bluuebunny/crossref_metadata_2025 huggingface dataset:
Bienzyme sensors based on “electrically wired” peroxidase
Authors: Ohara T. J. , Vreeke M. S. , Battaglini F. , Heller A.
Published: May 2005

Single‐layer and bilayer bienzyme electrodes based on the combination of a three‐dimensional (3‐D) redox epoxy network that electrically connects redox centers of bound horseradish peroxidase (HRP) to electrodes with a hydrogen peroxide generating enzyme, the redox centers of which are not connected to the redox‐epoxy network, are described. In the single‐layer electrodes, H2O2 generated by the first enzyme oxidizes the second enzyme HRP, which oxidizes the redox polymer network that is electrochemically reduced at 0 mV saturated calomel electrode (SCE). When the redox centers of the H2O2 generating enzyme are also electrically connected to the redox‐epoxy network, the substrate reduced redox centers are oxidized by the redox polymer network, thus lowering the cathodic current. Such attenuation is avoided in bilayer electrodes, where the H2O2 producing enzyme and the redox‐epoxy‐HRP network are not electrically connected.

The single‐layer bienzyme electrodes extend the range of amperometric biosensors based on directly redox‐epoxy “wired” enzymes to enzymes that are difficult to electrically connect to redox polymer networks and whose preferred or only cosubstrate is oxygen. For the difficult to wire enzyme‐choline oxidase, the cathodic current density in the single‐layer peroxidase and choline oxidase containing electrode is 80 μA cm−2 at 10 mM choline concentration, while the anodic current density of the directly wired enzyme is only 5 μA cm−2. Alcohol oxidase is not electrically connected to the wiring 3‐D redox‐epoxy network. The anodic current density of its redox‐epoxy wired electrodes is close to nil, while the cathodic current density, observed in alcohol oxidase and wired peroxidase containing single‐layer electrodes at 10 mM ethanol, is 5 μA cm−2. When well‐wired enzymes, such as glucose oxidase or lactate oxidase, are utilized in single‐layer electrodes, limiting cathodic current densities of 60 μA cm−2 are observed for both enzymes. These currents are much lower than those observed in the directly wired enzyme anodes.
Selectivity for phosphate and citrate with benzyltin‐based polymer membrane electrodes
Authors: Demeulenaere Robert L. , Onsrud Paula , Arnold Mark A.
Published: May 2005

Response properties are reported for membrane electrodes prepared by incorporating bis(p‐fluorobenzyl)tin dichloride, bis(p‐chlorobenzyl)tin dichloride, and tris(p‐chlorobenzyl)tin chloride into plasticized poly(vinyl chloride) membranes. These electrodes respond selectively to dibasic phosphate and tribasic citrate. The magnitude of response and the degree of selectivity are affected by the method of pretreating the membrane. The highest level of selectivity is obtained for tribasic citrate from electrodes based on bis(p‐fluorobenzyl)tin dichloride after conditioning with citrate. This electrode possesses a limit of detection for citrate of 0.4 μM at pH 7.0 with response times ranging from 2 to 5 minutes. Selectivity for citrate is demonstrated over phosphate, thiocyanate, iodide, bromide, nitrate, acetate, lactate, pyruvate, ascorbate, and chloride.
Miniaturized reference electrodes with microporous polymer junctions
Authors: Pedrotti Jairo J. , Angnes Lúcio , Gutz Ivano G. R.
Published: May 2005

The construction and evaluation of a simple and effective mini‐reference electrode is presented. Major innovations are the use of high density microporous polyethylene for the restricted flow junction between the internal and external electrolyte and a polypropylene disposable pipette tip as the electrode body. Low electrolyte bleeding, reduced size, relatively low electrical resistance and simplified procedure of construction are some of the advantages of the presented design. These characteristics make this electrode particularly suitable for use with small‐volume cells and flow cells, as well as applications where chloride contamination should be avoided. Other applications for the microporous membrane in electroanalysis are introduced.
Torulopsis candida based sensor for the estimation of biochemical oxygen demand and its evaluation
Authors: Sangeetha S. , Sugandhi G. , Murugesan M. , Murali Madhav V. , Berchmans Sheela , Rajasekar R. , Rajasekar Sumathi , Jeyakumar D. , Prabhakar Rao G.
Published: May 2005

A universal biochemical oxygen demand (BOD) sensor that can be used for the analysis of all types of organic pollutants is the objective of current researchers working in the area of biosensors. Development of such type of sensor involves the proper choice of a microorganism that can assimilate a wide spectrum of organic compounds. This study has led to the screening of many microorganisms and in this process we were able to identify Torulopsis candida as an attractive alternate for Trichosporon cutaneum. The BOD sensor employing T. candida was fabricated and evaluated in the batch mode as well as under flow conditions. The response time, linear range of estimation of BOD and storage life of the immobilized microbe and the assimilation spectrum have been studied.
Conducting polymer sensors for the amperometric detection of proteins in a flow system – the use of sulfonated dye counterions to induce selectivity
Authors: Lu W. , Wallace Gordon G.
Published: May 2005

In this article, we have demonstrated the feasibility of using dye binding interactions for the amperometric detection of proteins at conducting polymer coated electrodes with flow injection analysis. Incorporation of appropriate dyes into the conducting polymer during synthesis enables sensitive and selective responses to be obtained. The effects of eluent pH and applied potential on the responses obtained for a range of proteins have been investigated. These parameters can be used to modify selectivity and achieve sensitivity.
Voltammetric behavior of sulfadimetoxol using square‐wave and adsorptive stripping square‐wave techniques
Authors: Berzas J. J. , Rodriguez J. , Lemus J. M. , Castañeda G.
Published: May 2005

The voltammetric behavior of sulfadimetoxol (SDX) was studied by square‐wave techniques, leading to two methods for its determination in aqueous samples and veterinary formulations. The application of the square‐wave mode shows the determination of SDX between 1 × 10−7 M and 2 × 10−6 M at −0.60 V and for the stripping voltammetry of adsorbed SDX with an accumulation step of 15 s proved to be more sensitive, yielding signals five times larger than those obtained without the accumulation. The determination of SDX was done between 2 × 10−8 M and 5 × 10−7 M by stripping mode. The relative standard deviations obtained for concentration levels of SDX as low as 3 × 10−7 M with square‐wave was 3.4 % (n = 8) and for 2 × 10−7 M with stripping square‐wave was 3.1 % (n = 8). The methods were satisfactorily applied for determining SDX in four veterinary products.
Adsorption voltammetry of nandrolone phenylpropionate
Authors: Jin Wenrui , Zheng Yanhong , Chen Xia
Published: May 2005

The electrochemical behavior of nandrolone phenylpropionate (NP) at a hanging mercury drop electrode (HMDE) was investigated. The adsorption phenomena were observed by linear sweep voltammetry in NaOH. The electrode reaction was found to be a totally irreversible reduction of the adsorbed NP. In 1 × 10−7 mol/L NaOH, the detection limit and the linear range are 5 × 10−10 and 8 × 10−10–5 × 10−7 mol/L, respectively. The relative standard deviation of the method is 1.6 % for 1.7 × 10−7 mol/L NP. The method was applied to the determination of NP in clinical ampuls.
Calmodulin binding to nitrocellulose and Zetapore membranes during electrophoretic transfer from polyacrylamide gels
Authors: Rochette‐Egly Cécile , Daviaud Danièle
Published: May 2005

In 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

A 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

A 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

An 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

The 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

The 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

Bromoperoxidases 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

Here 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

Two‐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

Actins 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

Psoriasin 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

Capillary 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

A 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

Microdialysis 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

Capillary 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

The 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

N3′→ 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

A 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

Microdialysis (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.
Analysis of recombinant cytokines in human body fluids by immunoaffinity capillary electrophoresis
Authors: Phillips Terry M. , Dickens Benjamin F.
Published: May 2005

An immunoaffinity capillary electrophoresis (ICE) system for rapidly quantifying recombinant cytokines in human body fluids has been developed. Cytokines within biological fluids were labeled with a red light emitting fluorochrome and injected into the capillary. Selected cytokines were captured by immobilized antibodies on the internal surface of the capillary, and held while unbound materials were purged. The cytokines were then eluted electrophoretically in acidic buffer. Individual cytokine peaks were detected by on‐line laser‐induced fluorescence detection coupled to a computerized fiber‐optic spectrometer, and analyzed by integration of the eluted peaks. The comparison of the results of ICE to routine assays used for these cytokines demonstrates that ICE provides a fast and accurate procedure for defining these cytokines in complex biological samples. Immunoaffinity separations can be used for any material to which a specific antibody can be raised, making this procedure applicable to a wide range of molecules of biomedical interest.
Analysis of isoquinoline alkaloids in medicinal plants by capillary electrophoresis—mass spectrometry
Authors: Sturm Sonja , Stuppner Hermann
Published: May 2005

The technique of capillary electrophoresis – mass spectrometry (CE‐MS) was applied for determination of isoquinoline alkaloids in crude methanolic extracts of medicinal plants. For the CE separations ammonium formate buffer solutions (70 or 100 mM, pH 3.0 or 4.0) containing 10% methanol or 20–60% acetonitrile as additives were used. The applied voltage was 25 kV, the thermostating temperature was kept constant at 25°C. Coupling with the mass spectrometer was performed via an atmospherical pressure ionization (API) interface and the electrospray ionization technique (ESI). As sheath liquid 5 mM formic acid in acetonitrile at a flow rate of 3 μL/min was used. The spray voltage was 4.5 kV and the temperature of the heated capillary was chosen to be 200°C. Detection in the positive ionization mode resulted in mass spectra showing either the molecular ions [M]+ or the protonated molecular ions [M+H]+. The presented method allows detection and identification of isoquinoline alkaloids in crude methanolic extracts of medicinal plants as Eschscholzia californica CHAM. (Papaveraceae), Hydrastis canadensis L. (Ranunculaceae), Berberis vulgaris L. (Berberidaceae), Jateorhiza palmata (LAM.) MIERS (Menispermaceae) and Chelidonium majus L. (Papaveraceae).
Optimized determination of carbohydrate‐deficient transferrin isoforms in serum by capillary zone electrophoresis
Authors: Tagliaro Franco , Crivellente Federica , Manetto Giulia , Puppi Ivana , Deyl Zdenek , Marigo Mario
Published: May 2005

Carbohydrate deficient transferrin (CDT) is one of the most reliable markers of chronic alcohol abuse. It consists of a group of minor isoforms of human transferrin (the main iron transport serum protein) deficient in sialic acid groups (asialo, monosialo and disialo) with a pI >5.7, while the main isotransferrin (tetrasialo) has a pI of 5.4. The aim of the present work was to develop a capillary electrophoretic method to determine CDT in serum, suitable for routine use as a confirmatory technique of the current screening methods based on immunoassays. Serum samples (0.5 mL) were saturated with iron by incubation with 10 mM FeCl3 (9 μL) and 500 mM NaHCO3 (12 μL) for 30 min, then diluted 1/10 in water and injected by positive pressure (0.5 psi for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused‐silica capillaries (20 μm ID, 37 cm in length) and a buffer composed of 100 mM sodium tetraborate adjusted with boric acid to pH 8.3. Applied voltage was 10 kV and temperature 25°C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo‐, monosialo‐, disialo‐, trisialo‐ and tetrasialo‐transferrin were separated in human serum. The limit of detection (signal‐to‐noise ratio of 2) was about 0.3% for disialo‐transferrin, and 0.4% of trisialo‐transferrin, expressed as percentages of the terasialo‐transferrin peak area. Relative standard deviations (RSD) of absolute migration times were < 1%, while RSD of relative migration times (on the basis of tetrasialo‐transferrin) were < 0.1%. Intra‐day and day‐to‐day peak quantitation precision studies showed RDS ranging from 4 to 9% and from 13 to 24% for disialo‐ and trisialo‐transferrin, respectively. The results from 30 control subjects, including social drinkers, and 13 alcoholics showed disialo‐ and trisialo‐transferrin significantly increased in patients by a factor of about 4.5 (P < 0.0001).
Basal sediment evacuation by subglacial meltwater: suspended sediment transport from Haut Glacier d'Arolla, Switzerland
Authors: Swift Darrel A. , Nienow Peter W. , Hoey Trevor B.
Published: May 2005

Proglacial suspended sediment transport was monitored at Haut Glacier d'Arolla, Switzerland, during the 1998 melt season to investigate the mechanisms of basal sediment evacuation by subglacial meltwater. Sub‐seasonal changes in relationships between suspended sediment transport and discharge demonstrate that the structure and hydraulics of the subglacial drainage system critically influenced how basal sediment was accessed and entrained. Under hydraulically inefficient subglacial drainage at the start of the melt season, sediment availability was generally high but sediment transport increased relatively slowly with discharge. Later in the melt season, sediment transport increased more rapidly with discharge as subglacial meltwater became confined to a spatially limited network of channels following removal of the seasonal snowpack from the ablation area. Flow capacity is inferred to have increased more rapidly with discharge within subglacial channels because rapid changes in discharge during highly peaked diurnal runoff cycles are likely to have been accommodated largely by changes in flow velocity. Basal sediment availability declined during channelization but increased throughout the remainder of the monitored period, resulting in very efficient basal sediment evacuation over the peak of the melt season. Increased basal sediment availability during the summer appears to have been linked to high diurnal water pressure variation within subglacial channels inferred from the strong increase in flow velocity with discharge. Basal sediment availability therefore appears likely to have been increased by (1) enhanced local ice‐bed separation leading to extra‐channel flow excursions and[sol ]or (2) the deformation of basal sediment towards low‐pressure channels due to a strong diurnally reversing hydraulic gradient between channels and areas of hydraulically less‐efficient drainage. Copyright © 2005 John Wiley & Sons, Ltd.
Methods of calculating the physical acation of flame retardants
Authors: Sellman L.‐G. , Östaman B. A.‐L. , Back E. L.
Published: May 2005

The chemical mechanisms for the action of flame retardants are often mentioned in the literature but the physical modes of action are seldom. Discussed. This article presents one way to quantify their efficiency. The technique is based on literature data for the physical and thermal properties of flams retardants for temperatures from 25°C up to 1000°C. The prolongation of the time to ignition by heat absorption by the retardant and the amount of inert gas evolved by the retardant are calculated at a given radiation for a material flame‐proofed with a given amount of the flame retardant. The ability to form an insulating surface layer is considered but not quantified. It is assumed that a medium density wood fibre building boards is treated with 2 kg of flame retardant per m2. The flame retardants included are borates, boric acid, phosphates and silicates. The board is assumed to be irradiated with an intensity of 15 k W m−2. Under these conditions an untreated board ignites after 6–7 min. The time to ignition is prolonged by 1–5 min through heat absorption by the different retardants, and the amount of inert gases evolved may be as high as 2.6 m3 per m2 board. The formation of an insulating surface layer is more difficult to quantify. The results confirm the importance of the physical modes of action of flame retardants and the technique could form the basis for evaluating materials in simulated fire situations.
Some measurements of fire turbulence
Authors: Cox G.
Published: May 2005

It is important to be able to predict the rate of smoke and fire spread in buildings in order to optimize the design of escape routes and the siting of detectors and sprinklers. The available mathermatical procedures based on turbulence models require experimental guidance. This paper describes the measurement of some turbulence parameters in smoke and flame plumes moving under a ceiling. Time correlation techniques are applied to signals obtained from thermocouple and Langmuir probes. Space‐time correlations in the hot layer show similar trends to those in decaying isotropic turulence from wind tunnels but superimposed upon a coherent sine wave produced by vortex shedding at the fire. Data are also presented of the probability of chemical reaction at several locations in the hot layer.
Comparison between different reaction to fire test methods on wall‐lining and flooring materials
Authors: Vandevelde P.
Published: May 2005

For building control purposes of wall‐lining and flooring materials in Europe several empirical reaction to fire tests are used. This paper examines the correlation between a number of different tests looking at some 42 different materials, and comments thereon.
Histological and electron microscopic analysis of tissue response to synthetic composite bone graft in the canine
Authors: John Kenneth R. St. , Zardiackas Lyle D. , Terry Roy C. , Teasdall Robert D. , Cooke Shannon E. , Mitias Hanna M.
Published: May 2005

A Synthetic composite bone graft material, composed of fibrillar collagen, hydroxyapatite, and tricalcium phosphate, was evaluated in canine femoral shaft defects and compared to ungrafted defects and defects grafted with autogenous bone. The results of mechanical testing of the grafted femora in torsion at 1 year postoperatively were previously reported. This report details the histological and microscopic features of the graft sites. The results of this study suggest that the healing of the graft site is not yet complete at 1 year and that healing may be more complete in defects that received the synthetic graft as compared to ungrafted sites and autogenous bone graft. The bone tissue appears to respond to the graft particles as if they were bone particles. © 1995 John Wiley & Sons, Inc.
Biologic properties and possible uses of polymer‐like sponges
Authors: Barvicč M. , Kliment K. , Zavadil M.
Published: May 2005

The biologic properties of the sponge‐like polymers of ethylene glycol methacrylate gels and their host compatibility have been verified in subcutaneous and intraperitoneal application in rats. All three sample types tested, with different porosities, have been tolerated very well with the receptor and have caused no pathologic reaction. Subcutaneous implants are directly integrated in teh organism, cellular immigration taking place with vascularization of the surface layer of the implantate to a depth of some 500 μ during the second week after implantation, causing the sponge to become an integral component of the surrounding tissues. More substantial differences in the reaction to the presence of implantates of different porosity ahve not been observed. These properties directly predetermine the sponge‐like polymers for wider application in plastic and reconstructive surgery. In addition, there is the possibility of utilizing them as an auxiliary surgical material, e.g., for drying and cleaning the operative field, or as operation towels. Good results have been achieved also in mechanically stopping bleeding from parenchymatose organs by application of the sponge foil.
Gel‐fabric prostheses of the ureter
Authors: Kočvara S. , Kliment Ch. , Kubat J. , Štol M. , Ott Z. , Dvořak J.
Published: May 2005

In 14 canines the ureter was substituted with a new type of ureteral gel‐fabric prosthesis. The prosthesis is a tube of knitted fabric of polyester fiber case in a layer of hydrophilous glycol methacrylate gel. Anastomoses were carried out by inserting the prosthesis ends into the ureter lumen and by suturing the stump of the ureter to a special fixing ring on the prosthesis. Preliminary results of short‐term obsevations (4 months) are quite promising. The prosthesis is non‐irritating and conducts the urine satisfactorily.
Antithrombogenic effect of a graphite, benzalkonium chloride, and heparin coating on a flexible polyvinyl chloride plastic implant in the rabbit
Authors: Farb Richard B. , Kaplan Harold M.
Published: May 2005

In vivo and in vitro experiments described herein have demonstrated that the GBH coating is antithrombogenic when placed on PVC, as tested in the blood system of rabbits for periods up to 2 weeks. In vitro clotting times cannot be correlated with the in vivo thrombogenicity of the same implants. For example, dog and man have very similar in vitro clotting times, but very dissimilar in vivo prosthetic thrombosis problems. In vivo and in vitro tests are for the most part not comparable in that they do not measure the same things. However, both in vivo and in vitro tests proved the GBH‐treated vinyl to be antithrombogenic. It may be concluded from the available data that a flexible plastic can be treated with GBH and become antithrombogenic for a practicable period of time.
Toxicologic aspects of implants
Authors: Autian John
Published: May 2005

Man‐made polymeric materials are being utilized in a number of devices which will be implanted into animals and humans for various periods of time. The diversity of materials and the introduction of new materials into the medical and paramedical professions appear to be increasing at an accelerated rate each year. Even though great advancements have been made by the biomedical engineer and the surgeon in designing and implanting a prosthetic device in the body, less emphasis apparently has been given to the possible or potential hazardous aspects of the material. An implantable material may initiate a body response in two general ways: (1) release of an ingredient from the material to the biological environment and (2) alteration of the material by the biological environment, leading to degradation of the material with its various consequences. If serious future hazards are to be kept to a minimum, greater emphasis must be placed on well‐designed toxicity studies of materials which, in turn, could lead to standardized materials for implants.
Change in zeta potential of polar plastics by electrical polarization
Authors: Offner Franklin , Breger Lance
Published: May 2005

Tubing made of a polar plastic, polychlorotrifluoroethylene, was electrically polarized while heated. The zeta potential of tubes polarized negatively inside was consistently higher than that of unpolarized tubes. Tubes polarized positively had a much smaller and variable change in zeta potential. The change is apparently not related to the formation of electrets, but may be due to the orientation of pre‐existing dipoles.
The search for correlation between electrokinetic phenomena and blood thrombus formation on implant materials
Authors: Milligan H. L. , Davis J. , Edmark K. W.
Published: May 2005

Bioelectric charge transfer at the blood–wall interface may be a crucial factor affecting thrombosis on implant materials. A program of studies was conducted to determine the electrokinetic and other physical properties of a wide spectrum of materials including organic polymers, metals, inorganics, heterogeneous compounds, and animal tissue. From these tests, materials were selected for in‐vivo cannulation experiments. In this manner, a search was made to find a correlation between surface charge characteristics and thrombosis on cardiovascular implant materials. It was found that materials exhibiting a substantial positive charge were prone to rapid thrombosis. No clear correlation can be stated, as yet, regarding the thrombogenecity of highly charged negative surfaces or slightly charged surfaces. However, many heterogeneous materials have exhibited prolonged in‐vivo patency. It appears necessary to isolate surface effects from volumetric effects to seek the role of bioelectric charge transfer in blood thrombus formation.