments. By two‐dimensional (2‐D) gel electrophoresis, combined with immunoblotting or microsequencing, we have identified 12 major MC in human umbilical vein endothelial cells (HUVEC): (i) the endoplasmic reticulum‐located MC GRP78, GRP94, protein disulfide isomerase, and calreticulin; (ii) the mitochondrial MC HSP65 and GRP75; and (iii) the cytosolic/nuclear MC HSP27, HSC70, HSP70, HSP90, cyclophilin, and ubiquitin. To differentiate oxidative stress‐ and Ca2+ ‐mediated effects, HUVEC were exposed to 1) xanthine oxidase plus hypoxanthine to generate oxidative stress, 2) ionomycin plus ethylene glycol‐bis(β‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid (EGTA) to deplete intracellular Ca2+ stores, or 3) thrombin to increase cytosolic Ca2+. De novo protein synthesis after exposure was quantified by the incorporation of [35S]methionine. Image processing with the MELANIE system was used to create and compare the 2‐D maps of [35S]methionine‐labeled proteins under conditions 1)–3) with those of the controls. In a total of 24 2‐D gels, 9 different MC were detected in at least 5 out 6 experimental replicates and were subjected to numeric analysis. The statistics showed a > 10% increase in GRP78 (p < 0.05), HSP27, cyclophilin, and ubiquitin after oxidative stress. Ionomycin plus EGTA influenced the expression of GRP78 (p < 0.01), cyclophilin, and ubiquitin in the same direction, but in addition increased GRP94 (p < 0.01) and calreticulin. Thrombin upregulated calreticulin (p < 0.05), HSC70, and cyclophilin. In conclusion, the computer analysis of MC expression on 2‐D maps under different stress conditions reveals a differential correlation between the oxidative stress and the depletion of Ca2+ stores in HUVEC.
Analysis of proteins from human breast epithelial cells using two‐dimensional gel electrophoresis
Authors: Giometti Carol S. , Tollaksen Sandra L. , Chubb Cynthia , Williams Catherine , Huberman Eliezer
Published: May 2005

The human breast is a highly specialized, complex tissue comprised of a heterogeneous population of cells with varying functions. Interactions between the different cell types, changes in their relative abundance, state of differentiation and function in response to stimuli, as well as the alterations that lead to the aberrant growth associated with malignancy are poorly understood. Two‐dimensional gel electrophoresis is being used to compare the proteins found in different breast cells in order to identify the gene products that are common or specific to particular cell types so as to provide markers that will be useful in studies of normal breast cell differentiation and the dedifferentiation or blocked differentiation characteristic of cancer. Protein patterns have been obtained from cells prepared for electrophoresis immediately after isolation from human milk, from cells cultured for fewer than ten passages after isolation from healthy breast tissue removed during reduction mammoplasty, and from cells maintained in long‐term tissue culture after isolation from the pleural effusions of patients with breast carcinomas. Differential expression of cytokeratins 8, 18, and 19, shown previously to be predominantly expressed by epithelial cells in the luminal layer of breast tissue, was observed among the cells analyzed. Other non‐cytokeratin proteins were also found to be differentially expressed in subsets of both the normal and tumor cells. A composite human breast cell protein pattern was created which includes all the commonly and specifically expressed proteins found in this study. This pattern will be the basis for continuing studies of proteins in the human breast.
Repetitive treatment with serotonin modifies protein synthesis and protein phosphorylation in the central nervous system of Hirudo medicinalis
Authors: Garcia‐Gil Mercedes , Bottai Daniele , Romano Antonia , Fineschi Loredana , Bini Luca , Pallini Vitaliano , Brunelli Marcello
Published: May 2005

Serotonin (5HT) is the neurotransmitter involved in some forms of short‐term memory in the leech. Behavioral experiments have demonstrated that long‐term memory requires new protein synthesis. With the aim of studying the molecular mechanism underlying memory processes in the leech, we have analyzed the effect of 5HT on protein synthesis and protein phosphorylation. Segmental ganglia of the leech central nervous system haves been labeled, proteins have been separated by two‐dimensional‐electrophoresis and labeled proteins detected by autoradiography. Our findings indicate that repetitive treatment with 5HT produces either the persistence of phosphorylation or changes in protein synthesis in several proteins.
Two‐dimensional electrophoresis of intracellular and secreted protein synthesized by fetal bovine chondrocytes in high‐density culture
Authors: Freyria Anne‐Marie , Ronzière Marie‐Claire , Boutillon Marguerite‐Marie , Herbage Daniel
Published: May 2005

In order to study the mechanisms involved in the differentiation/dedifferentiation of chondrocytes, fetal bovine chondrocytes in high‐density cultures were treated with retinoic acid, an agent known to modify the chondrocyte phenotype (10 μmol/L between day 2 to day 5 of culture). The synthesis of intracellular and secreted proteins was studied by two‐dimensional electrophoresis in cell lysates and culture media after labeling with [35S]methionine for the last 14 h of culture. The proteins expressed in control and retinoic acid‐treated cells were identified by microsequencing after “in‐gel” tryptic digestion of the spot or by immunodetection with specific antibodies after two‐dimensional gel blotting. Intracellular protein modifications included one of 56.9 kDa and with an isoelectric point (pI) of 5.8 whose synthesis was previously reported to be up‐regulated by 75%. Microsequencing of two internal peptides did not reveal a known protein. Changes to the chondrocyte phenotype were also recorded in the culture medium, as a decrease in type II collagen synthesis and expression of the small proteoglycan, decorin. Several new spots were also observed after treatment with retinoic acid, including a large, diffuse spot, not yet characterized, with a mean molecular mass of 39 kDa and a pI of 4.5–5.0. Under our experimental conditions, retinoic acid induces morphological changes of the chondrocytes and dramatic changes in the synthesis of several intracellular and secreted proteins that predate the synthesis of collagen type I (the classical marker of chondrocyte dedifferentiation).
Two‐dimensional electrophoresis of acidic proteins isolated from ozone‐stressed Norway spruce needles (Picea abies L. Karst): Separation method and image processing
Authors: Davidsen Nina B.
Published: May 2005

The effect of ozone stress (< 10, 200 or 400 μg m−3) on the protein pattern of Picea abies L. Karst needles was studied by fumigating potted grafts from mature trees. Clonal trees of Norway spruce were fumigated in open‐top chambers for one growing season. Proteins, soluble and membrane bound, were extracted from the needles in a four‐step procedure: (i) grinding in liquid nitrogen and then in a pH 5.0 buffer with thiourea added to inhibit phenol oxidase and polyvinylpolypyrrolidone (PVP) added to bind phenolic compounds; (ii) precipitation of proteins with acetone at −20°C; (iii) solubilization in a modified lysis buffer; and (iv) dialysis against the modified lysis buffer. Proteins were separated by two‐dimensional electrophoresis according to O'Farrell's method. The changes in the silver‐stained protein pattern were evaluated by using image processing methods. Electrophoretograms from stressed and nonstressed trees are presented.
Genetic variability of carrot seed proteins analyzed by one‐ and two‐dimensional electrophoresis with immobilized pH gradients
Authors: Posch Anton , van den Berg Bartel M. , Burg Henk C. J. , Görg Angelika
Published: May 2005

The genetic variability of eight carrot populations representing commercially important morphological types was analyzed for seed protein polymorphism by one‐ and two‐dimensional electrophoresis using immobilized pH gradients (IPG) in order to assess the potential of both techniques in carrot hybrid breeding as well as genetic quality testing of carrot trade seeds. The digitized silverstained one‐ and two‐dimensional gels of the urea/detergent‐soluble seed proteins were subjected to computer analysis to estimate both the inter‐ and intrapopulation genetic variability. The results indicated that isoelectric focusing (IEF‐IPG) of seed proteins is a powerful tool for quick screening of the genetic variability present between and within carrot populations, while two‐dimensional polyacrylamide gel electrophoresis (IPG‐DALT) can be employed to establish detailed genetic relationships of carrot populations selected by initial IEF‐IPG screening in order to use genetic distant and homogeneous populations for successful carrot hybrid breeding. However, the applicability of IEF‐IPG in genetic quality testing of carrot seeds seems to be restricted to hybrid purity testing. IPG‐DALT is a far more powerful tool to profile unambiguously all carrot varieties commercially used in order to test the genetic quality of trade seeds, because this technique exhibits protein spots or spot combinations exclusively found in each of the eight varieties investigated.
Phosphorylation of herpes simplex virus type 1 Us11 protein is independent of viral genome expression
Authors: Simonin Denis , Diaz Jean‐Jacques , Kindbeiter Karine , Pernas Patrick , Madjar Jean‐Jacques
Published: May 2005

The Us11 protein is a true late gene product of herpes simplex virus type 1 (HSV‐1), whose exact function is unknown but which exhibits RNA‐binding properties and which is phosphorylated on serine residues. In order to determine whether the Us11 protein is phosphorylated by cellular kinase(s) or by virally encoded kinase(s), the Us11 gene has been cloned and transiently expressed in HeLa cells. In addition, HeLa‐derived cell lines have been selected for their ability to express Us11 protein constitutively. 32P‐Labeling and analysis by two‐dimensional electrophoresis of transiently and constitutively expressed Us11 protein demonstrated that, indeed, multiple phosphorylation of the protein occurs in absence of HSV‐1 genome expression, indicating that the protein behaves as a natural substrate for cellular kinase(s). In addition, a sequence heterogeneity of the Us11 protein, due to a difference in the number of SPREPR repeats, has been characterized between different strains of HSV‐1.
Manipulation of a single cell with microcapillary tubing based on its electrophoretic mobility
Authors: Kitagawa Shinya , Kawaura Chiyo , Hashimoto Osamu , Takahashi Tsutomu , Naoi Makoto , Tsuda Takao
Published: May 2005

Manipulation of a single cell of spherical shape, approximately 5–10 μm in diameter, was performed with capillary tubing and an electrostatic field. A single cell migrates with its electrophoretic mobility into capillary tubing against the flow of electroosmosis coming out of the capillary. After trapping the cell in the capillary, it is pulled out into the other microreservoir with the application of a reverse electric voltage. When we apply a negative voltage to the microreservoir itself, the cell in it can keep floating for a relatively long period due to electrostatic repulsion. The electrophoretic mobility of a single cell is also estimated.
Calculation of the isoelectric points of native proteins with spreading of pKa values
Authors: Henriksson Gunnar , Englund Ann‐Kristin , Johansson Gunnar , Lundahl Per
Published: May 2005

The isoelectric points (pI) of native proteins are important in several separation techniques. For estimating pI values the net charge of several proteins was calculated versus pH by use of the Henderson‐Hasselbalch equation. Amino acid composition, pKa values for amino acid side chains and for the N‐ and C‐terminal groups, and the presence of other charged groups were taken into account. A set of pKa values was chosen for amino acid residues with ionizable side chains. Each particular type of ionizable group was assumed to have pKa values distributed around the chosen value, thereby simulating the situation in proteins and polypeptides. The calculated pI values showed reasonably good agreement with experimental ones for most of 16 native proteins over a wide pH range (3.4–11) when charge contributions of heme groups, sialic acid residues, etc., were taken into account. The calculated pI for the human red cell glucose transporter (Glut 1) with one sialic acid residue was decreased from 8.8 to 8.5 by introducing pKa value spreading and became consistent with the experimental pI value of 8.4 ± 0.05 at 15°C determined in the presence of 6 M urea. The pI of the native Glut1 was lower, 8.0 ± 0.1, at 22°C. In general, the pI values for native proteins are affected by the three‐dimensional structure of the proteins, which causes greater differences between calculated and experimental pI values than in the case of polypeptides for which pI values are determined in the presence of urea.
Characterization of proteins by sequential isoelectric focusing on immobilized pH gradients and electrospray mass spectrometry
Authors: Breme Umberto , Breton Jerǒme , Visco Carlo , Orsini Gaetano , Righetti Pier Giorgio
Published: May 2005

The coupling of isoelectric focusing on immobilized pH gradients (IPG) with electrospray‐mass spectrometry (ES‐MS) was applied to the characterization of proteins according to two different and important properties, such as net surface charge and molecular mass. From a technical point of view, these methods are complementary, since ES‐MS requires ion‐free samples as usually supplied by isoelectric focusing on IPGs. This report describes the experiments carried out on model proteins to demonstrate the feasibility of the sequential application of these two techniques for the characterization of proteins. A minimum of 5 μg protein was needed for good signal by mass spectrometry. The following proteins were studied: myoglobin, truncated interleukin‐6‐mutein, recombinant cytochrome c551 and insulin‐like growth factor I. Extraction from the IPG matrix was carried out in 70% acetonitrile/30% water/0.05% trifluoroacetic acid either by passive diffusion or by centrifugation through a 0.22 μm Amicon membrane, with protein recoveries of 80–85%.
DNA separation with field inversion capillary electrophoresis
Authors: Heller Christoph , Pakleza Christophe , Viovy Jean Louis
Published: May 2005

We have built an automated pulsed field capillary electrophoresis system on the basis of a commercially available device. Using entangled polymer solutions as separating matrix, we demonstrate a considerable improvement of separation of double‐stranded DNA in the range of 1–50 kbp under pulsed field conditions. The influence of the main parameters, i.e. pulse frequency and electric field, is studied and the results are compared to existing electrophoresis theories.
Gene linkage of two‐dimensional polyacrylamide gel electrophoresis resolved proteins from isogene families in Saccharomyces cerevisiae by microsequencing of in‐gel trypsin generated peptides
Authors: Norbeck Joakim , Blomberg Anders
Published: May 2005

The total cellular extract of proteins from the yeast Saccharomyces cerevisiae was resolved by preparative two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) where 500 μg was loaded per gel, and a number of proteins in isogene families were selected for microsequencing analysis. Peptides were generated from resolved proteins by in‐gel trypsin digestion, and fractionated by reversed phase‐high performance liquid chromatography (RP‐HPLC). Subsequent sequencing of peptides yielded internal amino acid sequences which unambiguously identified the selected proteins spots as gene products from PDC1, ENO1 ENO2, ADH1, HXK2, TDH2, TDH3, SSB1 and SSB2. The chromatograms obtained from RP‐HPLC of related proteins were utilized to distinguish discriminating peptide fractions. With this approach two out of four amino acid differences between Ssb1p and Ssb2p were allocated. We estimate that by pooling five preparative gels, at least one hundred protein spots in the 2‐D pattern of S. cerevisiae will be obtained in sequencable amounts.
An new mode of size‐dependent separation of proteins by capillary electrophoresis in presence of sodium dodecyl sulfate and concentrated oligomeric dextran
Authors: Takagi Toshio , Karim Mohammad Rezaul
Published: May 2005

Capillary electrophoresis in presence of sodium dodecyl sulfate (SDS) and 10% w/v of oligomeric dextran with molecular weight of just above 1000 was found to be an efficient medium for size‐dependent separation of proteins (Karim, M. R., Janson, J.‐C., Takagi, T., Electrophoresis 1994, 15, 1531–1534). The separations were found to be highly reproducible, both with respect to the position and relative amounts of the peaks. No evidence for a network structure in the medium could be found on examination of the effect of dextran concentration and viscosity measurements. The separation mechanism appears to be different from sieving by a polymer network structure. In free solution, SDS‐protein complexes with molecular weights > 10000 migrate with almost the same mobilities, irrespective of their molecular weight (this behavior is called free draining). Such SDS‐complexes of proteins or peptides with molecular weights < 10000, however, tend to increase their negatively signed mobilities with decreasing molecular weight (Karim, M. R., Shinagawa, S., Takagi, T., Electrophoresis 1994, 15, 1141–1146). Our working hypothesis is that in presence of the oligomeric dextran, the range of deviation from the free‐draining electrophoretic behavior extends to higher molecular weights, resulting in a novel separation made.
Sodium dodecyl sulfate‐gel electrophoresis of proteins employing short capillaries
Authors: Manabe Takashi
Published: May 2005

The performance of capillary gel‐sodium dodecyl sulfate (SDS) electrophoresis for molecular‐mass‐dependent separation of proteins has been examined employing cross‐linked polyacrylamide gels and untreated fused‐silica capillaries of short length. An apparatus for capillary electrophoresis has been constructed, combining a UV detector equipped with a capillary holder, a power supply, and a micromanipulator for sample loading. The apparatus is capable of accomodating gel capillaries of different lengths and can be operated with two methods of sample loading: manual injection on top of the gel or electrokinetic injection. Standard proteins have been separated according to their molecular mass (ranging from 17000 to 116000) within 30 min, employing gel capillaries of effective lengths shorter than 10 cm and polyacrylamide gels ranging from 2.4 to 4.8%T (5%C). The results confirmed that gel capillaries of effective length less than 10 cm can be used for protein size‐separation by SDS electrophoresis, with much higher performance than is achieved in rod gel‐SDS electrophoresis.
The use of capillary electrophoresis for the determination of hemoglobin variants
Authors: Castagnola Massimo , Messana Irene , Cassiano Loredana , Rabino Rita , Rossetti Diana V. , Giardina Bruno
Published: May 2005

The application of capillary electrophoresis and related techniques for the detection of hemoglobin variants is described. Capillary zone electrophoresis (CZE) was applied for the analysis of intact tetrameric hemoglobin. CZE under denaturing conditions was used for the separation of globin chains. Both CZE and micellar electrokinetic capillary chromatography were applied for a fast and sensitive separation of tryptic digests and for the analysis of amino acid derivatives.
Differential expression of nucleophosmin and stathmin in human T lymphoblastic cell lines, CCRF‐CEM and JURKAT analyzed by two‐dimensional gel electrophoresis
Authors: Nakamura Kazuyuki , Fujimoto Masanori , Tanaka Tatehiko , Fujikura Yoshihisa
Published: May 2005

Two‐dimensional gel electrophoresis was used to study the expression of intracellular proteins in adherent cells of human T lymphoblastic cell line, CCRF‐CEM. The adherent cells grown in monolayer on a culture plate decreased the amount of proteins of Mr 37 000 and pI 4.7–4.9, and of 17 000 and pI 5.7. The proteins were identified to be nucleophosmin for the 37 000 protein and stathmin for the 17 000 protein by microsequencing their CNBr fragments. The amount of proteins was increased in CCRF‐CEM cells grown in floating mass to a comparable level of JURKAT cells which grew in floating mass throughout the culture. The adherent cells decreased their growth rate as compared with the cells in the floating mass. These results suggest that the adhesion of human T lymphoblastic cells modulates their morphology and proliferation via a concomitant decrease in the amount of nucleophosmin and stathmin.
Biological significance of minisatellites
Authors: Singh Lalji
Published: May 2005

Minisatellites are tandemly repeated, highly variable DNA sequences found in most higher eukaryotes. These contain a core sequence resembling the chi sequence of Escherichia coli, which is a binding site for recombination proteins. Based on this, a generalized function of minisatellites to provide binding sites for recombination proteins in eukaryotes has also been suggested. However, recent discoveries of trinucleotide repeat expansion mutations associated with at least four human genetic diseases, several short repeats acting as motifs for binding of various transcription factors, and several minisatellite‐binding nuclear proteins, which are expressed in specific tissues and bind to specific sequences, strongly suggest that different families of minisatellites may have different functions. A banded krait minor (Bkm) satellite DNA, consisting of highly conserved GATA repeats, which is arranged in a sex‐specific manner, is hypervariable. We have found a sex‐ and tissue‐specific factor designated as Bkm‐binding protein (BBP), which specifically binds to Bkm (GATA), in the germ cells of the heterogametic sex [ovary, in the case of female heterogamety (in snakes); and testis, in the case of male heterogamety (in mice, rats and humans)]. It is in these tissues that decondensation of the W and Y chromosomes occurs. We suggest that GATA repeats of Bkm bring about a coordinated decondensation of the W and Y sex chromosomes in the germ cells in response to BBP, which may serve as a “switch” for the activation of the genes present on the W and Y sex chromosomes. Since the number of GATA repeats, in tandem, necessary for the binding of BBP is flexible, there is no selection pressure on the maintenance of the exact length of the repeats, unless it is reduced below a threshold at which the binding is completely abolished.
Genetic typing with HUMTH01, HUMVWA31A and HUMFES/FPS short tandem repeat loci, D1S80 variable number tandem repeat locus and HLA‐DQα of recent and from XII–XIII centuries spongy bone
Authors: de Pancorbo Marian M. , Castro Azucena , Alonso Santos , Fernández‐Fernández Isabel , Barbero Carmen , García‐Orad Africa , Izaguirre Neskuts , Iriondo Mikel , de la Rúa Concepción
Published: May 2005

The genetic analysis of ancient populations through DNA from bone remains, requires the use of short sized loci that can be amplified by polymerase chain reaction (PCR) for which the short tandem repeat (STR) loci are most suitable. These techniques can also be applied to genetic identification in forensic casework. In this study three STR loci, HUMFES/FPS, HUMTH01 and HUMVWA31A, were selected to estimate their usefulness when applied to recent and ancient spongy bone DNA typing. In addition, loci D1S80 and HLA DQα were also tested in the analysis of recent spongy bone DNA. The recent remains studied were constituted by ten spongy bone samples of postmortem material from one individual buried for 1 year. The ancient remains are composed by 8 spongy bone samples from the heads of left femurs from a XII–XIII Centuries Basque Country population. Adequate amplification and typing results could only be obtained with cetyltrimethyl ammonium bromide (CTAB)‐extracted DNA, without any further purification after precipitation. Genotypes of the one year post‐mortem material and those of his son and his wife were obtained at the D1S80, HLA‐DQα, and STR loci. In all these systems, no exclusion was observed, with a combined probability of paternity of 0.9997. This demonstrates the reliability of the obtained results. The genetic typing of HUMTH01 in spongy bone from the XII–XIII Centuries Basque Country individuals was also performed. This will allow the genetic analysis on ancient bone remains and therefore, to carry out evolutionary population studies.
Genome fingerprinting of the silkworm, Bombyx mori, using random arbitrary primers
Authors: Nagaraja Ganachari M. , Nagaraju Javaregowda
Published: May 2005

The random amplified polymorphic DNA (RAPD) technique was used to study DNA profiling of thirteen silkworm genotypes. The genotypes included six diapausing and seven nondiapausing varieties that represent a high degree of divergence with respect to geographic origin, and morphological, qualitative, quantitative and biochemical characters. Two hundred sixteen amplified products were generated using 40 random primers. Genotype‐specific amplification products were identified. Amplification products specific to diapausing genotypes were also identified. Segregation of the RAPD marker was analyzed in a backcross population and found to be inherited as dominant Mendelian traits. Based on pairwise comparison of amplified products, the genetic similarity coefficient was calculated using the NeiLi similarity coefficient, and cluster analysis was performed by a hierarchical clustering technique. Silkworm genotypes were clustered into two groups, one consisting of six diapausing and the other of seven nondiapausing genotypes. The results of our study suggest that the RAPD technique could be used as a powerful tool to generate genetic markers that are linked to traits of interest in the silkworm.
Intra‐ and inter‐population diversity at short tandem repeat loci in diverse populations of the world
Authors: Deka Ranjan , Shriver Mark D. , Yu Ling M. , Ferrell Robert E. , Chakraborty Ranajit
Published: May 2005

To study the level of intra‐ and inter‐population variation at hypervariable DNA loci, we have characterized 15 human populations of diverse ethnic and geographic origins at six short tandem repeat loci by using the polymerase chain reaction. Even though the spectrum of allelic variation is quite broad and there are substantial differences in allele frequency distributions among populations, in general, populations within a major racial group show a greater degree of similarity. This observation is reflected in the analysis of gene diversity. When the total diversity is apportioned, the maximum variation becomes attributable to inter‐individual differences within a population; of the variation that is attributable to differences between populations within a racial group and differences between racial groups, the former is smaller than the latter. Separate analysis of gene diversity for each of the major population groups based on geographic and ethnic relationship shows that the total gene diversity is higher for the larger racial groups, namely, African, Caucasian and Mongoloid, than the American Indians and the Pacific Islanders. As expected, a reciprocal relationship between gene diversity and FST levels is observed. Higher values of FST in the American Indian and the Pacific Islanders may reflect smaller population size and a higher level of isolation. An analysis of genetic distance encompassing the populations belonging to the three major racial groups recognizes three distinct clusters – all the populations of African affiliation cluster together, as do the Caucasian affiliated and the Mongoloid groups, in two distinct clusters. Interestingly, three broadly classified cosmopolitan US populations, namely, US White, US Black and US Asian, cluster with their ancestrally related populations. This study dispels some of the concerns regarding the applicability of DNA typing data for forensic use.
Trinucleotide repeat expansion and human disease
Authors: Hummerich Holger , Lehrach Hans
Published: May 2005

Trinucleotide repeat expansions have been identified as the underlying mutation in an increasing number of human genetic diseases, such as fragile site syndromes, myotonic dystrophy and several neurodegenerative disorders including Huntington's disease. By an unknown mechanism, polymorphic GC‐rich triplet repeats expand in all these diseases. The expansions of a CCG repeat in fragile‐site‐associated disorders and the CTG repeat (in the 3′‐untranslated region of the myotonin kinase gene) causing myotonic dystrophy are very large, whereas small expansions of CAG repeats have been identified in the open reading frame of genes in a number of neurological genetic disorders.
DNA polymorphism analysis in five endangered species of Meconopsis (Himalayan poppy) using multi‐copy sequence‐based probes
Authors: Sulaiman Irshad M. , Hasnain Seyed E.
Published: May 2005

An attempt to understand the genetic basis of extinction of Meconopsis (Himalayan poppy), an endangered genus of ornamental value and confined to alpine Himalayas, is described. Multicopy DNA sequences were isolated from a Sau3A partial genomic plasmid library of M. simplicifolia and M. paniculata. Genetic polymorphism between five species of Meconopsis belonging to geographically isolated populations was evident, albeit at a low level, in genomic Southerns when probed with repetitive DNA clones isolated from this library. Intra‐population monomorphic band patterns for different populations of a species was evident with respect to a number of enzymes suggesting very low or no genetic variability and/or low mutation rate within a given population of Meconopsis.
Why ontogeny recapitulates phylogeny
Authors: Ohno Susumu
Published: May 2005

In development, mammalian embryos successively develop notochord, somites, gill pouches and three sets of kidneys: pronephros, mesonephros and metanephros. Hence, Ernst Haeckel's dictum “ontogeny recapitulates phylogeny.” The reason for this dictum appears to be the immortality of genes. Genes, once well entrenched, appear to linger for a long time even after the forfeiture of their raison d'ětre. The genes for dental enamel and dentin of the chicken are good examples. Furthermore, genes once doomed may resurrect themselves. For example, genes for red and green opsins of higher primates arose as alleles of the once doomed but resurrected red opsin locus.
An electrophoretic approach to the assessment of the spatial distribution of DNA double‐strand breaks in mammalian cells
Authors: Friedl Anna A. , Kraxenberger Alfred , Eckardt‐Schupp Friederike
Published: May 2005

An approach is presented making it possible to investigate whether breaks in fragmented mammalian chromosomal DNA were induced randomly and independently from each other. Genomic DNA isolated from mammalian cells irradiated with γ‐rays or restriction enzyme‐treated human DNA was resolved according to size using pulsed field gel electrophoresis, and the resulting DNA mass distributions were measured in ethidium bromide‐stained gels. The DNA profiles thus obtained were compared to the predictions on DNA fragment size distribution which follow from a so‐called random breakage model to test whether the experimental outcome is compatible with the assumption of a random localization of breaks. Comparisons of fragment distributions may be performed utilizing two equivalent representations that are linked by an adequate transformation. Considering either directly measurable DNA mass profiles in units of migration distances along a gel lane or transformed distributions in units of molecular length, we show for γ‐irradiated samples that the predictions derived from the employed models agree well with the observed data, thus allowing an immediate quantification of double‐strand breaks (DSB). Using restriction enzyme‐treated DNA as a paradigm, the disagreement of predicted and observed data shows the applicability of our approach to the detection of a non‐random distribution of DSB. Therefore, we suppose that our approach may also be useful to reveal a clustering of DSB, which is postulated to occur after damage induction by densely ionizing radiation. Furthermore, investigations on the spatial distribution of chemically or endogenously produced DSB, as well as residual DSB after repair, may be attempted.
Analysis of microsatellites by direct blotting electrophoresis and chemiluminescence detection
Authors: Mekus Frauke , Dörk Thilo , Deufel Thomas , Morral Núria , Tümmler Burkhard
Published: May 2005

We describe a fast and reliable method for the nonradioactive analysis of microsatellites. For three dinucleotide repeats within the cystic fibrosis transmebrane conductance regulator (CFTR) gene, the separation of polymerase chain reaction (PCR) products generated with biotinylated primers on a direct blotting electrophoresis system and subsequent chemiluminescence detection is shown. In direct blotting electrophoresis, the separation of DNA fragments depended linearly on size. The reproducible resolution allowed reliable assignment of allele lengths to a given signal. The nonradioactive detection protocol was advantageous compared to radioactive methods: samples could be analyzed within one day due to the fast signal development by 3‐(4‐meth‐oxyspiro{1,2‐dioxetane‐3,2′‐(5′‐chloro)tricyclo[3.3.1.1.3,7]decan}‐4‐yl)phenylphosphate disodium salt (CSPD). Variation of exposure times enabled differentiation between major bands and byproducts of comparable intensity that are due to the slippage of the Taq polymerase during PCR amplification.
Specific staining of glycine N‐methyltransferase
Authors: Santos Fátima , Amorim António , Kömpf Jost
Published: May 2005

Glycine N‐methyltransferase from rabbit, human, rat and pig livers was separated by isoelectric focusing and a specific functional staining method was developed through the detection of sarcosine produced from the methylation of glycine. Isozyme patterns obtained in the various species tested differ both in the number of bands and apparent isoelectric points. These differences may explain the contradictory data on the subunit structure and glycosylation status of the enzyme reported so far.
Capillary gel electrophoresis separation of high‐mannose type oligosaccharides derivatized by 1‐aminopyrene‐3,6,8‐trisulfonic acid
Authors: Guttman András , Pritchett Tom
Published: May 2005

In this paper we report the capillary gel electrophoresis separation of 1‐amino‐pyrene‐3,6,8‐trisulfonic acid (APTS) labeled oligosaccharides, released enzymatically from bovine pancreatic ribonuclease B. The released and labeled high‐mannose structures were identified by spiking the separated peaks with the appropriate commercially available individual oligosaccharides. Baseline separation of the three positional isomers of the mannose‐7 and mannose‐8 oligosaccharides was attained. Comparison of the electrophoretic mobilities of the high‐mannose type branched carbohydrates to the linear molecules of malto‐oligosaccharides (glucose oligomers) have been shown using different gel concentrations in the running buffer system. We observed that increasing gel concentration in the running buffer causes an increase in the relative mobility values of the high‐mannose type carbohydrate molecules compared to the linear glucose oligomers. Analysis of our data indicated that this increase in relative migration time was not due to sieving, but seemed to be related to the mannose content and hydrodynamic volume of the branched glycans as well as to the viscosity of the separation medium.
Fractionation of carrier ampholytes in multicompartment electrolyzers with isoelectric membranes
Authors: Bossi Alessandra , Righetti Pier Giorgio
Published: May 2005

Multicompartment electrolyzers with isoelectric membranes can be successfully utilized for the preparation of carrier ampholytes of narrow pH ranges from commercially available wider pH intervals. An example is given on the preparation of a 0.6 pH unit (pH 6.7–7.3) range from a standard pH 6–8 interval. A 10% Ampholine solution is focused in an electrolyzer equipped with the following isoelectric membranes: pI 6.0, 6.7, 7.3 and 8.0. The narrow pH 6.7–7.3 cut can be efficiently utilized for base‐line separation and quantitation of hemoglobin A from its glycated form, Hb A1c. This analysis is important for the screening and follow up of diabetic patients. The advantage of multicompartment electrolyzers are: (i) precision in the preparation of narrow pH cuts, due to the presence of membranes with defined pI values; (ii) ability to perform in both small‐ and large‐scale operations and (iii) absence of contaminants leaching from granulated supports, as typical of some previous techniques.
An updated two‐dimensional gel database of rat liver proteins useful in gene regulation and drug effect studies
Authors: Anderson N. Leigh , Esquer‐Blasco Ricardo , Hofmann Jean‐Paul , Meheus Lydie , Raymackers Jos , Steiner Sandra , Witzmann Frank , Anderson Norman G.
Published: May 2005

We have improved upon the reference two‐dimensional (2‐D) electrophoretic map of rat liver proteins originally published in 1991 (N. L. Anderson et al., Electrophoresis 1991, 12, 907–930). A total of 53 proteins (102 spots) are now identified, many by microsequencing. In most cases, spots cut from wet, Coomassie Blue stained 2‐D gels were submitted to internal tryptic digestion [2], and individual peptides, separated by high‐performance liquid chromatography (HPLC), were sequenced using a Perkin‐Elmer 477A sequenator. Additional spots were identified using specific antibodies.
Steady‐state models in electrophoresis: From isotachophoresis to capillary zone electrophoresis
Authors: Beckers Jozef L.
Published: May 2005

Although all electrophoretic techniques are closely allied and controlled by the same rules, we often distinguish between steady‐state and dynamic models in the modeling of electrophoretic processes, whereby steady‐state models are applied for isotachophoresis (ITP) and dynamic models are applied for other electrophoretic processes, wherein a steady‐state is not reached. This paper shows how, starting from a mathematical model for the steady‐state in ITP, mathematical models can be derived for several modifications of ITP and that even nonsteady‐state processes can be estimated by a repeated application of a steady‐state model. In this way all parameters in sample zones in capillary zone electrophoresis (CZE), through which temporal electropherograms can be simulated, can be calculated. Realistic simulations can be obtained for zone electrophoretic processes, for which the electrodispersive character is the dominating peak broadening mechanism, and simulated electropherograms resemble measured electropherograms concerning migration times, peak shapes, fronting or tailing character and the question of peaks and dips.
Capillary electrophoresis with polymeric separation media: Considerations for theory
Authors: Smisek David L.
Published: May 2005

The theory of capillary electrophoresis with polymeric separation media is reviewed. Some of the major theories of gel electrophoresis are examined in terms of the experimental observations that they support. The considerations and difficulties in applying these theories to capillary electrophoresis with polymeric separation media are investigated, particularly the use of high electric field strengths and low concentration polymer solutions. Recent advances in this area, including some empirically‐derived relationships and models currently found in the literature, and future directions for theory development are discussed.
Study of high‐field dispersion in micellar electrokinetic chromatography
Authors: Yu Lixin , Davis Joe M.
Published: May 2005

The anomalous dispersion of hydrophobic, highly retained analytes at high electric field strengths E in micellar electrokinetic chromatography (MEKC) is experimentally and theoretically quantified. A simple model for plate number N is proposed, in which only plug size and longitudinal diffusion govern dispersion. This model describes well the dispersion of acetone, 2′‐deoxyadenosine, and 2′‐deoxyadenosine‐5′‐monophosphate (dAMP) in MEKC buffers and also dAMP in capillary‐electrophoresis buffers. However, the model describes the dispersion of 1‐nitropyrene, pyrene, perylene, and the 4‐chloro‐7‐nitrobenzofuran (NBD) derivative of cyclohexylamine only at low E's; for these analytes, a nonequilibrium‐like dispersion causes N to decrease rapidly at high E's. Furthermore, the dispersion of NBD‐cyclohexylamine has a dependence on capillary diameter. Theoretical arguments are proposed, which show that this additional dispersion cannot be explained quantitatively by mass‐transfer kinetics, micellar polydispersity, hydrodynamic flow, Joule heating, or the radial variation of partition coefficient. In addition, experiments are performed suggesting that wall adsorption and sample inhomogeneity are not responsible for the efficiency loss. Other dispersion sources are suggested for future exploration.
Capillary isoelectric focusing: A routine method for protein analysis?
Authors: Schwer Christine
Published: May 2005

Although isoelectric focusing (IEF) in the slab gel format is a widely used technique in protein analysis, capillary isoelectric focusing (CIEF) is not yet a method used routinely, despite major advantages. This may be due to the fact that several methods for performing IEF in the capillary can be found in the literature, differing in the manner of mobilizing the focused protein zones for detection. We therefore discuss and compare the three most widely used CIEF techniques – using chemical, pressure, or electroosmotic flow (EOF) mobilization – with respect to linearity of the calibration curve, resolution, and reproducibility.
Chiral separation in capillary electrophoresis
Authors: Wren Stephen A. C.
Published: May 2005

A mathematical model which explains separation in chiral capillary electrophoresis is presented. The model is analysed to give a more general solution than previous work and is supported by further experimental work.
Electroosmotic control of chiral separation in capillary zone electrophoresis
Authors: Hong Shencai , Lee Cheng S.
Published: May 2005

The resolution in capillary zone electrophoresis (CZE), with the assumption of diffusion control only, is strongly dependent on the direction and magnitude of electroosmotic flow. In fact, excellent separation resolution will be obtained if the electroosmotic flow is in the opposite direction of the electrophoretic migration. By applying various radial electric potential gradients across the capillary wall, the direct control of the ζ potential and the electroosmotic flow results in a great enhancement of chiral resolution in cyclodextrin‐modified CZE. All 12 basic chiral drugs, including (+/−)‐ephedrine, (+/−)‐pseudoephe‐drine, (+/−)‐norephedrine, (+/−)‐epinephrine, (+/−)‐norepinephrine, and (+/−)‐isoproterenol, are separated and resolved within 22 min of the analysis time. There is no additional band broadening and dispersion introduced by the direct control of electroosmosis with the application of various radial electric potential gradients.
On the measurements of electrophoretic mobilities by means of capillary isotachophoresis at a constant voltage
Authors: Ermakov Sergey V. , Zhukov Michael Y. , Capelli Laura , Righetti Pier Giorgio
Published: May 2005

A method for measuring electrophoretic mobilities by means of isotachophoresis (ITP) at a constant voltage as described by H. Carchon and E. Eggermont (Electrophoresis, 1982, 3, 263–274) is analyzed. An error made in this work, disregarding the pH shift arising at the initial discontinuity on the leader‐terminator boundary, has been corrected. This method has been carefully studied and generalized for the presence of constant electroosmotic flow in a capillary. The limits of its applicability and the diffusionless ITP theory in general are discussed. A detailed study of the evolution of initial discontinuity (stationary boundary) showed some anomalies not reported previously, particularly nonmonotonic concentration profiles in the vicinity of stationary boundaries. Moreover, in some cases, diffusion effects and the contribution of H+ ions can also strongly influence the behavior of moving boundaries. Computer modelling (confirmed by experimental data) showed that these effects could lead to the decay of the ITP train, despite the fact that the steady state diffusionless ITP theory predicts its stability.