bluuebunny/crossref_metadata_2025_split huggingface dataset:
Calmodulin binding to nitrocellulose and Zetapore membranes during electrophoretic transfer from polyacrylamide gels
Authors: Rochette‐Egly Cécile , Daviaud Danièle 
Published: May 2005

AbstractIn the classical electrotransfer conditions from sodium dodecyl sulfate‐polyacrylamide gels, calmodulin fails to bind to nitrocellulose membranes. However, under conditions favorizing hydrophobic interactions such as a higher salt concentration, calmodulin can be immobilized on nitrocellulose sheets, thus allowing detection in a complex protein mixture.
Complete separation of anti‐hapten antibodies by two‐dimensional affinity electrophoresis
Authors: Takeo Kazusuke , Suzuno Ryosuke , Tanaka Tatehiko , Nakamura Kazuyuki 
Published: May 2005

AbstractA high resolution two‐dimensional affinity electrophoresis has been developed, using capillary isoelectric focusing as the first electrophoresis and slab gel affinity electrophoresis as second electrophoresis. By this method 1–2 μg of anti‐dinitrophenyl antibodies have been separated completely into several hundred homogeneous IgG spots. They are grouped into a number of families which are composed of several IgG spots of the same affinity to the hapten but of a different pI. It is suggested that each individual family is derived from one monoclonal antibody producing cell line.
Affinity electrophoresis for studies of mechanisms regulating glycosylation of plasma proteins
Authors: Mackiewicz Andrzej , Kushner Irving 
Published: May 2005

AbstractA model system for studies of mechanisms governing the alterations of glycosylation of plasma glycoproteins was developed. The system employs two human hepatoma cell lines, Hep 3B and Hep G2, as target cells and agarose affinity electrophoresis with lectins for studies of microheterogeneity of α1‐protease inhibitor (PI), a model glycoprotein synthesized by hepatocytes. As an example for the application of the system, the effect of cytokines on major microheterogeneity of plasma proteins is demonstrated. The results indicate that interleukin 6, transforming growth factor β1 and, to some extent, tumor necrosis factor α are directly involved in regulating the pattern of glycosylation of plasma proteins in vitro, but the major effect is obtained by using combinations of interleukin 6, transforming growth factor β1, tumor necrosis factor a and interleukin 1. In addition, the results underline the dissociation between alteration of gene expression and the changes in the pattern of plasma protein glycosylation.
Lipopolysaccharide‐protein interactions: Determination of dissociation constants by affinity electrophoresis
Authors: Borneleit Petra , Blechschmidt Bernd , Kleber Hans‐Peter 
Published: May 2005

AbstractAn affinity electrophoresis system is described to allow determination of dissociation constants of lipopolysaccharide (LPS)‐protein complexes. The LPS ligand is incorporated into polyacrylamide gels by addition to the polyacrylamide‐N, N′‐methyl‐enebisacrylamide polymerization mixture. Quantitative evaluation revealed formation of immobile protein‐ligand complexes. The method was applied both to R‐ and S‐ form LPS from Acinetobacter calcoaceticus. For a heat‐modifiable outer membrane protein with Mr 18 000 from strain 69V the dissociation constant was determined to be 0.5 mM (EDTA‐salt extracted R‐LPS) and 0.3 mM (phenol‐chloroform‐petrolether extracted R‐LPS). In comparison, for another A. calcoaceticus strain, CCM 5593, a higher dissociation constant of 1.0 mM (phenol‐chloroform‐petrolether extracted R‐LPS) ‐ indicative of lower affinity ‐ was obtained. When S‐LPS from A. calcoaceticus 69V was incorporated into the affinity gels, a dissociation constant of 0.02 mM was determined which indicates much stronger interactions than those exerted by R‐LPS forms.
Correlation of migration behavior in free‐flow zone electrophoresis and electrophoretic titration curve
Authors: Nath Sunil , Schütte Horst , Hustedt Helmut , Deckwer Wolf‐Dieter 
Published: May 2005

AbstractThe correlation of electrophoretic migration behavior in free‐flow zone electrophoresis (FFZE) and electrophoretic titration curve (ETC) has been explored. It is shown that the ETC of a protein or a mixture of proteins can be used to predict the fraction numbers at which those proteins elute in a preparative scale FFZE experiment. The ETC is a quick and effective way to choose optimal buffer conditions in FFZE. FFZE is employed to determine the isoelectric points (pI) of proteins whose pIs lie beyond the range of IEF 3–9 gels. It is found that separations in FFZE are governed by the net surface charge of the proteins.
Synthesis of thiomorpholino buffers for isoelectric focusing in immobilized pH gradients
Authors: Chiari Marcella , Righetti Pier Giorgio , Ferraboschi Patrizia , Jain Tikam , Shorr Robert 
Published: May 2005

AbstractThe two commercially available Immobilines having a pK of 6.2 (2‐morpholino ethyl acrylamide) and 7.0 (3‐morpholinopropylacrylamide) have been modified and two new buffers have been synthesized: 2‐thiomorpholinoethylacrylamide, pK 6.6, and 3‐thiomorpholinopropyl acrylamide, pK 7.4. The replacement of an oxygen with a sulfur atom in the morpholino ring is thus seen to shift the pK values of these two bases by +0.4 pH units. In formulations in which the two new bases replaced the standard morpholino derivatives, identical pH profiles and protein patterns were obtained. The reason for this work was to try to close the gap between the pK 7.0 and 8.5 species and to provide the users of immobilized pH gradients with more buffers in the neutral pH region. The two new thiomorpholino derivatives are an important step in this direction.
Native bromoperoxidases do not bind to nitrocellulose: Use of DEAE‐cellulose as an alternative in blotting
Authors: Jordan Peter , Vilter Hans 
Published: May 2005

AbstractBromoperoxidases were investigated by protein blotting after polyacrylamide gel electrophoresis, While the denatured proteins bound to nitrocellulose, the native enzymes did not. Instead, they could be transferred successfully to DEAE‐cellulose. Procedures for immunostainig and glycoprotein detection with concanavalin A on DEAE‐cellulose are described. The resultes indicate that binding of native proteins to nitrocellulose can not necessarily be assumed. DEAE‐cellulose is suitable both to investigate this phenomenon or as a substitute for nitrocellulose in blotting of native proteins.
Identification of isoprenyl modified proteins metabolically labeled with [3H]farnesyl‐ and [3H]geranylgeranyl‐pyrophosphate
Authors: Gromov Pavel S. , Madsen Peder , Celis Julio E. 
Published: May 2005

AbstractHere we describe a direct approach for two‐dimensional (2‐D) gel mapping of proteins that are modified by post‐translational isoprenylation in mammalian cells. Briefly, transformed human amnion cells (AMA) and transfected COS‐1 cells were metabolically labeled with either [3H]farnesyl‐pyrophosphate or [3H]geranylgeranyl‐pyrophosphate following treatment with lovastatin, which blocks the synthesis of mevalonic acid. The proteins were then separated by 2‐D gel electrophoresis and electrotransferred to nitrocellulose filters. The membranes were immersed in dimethyl ether, containing 10% of 2,5‐diphenyloxazole prior to fluorography. Over 40 [3H]farnesyl‐labeled proteins and over 25 [3H]geranylgeranylated proteins were identified on the 2‐D autoradiograms. Several [3H]farsenyl‐labeled proteins exhibited the same coordinates (Mr and pI) as their [3H]geranylgeranylated counterparts, raising the possibility that they may be substrates for both farnesyl and geranylgeranyl transferase(s). The approach offers high resolution of both farnesylated and geranylgeranylated proteins and it may serve as a powerful tool for the identification of hitherto unknown prenylated proteins as well as for the determination of prenylated protein levels, type of isoprenoid modification, and possible changes in protein prenyltransferase activity.
Two‐dimensional separations of the genome and proteome of neuroblastoma cells
Authors: Wimmer K. , Kuick Rork , Thoraval D. , Hanash Samir M. 
Published: May 2005

AbstractTwo‐dimensional (2‐D) electrophoretic methods have been available that allow separation of the protein constituents of a cell population. It has also become feasible to electrophoretically separate in two dimensions and to display DNA fragments derived from genomic digests. Through the appropriate choice of restriction enzymes, the functional component of the genome that encompasses CpG islands can be preferentially visualized in 2‐D gels. The same computerized approach for the analysis of 2‐D patterns can be applied to investigations at either the protein or DNA levels. Our group has utilized 2‐D electrophoresis to investigate both protein and DNA changes in cancer. The emphasis to date has been on the identification of proteins, the abundance of which is related to specific biological features of the tumors analyzed and of DNA fragments encompassed in genomic amplifications, as the latter commonly contain growth‐related genes. Findings derived from our analysis of neuroblastoma tumors and cell lines using 2‐D approaches are reviewed. Data for four proteins observed in 2‐D gels are presented because of our demonstrated association of these proteins with differentiation and proliferation properties of neuroblastoma. At the genomic level, the detection of amplifications using 2‐D gels has necessitated an understanding of the variability displayed by multi‐copy genomic fragments, which we have accomplished to a large part and which we present. An important benefit of 2‐D approaches is the efficiency of scale and the ease with which abundant proteins or multicopy genomic fragments can be detected, identified and quantitatively analyzed.
ADP‐ribosylation of actins in fibroblasts and myofibroblasts by botulinum C2 toxin: Influence on microfilament morphology and migratory behavior
Authors: Rønnov‐Jessen Lone , Petersen Ole William 
Published: May 2005

AbstractActins comprise six isoforms of which the nonmuscle isoforms β‐/γ‐actins are expressed by all eukaryotic cells. The expression pattern of one of the muscle actin isoforms, α‐sm actin, previously believed to be restricted to smooth muscle, has been broadened to encompass activated fibroblasts (myofibroblasts) as well. The significance of this molecular conversion has remained largely unknown. We have recently shown that a reduction in filamentous α‐sm actin by electroinjected specific antibodies or antisense oligodeoxynucleotides leads to increased motility in breast myofibroblasts (Rønnov‐Jessen L., Petersen, O. W. J. Cell Biol. 1996, 134, 67–80). In the present study we have expanded on the functional significance of actin isotypes in fibroblasts from the opposite point of view, namely filamentous nonmuscle actin. Nonmuscle actins in fibroblasts and myofibroblasts were ADP‐ribosylated by Clostridium botulinum C2 toxin. The substrate for C2 toxin is globular actin, which upon ribosylation cannot incorporate into microfilaments. The pattern of actin ADP‐ribosylation in (myo)fibroblasts in the presence of [32P]NAD was analyzed by isoelectric focusing, fluorography and immunoblotting. The influence of C2 toxin on microfilaments in intact cells was further assessed by immunofluorescence, and motility was measured in a mass migration assay and by computerized video time‐lapse microscopy. We show here that C2 toxin specifically ribosylates β‐ and γ‐actin in both fibroblasts and myofibroblasts. Whereas fibroblasts rapidly round up and stop migrating when filamentous β‐/γ‐actin is reduced by short‐term ADP‐ribosylation, myofibroblasts maintain their flattened morphology and a basic low motility.
Expression and divalent cation binding properties of the novel chemotactic inflammatory protein psoriasin
Authors: Vorum Henrik , Madsen Peder , Rasmussen Hanne H. , Etzerodt Michael , Svendsen Ib , Celis Julio E. , Honoré Bent 
Published: May 2005

AbstractPsoriasin is a novel chemotactic inflammatory protein that possesses weak similarity to the S100 family members of Ca2+‐binding proteins, and that is highly up‐regulated in hyperproliferative psoriatic keratinocytes. Here we have used the psoriasin cDNA to express recombinant human (rh) psoriasin in Escherichia coli as a fusion protein containing a hexa His tag and a factor Xa cleavage site in the NH2‐terminus. The protein was purified by affinity chromatography on Ni2+‐nitrilotriacetic acid agarose, digested with factor Xa, further purified by ion‐exchange chromatography and characterized by two‐dimensional (2‐D) gel electrophoresis and NH2‐terminal sequencing. The ability of rh psoriasin to bind Ca2+, Zn2+, and Mg2+ was determined by dialysis experiments. We found that rh psoriasin may bind at least seven molecules of Ca2+ in KCl and several molecules in NaCl, with an affinity for the first bound molecule of 1.3–1.6 × 104 M−1. This indicates that psoriasin may cooperatively bind several molecules of Ca2+ when present in the extracellular space, or putatively, if localized in subcellular compartments where the concentration of Ca2+ is relatively high. At least eight molecules of Zn2+ were bound in KCl and four in NaCl, with an affinity just below 1 × 104 M−1 for the first molecule. Thus psoriasin does not bind significant amounts of Zn2+ at physiological concentrations. Mg2+ and Ca2+ are bound anti‐cooperatively and binding of each of the ions (Ca2+, Zn2+, or Mg2+), is accompanied by conformational changes that move tyrosine residues to more hydrophobic areas.
Capillary electrophoresis in drug analysis
Authors: Thormann Wolfgang , Caslavska Jitka 
Published: May 2005

AbstractCapillary electrophoresis has become one of the advanced analytical methods for drugs in pharmaceutical, therapeutic, diagnostic and forensic applications. This review discusses key issues and provides key references to the topic of drug analysis using capillary electrophoresis. It gives readers a brief summery for the current status of the technology and serves as an editorial for the paper symposium “Capillary electrophoresis in drug analysis” (Electrophoresis 1998, 19, 2691–3054).
A literature assessment of sample pretreatments and limits of detection for capillary electrophoresis of drugs in biological fluids and practical investigation with some antimalarials in plasma
Authors: Taylor Robert B. , Toasaksiri Suwanna , Reid Raymond G. 
Published: May 2005

AbstractA literature survey on published reports of the determination of drugs in biological fluids shows that all methods of sample pretreatment have been used and that the limits of detection achieved vary widely, ranging from low ngcm−3 to μgcm−3. The most widely used injection method was hydrodynamic and, in the majority of cases, whenever low detection limits were achieved, this was a result of preconcentration during the sample pretreatment. Only a small proportion of the reported methods employed electrokinetic injection and utilised the field amplified sample injection (FASI) techniques. An experimental investigation of the alternative hydrodynamic and electrokinetic injection methods for a small set of antimalarial drugs is reported. It was found that electrokinetic injection with FASI from an acetonitrile‐water matrix produced dramatic improvements in detection limits. This improvement could not, however, be achieved when the drugs were in plasma using protein precipitation, liquid‐liquid extraction or solid phase extraction pretreatment methods. This highlights the importance of sample pretreatment in utilising the potential sensitivity of capillary electrophoresis with electrokinetic injection.
Assessment of pharmacodynamic and pharmacokinetic characteristics of drugs using microdialysis sampling and capillary electrophoresis
Authors: Denoroy Luc , Bert Lionel , Parrot Sandrine , Robert Frédéric , Renaud Bernard 
Published: May 2005

AbstractMicrodialysis sampling combined with capillary electrophoresis is emerging as a new approach in drug studies. It allows the continuous monitoring, in vivo or in vitro, of changes in free endogenous compounds as well as in drug substances, following the administration of pharmacological agents. The low volume requirement of capillary electrophoresis for injection allows the collection of dialysates during short sampling times, leading to a precise temporal description of drug‐induced biochemical changes or pharmacokinetics. Various protocols can be used for analyzing endogenous compounds and drug substances in microdialysis samples. Capillary electrophoresis with laser‐induced fluorescence detection often affords the high sensitivity level which is needed in most studies. Furthermore, the direct on‐line coupling of microdialysis, derivatization of samples, and electrophoretic analysis now brings a separation‐based biosensor, allowing a real‐time description of chemical events with a high molecular specificity. Microdialysis sampling combined with capillary electrophoresis has recently been used to assess pharmacodynamic and pharmacokinetic characteristics of various drugs in animal studies; it may also represent a new approach in clinical pharmacology in the near future.
Use of cyclodextrins in capillary electrophoresis: Resolution of tramadol enantiomers
Authors: Rudaz Serge , Veuthey Jean Luc , Desiderio Claudia , Fanali Salvatore 
Published: May 2005

AbstractCapillary zone electrophoresis was successfully applied to the enantiomeric resolution of racemic tramadol. Both uncoated and polyacrylamide‐coated capillaries were tested for method optimization using either negatively charged or native cyclodextrins (CD) added to the background electrolyte (BGE). The resolution was strongly influenced by the CD type and concentration as well as by the pH and the concentration of the BGE. Among the CDs tested, carboxymethylated‐β‐cyclodextrin allowed the baseline separation of tramadol enantiomers. After the method was optimized, it was validated in a coated capillary for enantiomeric analysis of tramadol enantiomers in pharmaceutical formulation, including specificity and elution order, linearity, accuracy and precision, determination of limit of detection (LOD) and quantification (LOQ), enantiomeric purity linearity, freedom from interference, and stability of sample solutions. Precision at the target concentration was less than 2%, with an accuracy higher than 99%. Furthermore, the method was able to detect 0.3% and to quantify 1% of the minor enantiomer in the presence of the major one at the target value.
Capillary electrophoresis separation of the new anti‐AIDS agents 9‐(2‐phosphonylmethoxyethyl)adenine and 9‐(2‐phosphonylmethoxyethyl)‐2,6‐diaminopurine in mixtures with some monoribonucleotides or the most common deoxynucleotides
Authors: Vargas Gabriela , Revillar Alma , Havel Josef , Holý Antonín 
Published: May 2005

AbstractThe present work describes an electrophoretic method for the separation and determination of the new antivirals, 9‐(2‐phosphonylmethoxyethyl)adenine (PMEA) and 9‐(2‐phosphonylmethoxyethyl)‐2,9‐diaminopurine (PMEDAP) in model mixtures with some monoribonucleotide isomers (3′‐AMP, 2′‐CMP, 3′‐CMP, 3′‐GMP, 2′‐GMP, 3′‐UMP, 5′‐GMP, and 5′‐UMP) or with the most common deoxynucleotides (dCMP, dCDP, dCTP, dTMP, dTDP, dTTP, dGMP, dGDP, dGTP, dAMP, dADP, dATP). A fused‐silica capillary tube, 75 μm ID, 67.8 cm total length (60.3 cm length to the detector), with detection at 210 nm was employed. A hydrodynamic injection for 10 s (1.5 psi vacuum) was utilized to introduce the sample, and 30 kV voltage was applied for the separation. The complete separation of PMEA and PMEDAP from the mononucleotide isomers and deoxynucleotide mixtures is possible in less that 10 min and 25 min, respectively, using 20 mM borate buffer, pH 9.9, with the addition of 10 mM β‐cyclodextrin. Efficiencies of more than 120 000 and resolution higher than 1.9 were reached for each of the compounds studied. This capillary electrophoretic procedure opens the possibility for future determination of PMEA and PMEDAP in cell pool samples.
Analysis of an oligonucleotide N3′→ P5′ phosphoramidate/phosphorothioate chimera with capillary gel electrophoresis
Authors: DeDionisio Lawrence A. , Raible Anette M. , Nelson Jeffrey S. 
Published: May 2005

AbstractN3′→ P5′ phosphoramidate/phosphorothioate chimeric oligonucleotides (ODNs) are presently under investigation as potential antisense drugs. Within the field of antisense research, “second generation” chimeric ODNs have exhibited improved characteristics relative to oligonucleotides with uniformly modified backbones. The ODN of interest for this study consisted of a chemically synthesized 18‐mer of mixed nucleotide base sequence with a backbone consisting of eight central phosphorothioate linkages flanked by four N3′→ P5′ phosphoramidate (amidate) linkages on the 5′‐end and five amidate linkages ont he 3′‐end. This chimera presents analytical challenges due to the central phosphorothioate region. Here, we present a capillary gel electrophoresis (CGE) method for the analysis of the above N3′→ P5′ phosphoramidate/phosphorothioate chimera. CGE was used to analyze the product prior to its purification by reversed phase ‐ high performance liquid chromatography (RP‐HPLC), and each fraction collected from the purification was similarly analyzed. An internal standard was utilized to determine the relative mobility of our product, and polyacrylamide gel electrophoresis (PAGE) analysis was used to verify CGE results.
On‐line dialysis solid‐phase extraction coupled to capillary electrophoresis
Authors: Veraart J. Rob , van Hekezen Judith , Groot Marjolein C. E. , Gooijer Cees , Lingeman Henk , Velthorst Nel H. , Brinkman Udo A. Th. 
Published: May 2005

AbstractA fully automated dialysis solid‐phase extraction (SPE) sample preparation procedure is coupled on‐line to capillary electrophoresis (CE) for the first time. The system is used to determine sulfonamides in serum and urine. The dialysis unit serves to remove proteins and particulate matter. Reconcentration of the analytes is performed with a small SPE column while (in)organic salts and other interferences are removed simultaneously. Finally, the analytes are desorbed and injected, via a homemade interface, into the CE system. Limits of detection (LOD) of 0.05‐0.1 and 0.05‐0.3 μg/mL are obtained in urine and serum, respectively. The within‐day and between‐day precisions are in the range of 2‐6% and 3‐8%, respectively, for a concentration of five times the LOD. The dialysis SPE‐CE system was used over a period of six months for the analysis of over 500 serum and urine samples without problems such as clogging of the CE capillary or SPE column.
Analysis of microdialysates from cancer patients by capillary electrophoresis
Authors: Mader Robert M. , Brunner Martin , Rizovski Blanka , Mensik Christa , Steger Guenther G. , Eichler Hans‐Georg , Müller Markus 
Published: May 2005

AbstractMicrodialysis (MD) is an innovative clinical technique for measuring interstitial tissue pharmacokinetics and plasma‐to‐tissue transfer rates of drugs in humans. However, microdialysis requires the availability of specialized analytical techniques. Capillary electrophoresis (CE), which enables concentration measurements of small volume samples, theoretically constitutes an ideal analytical technique for measuring drug concentrations in microdialysates. In the present experiments, we aimed at assessing the potential utility and limitations of CE for analysis of microdialysates in a clinical situation. Microdialysates were obtained from primary breast cancer patients who received chemotherapy including 5‐fluorouracil (5‐FU; 600 mg/m2). Subsequently, 5‐FU concentrations were measured in tumor ‐ and subcutaneous adipose tissue ‐ microdialysates by CE. By combining MD and CE, complete time versus concentrations profiles could be obtained for 5‐FU in the interstitial tumor space and important clinical questions could be addressed. We conclude that the combination of MD and CE leads to important and previously inaccessible information about the drug distribution process in a clinical setting.