bluuebunny/crossref_metadata_2025 huggingface dataset:
Early development of children at familial risk for Dyslexia—follow‐up from birth to school age
Authors: Lyytinen H. , Ahonen T. , Eklund K. , Guttorm T. , Kulju P. , Laakso M.‐L. , Leiwo M. , Leppänen P. , Lyytinen P. , Poikkeus A.‐M. , Richardson U. , Torppa M. , Viholainen H.
Published: July 2004

We review the main findings of the Jyväskylä of Dyslexia (JLD) which follows the development of children at familial risk for dyslexia (N = 107) and their controls (N = 93). We will illustrate the development of these two groups of children at ages from birth to school entry in the skill domains that have been connected to reading and reading disability in the prior literature. At school entry, the highest score on the decoding task among the poorer half (median) of the at risk children—i.e. of those presumably being most likely genetically affected—is 1 SD below the mean of the control group. Thus, the familial risk for dyslexia shows expected consequences. Among the earliest measures in which group differences as well as significant predictive associations with the first steps in reading have emerged, are indices of speech processing in infancy. Likewise, various measures of early language including pronunciation accuracy, phonological, and morphological skills (but not performance IQ) show both group differences and predictive correlations, the majority of which become stronger as the reliability of the measures increases by age. Predictive relationships tend to be strong in general but higher in the at risk group because of its larger variance in both the predictor variables and in the dependent measures, such as early acquisition of reading. The results are thus promising in increasing our understanding needed for early identification and prevention of dyslexia. Copyright © 2004 John Wiley & Sons, Ltd.
Antibody‐mediated activation of the classical pathway of complement may compensate for mannose‐binding lectin deficiency
Authors: Roos Anja , Garred Peter , Wildenberg Manon E. , Lynch Nicholas J. , Munoz Jeric R. , Zuiverloon Tahlita C.M. , Bouwman Lee H. , Schlagwein Nicole , van den Houten Francien C. Fallaux , Faber‐Krol Maria C. , Madsen Hans O. , Schwaeble Wilhelm J. , Matsushita Misao , Fujita Teizo , Daha Mohamed R.
Published: July 2004

Deficiency of mannose‐binding lectin (MBL), a recognition molecule of the lectin pathway of complement, is associated with increased susceptibility to infections. The high frequency of MBL deficiency suggests that defective MBL‐mediated innate immunity can be compensated by alternative defense strategies. To examine this hypothesis, complement activation by MBL‐binding ligands was studied. The results show that the prototypic MBL ligand mannan can induce complement activation via both the lectin pathway and the classical pathway. Furthermore, antibody binding to mannan restored complement activation in MBL‐deficient serum in a C1q‐dependent manner. Cooperation between the classical pathway and the lectin pathway was also observed for complement activation by protein 60 from Listeria monocytogenes. MBL pathway analysis at the levels of C4 and C5b–9 in the presence of classical pathway inhibition revealed a large variation of MBL pathway activity, depending on mbl2 gene polymorphisms. MBL pathway dysfunction in variant allele carriers is associated with reduced MBL ligand binding and a relative increase of low‐molecular‐mass MBL. These findings indicate that antibody‐mediated classical pathway activation can compensate for impaired target opsonization via the MBL pathway in MBL‐deficient individuals, and imply that MBL deficiency may become clinically relevant in absence of a concomitant adaptive immune response.
Peritoneal B cells govern the outcome of diabetes in non‐obese diabetic mice
Authors: Kendall Peggy L. , Woodward Emily J. , Hulbert Chrys , Thomas James W.
Published: July 2004

Type 1 diabetes mellitus (T1DM) results from autoimmune destruction of insulin‐producing beta cells in the pancreatic islets. Although T1DM is mediated by T lymphocytes, B lymphocytes are essential for insulitis and disease progression in the non‐obese diabetic mouse model. We find that B cells invading the pancreas phenotypically resemble B1a B cells in the peritoneal cavity, including the presence of CD5+. To investigate the link between the peritoneal cavity and lymphocytes invading the pancreas, we used intraperitoneal hypotonic lysis to target these cells. B1a cells were eliminated from the peritoneal compartment by this treatment and were quickly replaced by B2 cells. Both B1a and B2 B cells were concordantly redistributed away from insulitis lesions, while pancreatic T cells showed little change. As a consequence of these events, the onset of diabetes was significantly delayed. These findings indicate that simple perturbations of the B cell‐enriched peritoneal compartment can affect the disease process in the pancreas even after islet invasion has begun.
Partial activation precedes apoptotic death in T cells harboring an IAN gene mutation
Authors: Lang Julie A. , Kominski Douglas , Bellgrau Donald , Scheinman Robert I.
Published: July 2004

The Biobreeding diabetes‐prone rat suffers from a profound peripheral lymphopenia and yet succumbs to a T cell‐dependent autoimmune disease. Lymphopenia segregates with a mutated chromosomal locus, termed lyp, recently identified as a frameshift mutation in IAN4. Others have correlated loss of IAN4 function with decreased mitochondrial integrity resulting in T cell apoptosis. Here we report that IAN4–/– T cells enter a state similar to that of partial activation wherein they down‐regulate CD62L and undergo incomplete blasting yet do not progress through mitosis. When given a strong stimulus, this partial activation phenotype can be overcome. This phenotype can be recapitulated in wild‐type T cells through suboptimal stimulation. The phenotype is not simply a reaction to the lymphopenic environment, as spontaneous CD62L down‐regulation occurs in mature single‐positive medullary thymocytes that develop within a non‐lymphopenic environment, and normal T cells do not undergo similar blasting when parked in a lymphopenic environment. Finally, we show that IAN4–/– T cells are more readily triggered via TCR stimulation. Thus, in addition to their role in apoptosis, IAN family members may also play a role in regulating the T cell activation state through modulation of TCR signaling strength.
Tc2 cells respond to soluble antigen in the respiratory tract and induce lung eosinophilia and bronchial hyperresponsiveness
Authors: Sawicka Elzbieta , Noble Alistair , Walker Christoph , Kemeny David M.
Published: July 2004

The key role of T helper 2 (Th2) cells in asthma is well established. In contrast, the function of CD8+ T cells producing a distinct cytokine profile similar to Th2 cells is largely unknown. To analyze a potential role of CD8+ T cell subsets, allergen‐specific, in vitro‐differentiated T cytotoxic (Tc1 or Tc2) cells from T cell receptor transgenic OT‐I mice were adoptively transferred into naive C57BL/6 mice. Subsequent allergen challenge of mice injected with Tc1 cells (producing IFN‐γ but no IL‐4) resulted in a neutrophilic airway inflammation without induction of bronchial hyperresponsiveness (BHR). In contrast, the inflammatory response of recipients of adoptively transferred Tc2 cells (producing high levels of IL‐4 but little IFN‐γ) was characterized by significantly increased numbers of eosinophils and induction of BHR to methacholine. The response of antigen‐specific CD8+ T cells to soluble antigen was also observed in an in vitro system. A low concentration of antigen was shown to favor the generation of Tc2 cells, whereas high antigen load resulted in the differentiation of Tc1 cells. Thus, allergen‐specific Tc2 cells respond to inhaled soluble antigen, produce an inflammatory response qualitatively similar to Th2 cells and therefore may exacerbate the Th2‐driven airway inflammation in asthma.
Complement activation by necrotic cells in normal plasma environment compares to that by late apoptotic cells and involves predominantly IgM
Authors: Ciurana Caroline L. F. , Zwart Bas , van Mierlo Gerard , Hack C. Erik
Published: July 2004

Necrotic cells are generally considered to stimulate inflammation, whereas apoptotic cells should not. However, apoptotic cells have pro‐inflammatory properties since they can activate complement. To what extent this activation compares to that by necrotic cells is not known. We compared complement activation by necrotic cells and apoptotic cells in plasma. Jurkat cells were made apoptotic or necrotic by incubation with etoposide or by heat shock, respectively. Cells incubated in recalcified plasma were tested for C3 and C4 fixation and fluid phase generation of complement activation products. Fixation of C3 and C4 to necrotic cells occurred mainly via the classical pathway, independent from the method of necrosis induction and the cell type. Depletion of IgM from plasma almost completely abrogated complement fixation by necrotic cells, which was restored by supplementation with purified IgM. Complement activation by late apoptotic cells was comparable to that by necrotic cells regarding the extent and dependence on IgM. Moreover, incubation of plasma with necrotic or late apoptotic cells led to the generation of comparable amounts of complement activation products. These results indicate that late apoptotic and necrotic cells employ similar complement activation mechanisms in the plasma environment.
Self‐Assembly of Stereoselective, Mononuclear, Triple‐Stranded Helicate Complexes ML3 with Trivalent Scandium and Lanthanide Metal Ions − Quantitative Transmetallation of the Lanthanide(III) Ion by Sodium(I) Ions
Authors: Capó Magdalena , Gonzalez José , Adams Harry
Published: July 2004

Mononuclear triple‐stranded helicate complexes ML3 (M = Sc3+, La3+, Sm3+, Yb3+) were self‐assembled and structurally characterized by NMR spectroscopy and high‐resolution mass spectrometry. The solid‐state structure of the ytterbium complex was determined by X‐ray crystallography. The core lanthanide metal ion was replaced quantitatively by three sodium ions. Homo‐ and heterotrinuclear complexes containing the alkali metal ions Li+ and Na+ were obtained from the mononuclear lanthanide complexes. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Chemical Synthesis of GPIs and GPI‐Anchored Glycopeptides
Authors: Guo Zhongwu , Bishop Lee
Published: July 2004

This microreview summarizes some recent progress in the study of glycosylphosphatidylinositols (GPI), focusing on the strategies established for the synthesis of optically pure myo‐inositol derivatives and complex GPIs and GPI conjugates. Although in the past decade many GPIs have been achieved by linear, convergent and solid‐phase synthesis, the GPIs with 2‐O‐acylated inositol residues and the GPI conjugates of peptides and glycopeptides have not been accomplished until recently. The successful coupling of peptides and glycopeptides to GPIs brings new promises to this area, but the scope of the relevant designs, in particular their application to more complex structures such as GPI‐anchored glycoproteins, remains untested. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Stereo‐ and Regiocontrol in the Formation of Lactams by Rhodium‐Carbenoid C−H Insertion of α‐Diazoacetamides
Authors: Gois Pedro M. P. , Afonso Carlos A. M.
Published: July 2004

The dirhodium(II)‐catalysed intramolecular C−H insertion reaction of α‐diazoacetamides is a powerful methodology for the preparation of highly valuable heterocyclic compounds such as lactams. In this Microreview we present lactam formation by intramolecular C−H insertion of α‐diazoacetamides, focusing on the effects that have a profound impact on the reaction pathway. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Catalytic Effect on Silver Electrodeposition of Gold Deposited on Carbon Electrodes
Authors: de la Escosura‐Muñiz Alfredo , Begoña González‐García María , Costa‐García Agustín
Published: July 2004

A new methodology, based on silver electrocatalytic deposition and designed to quantify gold deposited onto carbon paste electrode (CPE) and glassy carbon electrode (GCE), has been developed in this work. Silver (prepared in 1.0 M NH3) electrodeposition at −0.13 V occurs only when gold is previously deposited at an adequate potential on the electrode surface for a fixed period of time. When a CPE is used as working electrode, an adequate oxidation of gold is necessary. This oxidation is carried out in both 0.1 M NaOH and 0.1 M H2SO4 at oxidation potentials. When a GCE is used as working electrode, the oxidation steps are not necessary. Moreover, a cleaning step in KCN, which removes gold from electrode surface, is included. To obtain reproducibility in the analytical signal, the surface of the electrodes must be suitably pretreated; this electrodic pretreatment depends on the kind of electrode used as working electrode. Low detection limits (5.0×10−10 M) for short gold deposition times (10 min for CPE and 5 min for GCE) were achieved with this novel methodology. Finally, sodium aurothiomalate can be quantified using silver electrocatalytic deposition and GCE as working electrode. Good linear relationship between silver anodic stripping peak and aurothiomalate concentration was found from 5.0×10−10 M to 1.0×10−8 M.
A New Nitric Oxide Gas Sensor Based on Reticulated Vitreous Carbon/Nafion and Its Applications
Authors: Sun Jie , Hauser Peter C. , Zhelyaskov Valentine , Lin Jie , Broderick Mark , Fein Harry , Zhang Xueji
Published: July 2004

Reticulated vitreous carbon (RVC), and Nafion membrane are used to fabricate a composition electrode to measure nitric oxide (NO) concentration amperometrically in the gas phase. Limit of detection was found to be 6 ppb at an applied voltage of 0.66 V (vs. mercury sulfate reference electrode) with average response time of less than 30 seconds. The response of the sensor was linearly dependent on the concentration over the whole tested range from 19 ppb‐50 ppm of NO. Simplicity in electrode fabrication and consistent performance between individual sensors make RVC and Nafion attractive materials for detecting very low levels of nitric oxide gas in routine analysis.
Bioelectrocatalysis of Oxygen Reduction Reaction by Laccase on Gold Electrodes
Authors: Gupta Gautam , Rajendran Vijaykumar , Atanassov Plamen
Published: July 2004

Direct electron transfer (DET) reaction of oxygen electroreduction catalyzed by enzyme laccase on monolayer modified gold electrodes was studied. Three different monolayers were investigated, from which 4‐aminothiophenol was found to be optimal for the direct electron transfer to take place. The electrocatalytic reduction of the oxygen at the electrode surface was found to depend significantly on the method of immobilization. Fungal laccase from Coriolus hirsitus modified with sodium‐periodate demonstrated more anodic onset potential for oxygen reduction than the tree laccase from Rhus vernicifera. Physical immobilization of enzyme did not result in any manifestation of bioelectrocatalytic activity. A maximum anodic shift in reduction potential of 300 mV was observed for fungal laccase covalently coupled on the electrode surface.
Catalytic Oxidation of Ascorbic Acid on 2D and 3D Monolayers of 4‐Hydroxythiophenol
Authors: Stolarczyk Krzysztof , Bilewicz Renata
Published: July 2004

Monolayers of 4‐hydroxythiophenol (4‐HTP) immobilized on gold electrode (2D SAMs) or gold nanoclusters (3D SAMs) lead to catalytic oxidation of ascorbic acid (AA). The catalytic role of the modified clusters was revealed by comparing the electrodes covered by 1,9‐nonanedithiol and the same nonanedithiol monolayer decorated by 4‐HTP protected clusters. The 4‐HTP protected gold nanoclusters supported on a metal surface using a monolayer of 1,9‐nonanedithiol as the bridge, transferred charge to ascorbic acid (AA) molecule in the solution more efficiently than when the same 4‐HTP monolayer was formed directly on the gold electrode.
Two‐step elution of human serum proteins from different glass‐modified bioactive surfaces: A comparative proteomic analysis of adsorption patterns
Authors: Magnani Agnese , Barbucci Rolando , Lamponi Stefania , Chiumento Antonio , Paffetti Alessandro , Trabalzini Lorenza , Martelli Paola , Santucci Annalisa
Published: July 2004

Plasma protein adsorption patterns on surfaces may give vital information to evaluate biocompatibility of biomaterials designed for direct blood‐contacting applications or tissue integration. Adsorption of human serum proteins on four different types of biomaterials (glass, aminosilanized glass, hyaluronan and sulfated hyaluronan) was analyzed by two‐dimensional electrophoresis. Desorption of proteins from the surfaces was first classically achieved by sodium dodecyl sulfate (SDS) elution. We introduced a second elution step (by use of isoelectric focusing (IEF) sample buffer consisting of urea, 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propansulfonate, and dithioerythritol) which allows more stringent elution conditions and is a tool to evaluate the protein adsorption strength to biomaterials. Moreover, the two‐step elution may discriminate between irreversible and reversible adsorption of plasma proteins for biomaterials, thus helping to elucidate the structure of protein multilayers which form a complex system at the surfaces. The IEF sample buffer proved not to alter the biomaterial structure and integrity. Hydrophobic bonds resulted to be the main strength driving protein adsorption onto our biomaterials. Apolipoproteins were the most important proteins interacting with the surfaces suggesting that high‐density lipoprotein (HDL) particles could play a role in biocompatibility due to their beneficial effects on endothelial cells.
Capillary electrophoresis analysis of fosfomycin in biological fluids for clinical pharmacokinetic studies
Authors: Petsch Martina , Mayer‐Helm Bernhard X. , Sauermann Robert , Joukhadar Christian , Kenndler Ernst
Published: July 2004

A feasible capillary zone electrophoresis (CZE) method with indirect UV and contactless conductivity detection was developed for the determination of fosfomycin, an antibiotic, in human plasma and microdialysis samples. Samples were collected from test persons during a clinical trial. The background electrolytes used consisted of 25 mM benzoic acid and 0.5 mM hexadecyltrimethylammonium bromide, adjusted with tris(hydroxymethyl)aminomethane solution to pH 6.95 for plasma, and to pH 8.05 for microdialysis samples. CZE separations of the anionic analyte were carried out with reversed electroosmotic flow directed towards the anode. The limit of detection was between 0.6 and 2 μg/mL, depending on the matrix and the detection method. No sample preparation was needed for microdialysis samples; for plasma samples, proteins were precipitated with methanol (1+2, v+v), and the supernatant was analyzed. The yield determined with spiked samples was about 100%, the reproducibility of the entire method, expressed by the RSD% of three independent determinations of fosfomycin in triplicate after spiking Ringer's solutions and plasma samples, respectively, was better than 8%. The method is thus well‐suited for clinical studies for the determination of the antibiotic in biological fluids.
Capillary electrophoresis of biological particles: Viruses, bacteria, and eukaryotic cells
Authors: Kremser Leopold , Blaas Dieter , Kenndler Ernst
Published: July 2004

A review about the application of electrophoretic methods in the capillary format for the investigation of large biological assemblies like viruses, bacteria, yeast or entire mammalian cells is given. These entities are of a size ranging between some nanometers and tens of micrometers. They can form colloidal solutions or dispersions and move under the influence of an electric field. They are separated by zone electrophoresis according to their different electrophoretic velocity, and characterized by the electrophoretic mobility, which is easily determinable in free solution in capillaries or in other microdevices. As the charge of these particles, when being amphoteric, is pH‐dependent, isoelectric focusing can also be carried out and the capillary format is increasingly being employed for their separation and determination of pI values. Furthermore, interactions with ligands can be assessed by various modes of affinity capillary electrophoresis. Capillary zone electrophoresis has thus become a valuable tool for investigation of large macromolecular assemblies in the field of biochemistry, clinical chemistry, toxicology, and nutrition chemistry amongst many others.
Integrated on‐chip derivatization and electrophoresis for the rapid analysis of biogenic amines
Authors: Beard Nigel P. , Edel Joshua B. , deMello Andrew J.
Published: July 2004

We demonstrate the monolithic integration of a chemical reactor with a capillary electrophoresis device for the rapid and sensitive analysis of biogenic amines. Fluorescein isothiocyanate (FITC) is widely employed for the analysis of amino‐group containing analytes. However, the slow reaction kinetics hinders the use of this dye for on‐chip labeling applications. Other alternatives are available such as o‐phthaldehyde (OPA), however, the inferior photophysical properties and the UV λmax present difficulties when using common excitation sources leading to a disparity in sensitivity. Consequently, we present for the first time the use of dichlorotriazine fluorescein (DTAF) as a superior in situ derivatizing agent for biogenic amines in microfluidic devices. The developed microdevice employs both hydrodynamic and electroosmotic flow, facilitating the creation of a polymeric microchip to perform both precolumn derivatization and electrophoretic analysis. The favorable photophysical properties of the DTAF and its fast reaction kinetics provide detection limits down to 1 nM and total analysis times (including on‐chip mixing and reaction) of <60 s. The detection limits are two orders of magnitude lower than current limits obtained with both FITC and OPA. The optimized microdevice is also employed to probe biogenic amines in real samples.
Capillary electrochromatography of peptides and proteins
Authors: Li Yan , Xiang Rong , Wilkins James A. , Horváth Csaba
Published: July 2004

This paper reviews recent progress in bioanalysis using capillary electrochromatography (CEC), especially in the field of separation of proteins and peptides. Fundamentals of CEC are briefly discussed. Since most of the recent developments on CEC have focused on column technology, i.e., design of new stationary phases and development of new column configurations, we describe here a variety of column architectures along with their advantages and disadvantages. Newly emerged column technologies in CEC for high speed and high efficiency separation are also discussed. Different analytical platforms of CEC such as pressure‐assisted CEC or voltage‐assisted micro‐ high‐performance liquid chromatography (HPLC), CEC with different detection techniques, CEC on microchip platforms and multidimensional electrochromatography with their applications in peptide and protein analysis are presented.
Which genetic marker for which conservation genetics issue?
Authors: Wan Qiu‐Hong , Wu Hua , Fujihara Tsutomu , Fang Sheng‐Guo
Published: July 2004

Conservation genetics focuses on the effects of contemporary genetic structuring on long‐term survival of a species. It helps wildlife managers protect biodiversity by identifying a series of conservation units, which include species, evolutionarily significant units (ESUs), management units (MUs), action units (AUs), and family nets (FNs). Although mitochondrial DNA (mtDNA) evolves 5–10 times faster than single‐copy nuclear DNA (scnDNA), it records few traces of contemporary events. Thus, mtDNA can be used to resolve taxonomic uncertainties and ESUs. Variable number of tandem repeats (VNTRs) evolve 100–1000 times faster than scnDNA and provide a powerful tool for analyzing recent and contemporary events. VNTR analysis techniques include polymerase chain reaction (PCR)‐based microsatellite assays and oligonucleotide probing. Size homoplasy problems in PCR‐based microsatellite assays can strongly affect the inference of recent population history. The high homozygosity in endangered species is reflected in a relatively low number and level of variability in microsatellite loci. This combined with “allelic dropout” and “misprinting” errors contributes to the generation of highly biased genetic data following analyses of natural populations. Thus, in conservation genetics, microsatellites are of limited use for identifying ESUs, MUs, and AUs. In contrast to PCR‐based microsatellite analysis, oligonucleotide probing avoids errors resulting from PCR amplification. It is particularly suitable for inferring recent population history and contemporary gene flow between fragmented subpopulations. Oligonucleotide fingerprinting generates individual‐specific DNA banding patterns and thus provides a highly precise tool for monitoring demography of natural populations. Hence, DNA fingerprinting is powerful for distinguishing ESUs, MUs, AUs, and FNs. The use of oligonucleotide fingerprinting and fecal DNA is opening new areas for conservation genetics.
Sequencing of oligosaccharides derivatized with benzylamine using electrospray ionization‐quadrupole time of flight‐tandem mass spectrometry
Authors: Morelle Willy , Michalski Jean‐Claude
Published: July 2004

Oligosaccharides were derivatized by reductive amination using benzylamine and analyzed by nanoelectrospray ionization‐quadrupole time of flight‐tandem mass spectrometry (nanoESI‐QTOF‐MS/MS) in the positive ion mode. The major signals were obtained under these conditions from the [M+H]+ions for all benzylamine‐derivatized oligosaccharides. To obtain structural information from these derivatized oligosaccharides, MS/MS was applied. Protonated molecular ions underwent extensive fragmentation, even under low‐energy collision‐induced dissociation. MS/MS spectra of [M+H]+ions are characterized by simple fragmentation patterns which result from cleavage of the glycosidic bonds and thus allow a straightforward interpretation. Fragmentation of the [M+H]+ions gave predominantly B‐ and Y‐type glycosidic fragments. A systematic study of various oligosaccharides showed that information on sugar sequence and branching could easily be obtained. Predictable and reproducible fragmentation patterns could be obtained in all cases. This derivatization procedure and mass spectrometric methodology were applied successfully to neutral and acidic glycans released from 10 μg of glycoproteins separated by gel electrophoresis. Moreover, the derivatives retain their sensitivity to exoglycosidases. Thus a series of sequential on‐target exoglycosidase treatments combined with matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) was found to be useful for the determination of structural features of the glycans released from proteins separated by gel electrophoresis such as the monosaccharide sequence, branching pattern, and anomeric configurations of the corresponding glycosidic linkages. Our strategy can be used successfully to assign the major glycans released from proteins separated by gel electrophoresis.
Aberrant expression of acute‐phase reactant proteins in sera and breast lesions of patients with malignant and benign breast tumors
Authors: Doustjalali Saeid R. , Yusof Rohana , Yip Cheng‐Har , Looi Lai‐Meng , Pillay Bhanumathy , Hashim Onn H.
Published: July 2004

We have analyzed unfractionated sera of newly diagnosed patients (n = 10) with breast carcinoma (BC), prior to treatment, and patients (n = 5) with fibrocystic disease of the breast (FDB) by two‐dimensional gel electrophoresis (2‐DE) and silver staining. The patients' 2‐DE serum protein profiles obtained were then subjected to image analysis and compared to similar data generated from sera of normal healthy female controls (n = 10) of the same range of age. The relative expression of α1‐antichymotrypsin (ACT), clusterin, and complement factor B was significantly higher in all BC patients as compared to normal controls. However, the expression of α1‐antitrypsin (AAT) in BC patients was apparently lower than that of the controls. Similar differential expression of ACT was detected in the FDB patients. The aberrant expression of the serum acute‐phase proteins of patients with BC and FDB was confirmed by competitive enzyme‐linked immunosorbent assay (ELISA). Similar altered proteins expression was also observed from immunohistochemical studies of malignant (n = 5) and benign (n = 5) breast lesions of the respective patients performed using antisera to the aberrantly expressed proteins. However, the malignant breast lesions were instead positively stained for AAT. The differential expression of the serum proteins was apparently abrogated when a six‐month follow‐up study was performed on nine of the BC patients subsequent to treatment.
Capillary electrophoresis sequencing of small ssDNA molecules versus the Ogston regime: Fitting data and interpreting parameters
Authors: Kopecka Katerina , Drouin Guy , Slater Gary W.
Published: July 2004

We study the mobility of short ssDNA fragments (approximately 30–500 bases) separated by capillary electrophoresis in entangled polymer solutions. Although this corresponds to what is commonly called the Ogston regime, the corresponding sieving concept has never been defined properly nor tested quantitatively. We consider three formulas that have been suggested to fit data in this range of ssDNA sizes, and we discuss how their free parameters are related to actual physical parameters. We test these formulas with new data obtained in our laboratory using a commercial poly‐N,N‐dimethylacrylamide sieving matrix. Our results show that all three formulas provide decent fits. However, the traditional Ogston equation produces fitting parameters that appear to lack physical meaning. Surprisingly, all three approaches predict that the effective pore size and fiber radius are almost equal. This is the first step towards the development of a systematic approach to optimizing sequencing systems for this size range.
Gene expression analysis of an integrin family of genes by systematic multiplex reverse transcription‐polymerase chain reaction
Authors: Yamamoto Miyako , Yamamoto Ami , Leung Patricia C. , Yamamoto Fumiichiro
Published: July 2004

We have established the systematic multiplex reverse transcription‐polymerase chain reaction (SM RT‐PCR) system of 26 members of the integrin family of genes, and used this system to investigate their expression in 25 different kinds of human adult tissues. A hierarchical clustering analysis of the tissue expression data illustrated clustering of functionally related tissues. Although the correlation was weak, bone marrow, thymus, and spleen were clustered, potentially confirming the role of certain integrin molecules in the immune response. We also investigated changes in integrin gene expression in cancer. Several differences were observed between normal and tumor, as well as between normal and cancer cell lines. The SM RT‐PCR experiments for the study of alternative splicing showed that most of the integrin genes analyzed exhibited fixed ratios of differentially spliced transcripts probably due to a tissue‐independent mechanism of splicing predetermined by the nucleotide sequences around the splicing donor and acceptor sites.
Evaluation and validation of the ABI 3700, ABI 3100, and the MegaBACE 1000 capillary array electrophoresis instruments for use with short tandem repeat microsatellite typing in a forensic environment
Authors: Koumi Pieris , Green Helen E. , Hartley Susan , Jordan Darren , Lahec Sharon , Livett Richard J. , Tsang Kam W. , Ward David M.
Published: July 2004

The demand for high‐throughput DNA profiling has increased with the introduction of national DNA databases and has led to the development of automated methods of short tandem repeat (STR) profile production; however, a potential bottleneck still exists at the gel electrophoresis stage. Capillary electrophoresis sequencers capable of processing 96 samples with considerably reduced manual intervention are now available. In this paper, we compare the ABI Prism 377 slab‐gel sequencer currently used by the Forensic Science Service with three leading capillary array electrophoresis instruments: the ABI Prism 3700, the Amersham MegaBACE 1000 and the 16‐capillary ABI Prism 3100. We describe the experiments used to evaluate and validate these platforms for forensic use with the STR multiplex Ampf/STR SGMplus [1, 2], along with comparative data from the ABI Prism 377 sequencer.
Polymer solutions and entropic‐based systems for double‐stranded DNA capillary electrophoresis and microchip electrophoresis
Authors: Xu Feng , Baba Yoshinobu
Published: July 2004

We give an overview of recent development of low‐viscosity polymer solutions and entropic trapping networks for double‐stranded DNA (dsDNA) separations by conventional capillary electrophoresis and microchip electrophoresis. Theoretical models for describing separation mechanisms, commonly used noncross‐linked polymer solutions, thermoresponsive (viscosity‐adjustable) polymer solutions, and novel entropic trapping networks are included. The thermoresponsive polymer solutions can be loaded at one temperature into microchannels at lower viscosities, and used in separation at another temperature at entanglement threshold concentrations and higher viscosities. The entropic‐based separations use only arrays of regular obstacles acting as size‐separations and do not need viscous polymer solutions. These progresses have potential in integration to automated capillary and microfluidic chip systems, enabling better reusability of separation microchannels, much shorter DNA separation times, and higher reproducibility due to less matrix degradation.
Separation and investigation of structure‐mobility relationships of insect oostatic peptides by capillary zone electrophoresis
Authors: Šolínová Veronika , Kašička Václav , Koval Dušan , Hlaváček Jan
Published: July 2004

Capillary zone electrophoresis (CZE) has been applied to qualitative analysis, separation, and physicochemical characterization of synthetic insect oostatic peptides (IOPs) and their derivatives and fragments. Series of homologous IOPs were separated in three acidic background electrolytes (BGEs; pH 2.25, 2.30, 2.40) and an alkaline BGE (pH 8.1). Best separation was achieved in acid BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The effective electrophoretic mobilities, μep, of all IOPs in four BGEs were determined and several semiempirical models correlating effective mobility with charge‐to‐size ratio (μep versus q/Mrk) were tested to describe the migration behavior of IOP in CZE. None of models was found to be unambiguously applicable for the whole set of 20 IOPs differing in size (dipeptide – decapeptide) and charge (−2 to +0.77 elementary charges). However, a high coefficient of correlation, 0.9993, was found for the subset of homologous series of IOPs with decreasing number of proline residues at C‐terminus, H‐Tyr‐Asp‐Pro‐Ala‐Prox‐OH, x = 6 – 0, for the dependence of μep on q/Mrk with k = 0.5 for IOPs as anions in alkaline BGE and with k = 2/3 for IOPs as cations in optimized acidic Tris‐phosphate BGE. From these dependences the probable structure of IOPs in solution could be predicted.
Refining the results of a whole‐genome screen based on 4666 microsatellite markers for defining predisposition factors for multiple sclerosis
Authors: Gödde René , Nigmatova Viktoria , Jagiello Peter , Sindern Eckhart , Haupts Michael , Schimrigk Sebastian , Epplen Jörg T.
Published: July 2004

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system with a complex genetic background. In order to identify loci associated with the disease, we had performed a genome screen initially using 6000 microsatellite markers in pooled DNA samples of 198 MS patients and 198 controls. Here, we report on the detailed reanalysis of this set of data. Distinctive features of microsatellites genotyped in pooled DNA causing false‐positive association or masking existing association were met by improved evaluation and refined correction factors in the statistical analyses. In order to assess potential errors introduced by DNA pooling and genotyping, we resurveyed the experiment in a subset of microsatellite markers using de novo‐composed DNA pools. True MS associations of markers were verified via genotyping all individual DNA samples comprised in the pools. Microsatellites share characteristically superb information content but they do not lend themselves to automation in very large scale formats. Especially after DNA pooling many artifacts of individual marker systems require special attention and treatment. Therefore, in the near future comprehensive whole‐genome screens may rather be performed by typing single nucleotide polymorphisms on chip‐based platforms.
Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser‐induced fluorescence detection
Authors: Cunliffe Jennifer M. , Liu Zhen , Pawliszyn Janusz , Kennedy Robert T.
Published: July 2004

Affinity probe capillary isoelectric focusing (CIEF) with laser‐induced fluorescence was explored for detection of Ras‐like G proteins. In the assay, a fluorescent BODIPY® FL GTP analogue (BGTPγS) and G protein were incubated resulting in formation of BGTPγS‐G protein complex. Excess BGTPγS was separated from BGTPγS‐G protein complex by CIEF using a 3–10 pH gradient and detected in whole‐column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5–5 pH gradient. In this case, analyte peaks passed the detector in ∼ 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPγS‐Ras and BGTPγS‐Rab3A complexes. Formation of specific complexes was confirmed by adding GTPγS to samples containing BGTPγS‐G protein. GTPγS competed with BGTPγS for G protein binding sites resulting in decreased BGTPγS‐G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.
Comprehensive two‐dimensional chromatography and capillary electrophoresis coupled with tandem time‐of‐flight mass spectrometry for high‐speed proteome analysis
Authors: Zhang Jie , Hu Hualing , Gao Mingxia , Yang Pengyuan , Zhang Xiangmin
Published: July 2004

A comprehensive two‐dimensional capillary liquid chromatography and capillary zone electrophoresis system coupled with tandem matrix assisted laser desorption/ionization‐time of flight‐time of flight‐mass spectrometry (MALDI‐TOF‐TOF‐MS) proteomics analyzer is presented. Protein/peptide samples were separated by capillary high‐performance liquid chromatography (cHPLC). The effluents from cHPLC (the first dimension) were continuously transferred into capillary zone electrophoresis (CZE, the second dimension) through a novel valve‐free hydrodynamic sampling interface. The CZE effluents were mixed with α‐cyano‐4‐hydroxycinnamic acid (CHCA) matrix sheath flow via CE‐MALDI interface, and then directly deposited on the MALDI target at a 3 s time‐interval for further MS analysis. The high efficiency of the overall system was demonstrated by analysis of proteins in D20 (human hepatocellular carcinoma model in nude mice with high metastatic potential) liver cancer tissue. More than 300 proteins were identified, which proved the system potential for high‐throughput analysis and application in proteomics.
Separation of double‐stranded DNA fragments by capillary electrophoresis: Impacts of poly(ethylene oxide), gold nanoparticles, ethidium bromide, and pH
Authors: Chiou Shin‐Huei , Huang Ming‐Feng , Chang Huan‐Tsung
Published: July 2004

The separation of DNA by capillary electrophoresis using poly(ethylene oxide) (PEO) containing gold nanoparticles (GNPs) is presented. The impacts of PEO, GNPs, ethidium bromide (EtBr), and pH on the separation of double‐stranded DNA have been carefully explored. Using a capillary dynamically coated with 5.0% poly(vinylpyrrolidone) and filled with 0.2% PEO containing 0.3×GNPs (the viscosity less than 15 cP), we have demonstrated the separation of DNA markers V and VI within 5 min at pH 8.0 and 9.0. In terms of resolution and reproducibility, GNPs have a greater impact on the separation of DNA at pH 9.0. Resolution improvements for large DNA fragments (> 300 base pairs, bp) are greater than those for small ones in the presence of GNPs. It is important to point out that reproducibility is excellent (relative standard deviations for the migration times less than 0.5%) and thus no further dynamic coating is required in at least 20 consecutive runs in the presence of GNPs. Using 0.2% PEO (pH 9.0) containing 0.3×GNPs, the separation of DNA fragments ranging in size from 21 to 23130 bp was accomplished in 7 min. The results presented in this study show the advantage of PEO containing GNPs for DNA separation, including rapidity, high resolving power, excellent reproducibility, and ease of filling capillaries.
Matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry of lipopolysaccharide species separated by slab‐polyacrylamide gel electrophoresis: High‐resolution separation and molecular weight determination of lipooligosaccharides from Vibrio fischeri strain HMK
Authors: Pupo Elder , Phillips Nancy J. , Gibson Bradford W. , Apicella Michael A. , Hardy Eugenio
Published: July 2004

We recently demonstrated that the combined use of lipopolysaccharide (LPS) reverse staining and high‐efficiency passive elution techniques can be successfully used as a suitable interface between LPS slab‐gel separation and electrospray ionization‐mass spectrometry (ESI‐MS) of LPS‐derived oligosaccharides. Here, we extend our micropurification strategy for the analysis of O‐deacylated LPS forms from Vibrio fischeri HMK after recovery from single reverse‐stained LPS bands using matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS). The quantities (30–40 μg) obtained from the two gel‐resolved LPS bands were sufficient to allow MALDI‐TOF‐MS detection of O‐deacylated LPS glycoforms at m/z 3767.1, 3890.1 for the high‐molecular‐weight or at m/z 2522.5, 2645.4, 2725.7, and 2848.7 for the low‐molecular‐weight LPS band. These LPS band heterogeneities resulted not only from variations in the oligosaccharide region of the LPS but also from two phosphorylation states of the lipid A (diphosphoryl and diphosphoryl plus a single phosphoethanolamine substitution). On the other hand, MALDI‐TOF mass spectra of the separated LPS bands displayed reduced heterogeneity and increased signal‐to‐noise ratios as compared to spectra of the unpurified LPS. Furthermore, micropurification of LPS bands prior MALDI‐TOF‐MS led to a higher sensitivity of detection of less abundant low‐molecular‐weight LPS glycoforms. Taken together, this and our previous study on gel‐micropurified LPS using ESI definitively show how one can unambiguously determine the different molecular species contained within each gel‐separated LPS band, their relative abundance and oligosaccharide sequences.
Editorial: Electrophoresis 14/2004
Authors: Rizzi Andreas
Published: July 2004

No abstracts.
Spontaneous mutant frequency and mutation spectrum for gene A of ΦX174 grown in E. coli
Authors: Raney Jessica L. , Delongchamp Robert R. , Valentine Carrie R.
Published: July 2004

The use of transgenic targets for measuring mutant frequencies in mammalian tissue requires an estimate of the mutant frequency that results from recovery of the transgene in bacterial recovery systems. In this study, we have determined the spontaneous mutant frequency, estimated the mutation rate, and ascertained the mutation spectrum for gene A of ΦX174 grown in E. coli strain CQ2 from 156 small independent cultures. The mutant frequency of 12 of the 156 cultures was 17 ± 1.0 × 10−6 and the estimated mutation rate per gene replication was 7.4 ± 2.3 × 10−6. The mutant frequency and spectrum from E. coli were not significantly different from that of solvent‐treated embryonic mouse cells in culture, 19 ± 0.5 × 10−6 (Valentine CR et al. [2002]: Environ Mol Mutagen 39:55–68), indicating that those spontaneous mutants were primarily derived from E. coli. The E. coli spectrum was heavily weighted toward two major target sites (hot spots), 4225A→G (56%) and 4218G→A or C (20%). Four new target sites and one new mutational event were recovered by the gene A forward assay. A mutant spectrum from an expanded phage stock was also determined to assess the effects of propagating the virus. This mutant frequency was higher (6 × 10−4), contained more double mutants (15% compared to 0.6%), and had a significantly different spectrum from the spectrum for independent cultures (fewer A:T→G:C and G:C→C:G changes and more G:C→A:T; P < 0.002). The E. coli mutation spectrum will be useful for determining the origin of gene A mutation in tissues of ΦX174 transgenic mice. Environ. Mol. Mutagen. 44:119–127, 2004. Published 2004 Wiley‐Liss, Inc.
Long‐term growth of a valley‐bottom gully, western Iowa
Authors: Thomas John T. , Iverson Neal R. , Burkart Michael R. , Kramer Larry A.
Published: July 2004

Growth of a permanent, valley‐bottom gully from 1964 to 2000 was determined annually from survey and sediment‐discharge data and compared with runoff and baseﬂow discharges. Data were analysed to test the hypothesis that rates of gully growth decay exponentially with time in response to shrinking catchment area caused by gully enlargement. Also, monthly values of growth rates and runoff, averaged over the 36‐year record, were analysed with mass‐wasting data to determine the extent to which colluvium availability affected growth rates seasonally.

From 1964 to 2000, the gully volume increased by 9200 m3, accounting for 34 per cent of sediment yield from the watershed. There were tight power‐law relationships between annual growth rates and annual runoff, with runoff exponents of 1·57 and 1·30 for headward and volumetric growth, respectively. Increases in gully length, area, and volume were ﬁtted successfully assuming an exponential decay in growth rate with time. Rather than being due to a decrease in catchment area, however, the decline in growth rate was caused by a 77 per cent decrease in the ratio of runoff to baseﬂow, which also widened the gully and reduced the mean slope of its banks. Order‐of‐magnitude seasonal changes in erosion efﬁciency, deﬁned as the fraction of stream power used to evacuate sediment from the gully, were roughly correlated with colluvium availability, as indicated by seasonal changes in the number of bank mass‐wasting events. No more than 2·2 per cent of stream power was used to evacuate sediment during any month. This study demonstrates the danger of attributing declining rates of gully growth to a shrinking catchment area if corroborative runoff and baseﬂow data are not available. Moreover, it illustrates that stream power alone provides only a rough and physically indirect measure of erosion potential. Copyright © 2004 John Wiley & Sons, Ltd.
Parallel adaptive refinement for unsteady flow calculations on 3D unstructured grids
Authors: Waltz Jacob
Published: July 2004

A parallel adaptive refinement algorithm for three‐dimensional unstructured grids is presented. The algorithm is based on an hierarchical h‐refinement/derefinement scheme for tetrahedral elements.The algorithm has been fully parallelized for shared‐memory platforms via a domain decomposition of the mesh at the algebraic level. The effectiveness of the procedure is demonstrated with applications which involve unsteady compressible fluid flow. A parallel speedup study of the algorithm also is included. Published in 2004 by John Wiley & Sons, Ltd.