bluuebunny/crossref_metadata_2025 huggingface dataset:
A flow injection‐capillary electrophoresis system with high‐voltage contactless conductivity detection for automated dual opposite end injection
Authors: Kubáň Pavel , Kubáň Petr , Hauser Peter C. , Kubáň Vlastimil
Published: January 2004

The system comprises two flow injection‐capillary electrophoresis interfaces into which the opposite ends of the separation capillary are inserted. The electrolyte solution flows through both interfaces by use of hydrostatic pressure. The injection of the samples into the electrolyte flow is accomplished by a rotary‐type chromatographic valve at the grounded side and by a pinch‐valve injector at the high‐voltage side that provides sufficient isolation from the high electric field. The system allows a fully automated dual‐injection sequence of samples from both capillary ends and simultaneous electrophoretic separation of anions and cations in the samples. The analytes are detected by a high‐voltage contactless conductometric detector positioned approximately in the middle of the separation capillary. The parameters of the system were evaluated. The repeatability of the flow injection‐capillary electrophoresis system for the simultaneous determination of anions and cations was evaluated for ten consecutive injections and relative standard deviation (RSD) values for peak areas were better than 1.0%. The sample throughput for total ionic analysis was estimated to be 25 samples per hour. The system was used for automated simultaneous analysis of anions and cations in various real samples. Using a short separation capillary, rapid total ionic analysis in less then 1 min is demonstrated.
Comparative proteomics of human endothelial cell caveolae and rafts using two‐dimensional gel electrophoresis and mass spectrometry
Authors: Sprenger Richard R. , Speijer Dave , Back Jaap Willem , de Koster Chris G. , Pannekoek Hans , Horrevoets Anton J. G.
Published: January 2004

The human endothelial cell plasma membrane harbors two subdomains of similar lipid composition, caveolae and rafts, both crucially involved in various essential cellular processes like transcytosis, signal transduction and cholesterol homeostasis. Caveolin‐enriched membranes, isolated by either cationic silica or buoyant density methods, were explored by comparing large series of two‐dimensional (2‐D) maps and subsequent identification of over 100 protein spots by matrix‐assisted laser desorption/ionization (MALDI) peptide mass fingerprinting. Improved representation and identification of membrane proteins and valuable information on various post‐translational modifications was achieved by the presented optimized procedures for solubilization, destaining and database searching/computing. Whereas the cationic silica purification yielded predominantly known endoplasmic reticulum residents, the cold‐detergent method yielded a large number of known caveolae residents, including caveolin‐1. Thus, a large part of this subproteome was established, including known (trans‐)membrane, signal transduction and glycosyl phosphatidylinositol (GPI)‐anchored proteins. Several predicted proteins from the human genome were isolated for the first time from biological samples, including SGRP58, SLP‐2, C8ORF2, and XRP‐2. These findings and various optimized procedures can serve as a reference to study the differential composition of endothelial cell caveolae and rafts, known to be involved in pathologies like cancer and cardiovascular disease.
Profiling and comprehensive expression analysis of ABC transporter solute‐binding proteins of Bacillus subtilis membrane based on a proteomic approach
Authors: Bunai Keigo , Ariga Masanori , Inoue Taro , Nozaki Manabu , Ogane Shinya , Kakeshita Hiroshi , Nemoto Tadashi , Nakanishi Hiroshi , Yamane Kunio
Published: January 2004

We analyzed ABC transporter solute‐binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach. We prepared a washed cell membrane fraction that was insoluble in 134 mM nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner. The membrane proteins were resolved by three two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) or two one‐dimensional (1‐D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile. Thereafter, matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins. We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY. Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome. We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis. We compared the 2‐D gel separation profiles of submembrane fractions solubilized by 1% n‐dodecyl‐β‐D‐maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7. The results suggested that YcdH, YtmK and YurO are binding proteins for Mn++, glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium).
Quantitative and qualitative profiling of mitochondrial DNA length heteroplasmy
Authors: Lee Hwan Young , Chung Ukhee , Yoo Ji‐Eun , Park Myung Jin , Shin Kyoung‐Jin
Published: January 2004

Quantitative and qualitative analysis of mitochondrial DNA length heteroplasmy for the first hypervariable segment (HV1) and second hypervariable segment (HV2) regions were performed using size‐based separation of fluorescently‐labeled polymerase chain reaction (PCR) products by capillary electrophoresis. In this report, the relative proportions of length heteroplasmies in individuals were determined, and each length variant in the heteroplasmic mtDNA mixture was identified. The study demonstrated that 36% and 69% of Koreans show length heteroplasmy in the HV1 and HV2 regions, respectively. Electropherograms revealed that length heteroplasmy in the HV1 region resulted in over 5 length variants in an individual. The peak patterns of length heteroplasmy in the HV1 region were classified into five major types. In the HV2 region, length heteroplasmy resulted in 3–6 length variants in an individual, and showed seven variant peak patterns. The increased knowledge concerning mtDNA length heteroplasmy is believed to not only offer a useful means of determining genetic identity due to increased mitochondrial DNA haplotype diversity by allowing mtDNAs to be classified into several peak patterns, but also represent a promising tool for the diagnosis of several common diseases which are etiologically or prognostically associated with mtDNA polymorphisms.
Resolution of 8‐aminonaphthalene‐1,3,6‐trisulfonic acid‐labeled glucose oligomers in polyacrylamide gel electrophoresis at low gel concentration
Authors: Cabanes‐Macheteau Marion , Chrambach Andreas , Taverna Myriam , Buzás Zsuzsanna , Berna Patrick
Published: January 2004

A discontinuous Tris‐Cl/acetate (OAc) buffer system, unprecedently containing OAc as the trailing constituent, and operative in polyacrylamide gel electrophoresis (PAGE) at low polyacrylamide concentration (T = 4.8%) is described in the paper. The characteristics of the electrophoretic system are illustrated by the resolution of fluorescent 8‐aminonaphthalene‐1,3,6‐trisulfonic acid (ANTS)‐labeled malto‐oligosaccharides and dextran homopolymers. In this buffer system, the resolving phase is constituted by Tris‐OAc behind a moving boundary formed between the leading chloride ion of Tris‐HCl gel buffer and the trailing OAc ion provided by a catholyte of NH4OAc. In contrast with the results obtained with Tris‐CI/glycinate buffer commonly used in electrophoresis, or with Tris‐CI/borate, the best resolution of the glucose oligomers containing 1–4 glucose units in Tris‐OAc, pH 8.8, ionic strength of 0.08, was obtained at 4.8% polyacrylamide concentration, using 0.5 M NH4OAc, pH 9.5 as the catholyte. Under those conditions, the ANTS‐glucose oligomers were separated with mobilities decreasing from glucose to maltohexaose. The linear Ferguson plots (log relative mobility, Rf, vs.%T) of the glucose oligomers show that the surface net charge of those oligomers is inversely related to their sizes, given by the slopes, KR, of the plots. The molecular weight of the oligomers is directly but nonlinearly related to KR. The novel electrophoretic system illustrated here for separation of short ANTS‐saccharides can be potentially applied to the resolution of other biomolecules such as rapidly migrating DNA, peptides or proteins.
Alternative sample preparation prior to two‐dimensional electrophoresis protein analysis on solid lipid nanoparticles
Authors: Göppert Torsten M. , Müller Rainer H.
Published: January 2004

The proteins adsorbing onto the surface of intravenously injected drug carriers are regarded as a key factor determining the organ distribution. Depending on the particle surface properties, certain proteins will be preferentially adsorbed, leading to the adherence of the particle to cells with the appropriate receptor. Therefore, the knowledge of the protein adsorption pattern and the correlation to in vivo behavior opens the perspective for the development of intravenous colloidal carriers for drug targeting. After incubation in plasma, the adsorbed proteins were analyzed using two‐dimensional polyacrylamide gel electrophoresesis (2‐D PAGE, 2‐DE). The purpose of the present study was to develop an alternative separation method to separate solid lipid nanoparticles (SLN) carriers from plasma by gel filtration prior to 2‐D PAGE. Via the specific absorption coefficients and a two‐equation system, elution fractions were identified being practically plasma‐free. This allows protein analysis on SLN which are typically in density too close to the density value of water to be separated by the standard centrifugation method. The SLN used for establishing the gel filtration were prepared in a way that they had a sufficiently low density to be additionally separated by centrifugation. The adsorption patterns obtained after separation with both methods were qualitatively and quantitatively identical, showing the suitability of the gel filtration.
Feasibility of nonvolatile buffers in capillary electrophoresis‐electrospray ionization‐mass spectrometry of proteins
Authors: Eriksson Jonas H. C. , Mol Roelof , Somsen Govert W. , Hinrichs Wouter L. J. , Frijlink Henderik W. , de Jong Gerhardus J.
Published: January 2004

The combination of capillary electrophoresis (CE) and electrospray ionization‐mass spectrometry (ESI‐MS) via a triaxial interface was studied as a potential means for the characterization of intact proteins. To evaluate the possibility to use a nonvolatile electrolyte for CE, the effect of sodium phosphate and ammonium borate on the MS signal of the proteins insulin, myoglobin, and bovine serum albumin (BSA) was investigated by employing infusion experiments, and compared to the effect of ammonium formate and formic acid. The study shows that with formic acid (50 mM, pH 2.4) the most intense protein signals were obtained, while the use of sodium phosphate buffer (5 and 10 mM, pH 7.5) almost completely diminished the MS response. Ammonium formate and ammonium borate (up to 100 mM, pH 8.5) also caused protein ion suppression, but especially with the borate buffer significant MS intensity remained. MS analysis of myoglobin revealed the loss of the heme group when an acidic CE electrolyte was used. Using a background electrolyte containing 25 mM ammonium borate (pH 8.5), it is demonstrated that a CE separation of a protein test mixture can be monitored with ESI‐MS without degrading the MS performance allowing molecular weight determinations of the separated compounds. In the presence of borate, detection limits were estimated to be 5–10 μM (ca. 100 fmol injected). The usefulness of the CE‐MS system employing a borate buffer is indicated by the analysis of a stored sample of BSA revealing several degradation products. A sample of placental alkaline phosphatase (PLAP), a potential therapeutic agent, was also analyzed by CE‐MS indicating the presence of a protein impurity. Probably due to insufficient ionization of the PLAP (a complex glycoprotein), no MS signals of the intact protein were observed.
Zeta potential determination by streaming current modelization and measurement in electrophoretic microfluidic systems
Authors: Renaud Louis , Kleimann Pascal , Morin Pierre
Published: January 2004

Electrophoresis in capillary and microfluidic systems, used in analytical chemistry to separate charged species, are quite sensitive to surface phenomena in terms of separation performances. In order to improve theses performances, new surface functionalization techniques are required. There is a need for methods to provide fast and accurate quantification about surface charges at liquid/solid interfaces. We present a fast, simple, and low‐cost technique for the measurement of the zeta‐potential, via the modelization and the measurement of streaming currents. Due to the small channel cross section in microfluidic devices, the streaming current modelization is easier than the streaming potential measurement. The modelization combines microfluidic simulations based on the Navier‐Stokes equation and charge repartition simulations based on the Poisson‐Boltzmann equation. This method has been validated with square and circular cross section shape fused‐silica capillaries and can be easily transposed to any lab‐on‐chip microsystems.
Analytical and preparative native polyacrylamide gel electrophoresis: Investigation of the recombinant and natural major grass pollen allergen Phl p 2
Authors: Suck Roland , Petersen Arnd , Weber Bernhard , Becker Wolf‐Meinhard , Fiebig Helmut , Cromwell Oliver
Published: January 2004

Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blot are amongst the most popular methods for allergen characterization, such as comparison of recombinant allergens with their natural counterparts. Native PAGE was evaluated as a possible robust and simple method offering high‐resolution capacity for characterization of the major grass pollen allergen Phl p 2. Analytical separation of recombinant Phl p 2 provided a superior quality control in terms of homogeneity and, after Western blotting, immunoglobulin E (IgE) reactivity. Separation of natural Phl p 2 identified two major isoforms which were shown to have different N‐terminal sequences and IgE‐binding properties. After isolation using preparative native PAGE in combination with electrodialysis, both isoforms were investigated by specific proteolysis and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The results demonstrate differences in the primary structures and that the recombinant counterpart corresponds exactly to one isoform. Analytical and preparative native PAGE thus proved to be powerful tools for the investigation of allergen isoforms and quality control of recombinant counterparts.
Study of the development of thermoresistance in human pancreatic carcinoma cell lines using proteome analysis
Authors: Poland Julia , Urbani Andrea , Lage Hermann , Schnölzer Martina , Sinha Pranav
Published: January 2004

In order to find candidate proteins that are potentially associated with the thermoresistant phenotype in combination with drug resistance, we analyzed the differential protein expression in vitro in the human pancreatic cancer cell line EPP85‐181‐P and classical and atypical multidrug‐resistant variants and their thermoresistant counterparts using proteomics. This study identifies sets of proteins that may lead to the development of thermoresistance. These results provide a fundamental basis to elucidate the molecular mechanism of thermoresistance and chemoresistance phenomena that may assist the therapy of inoperable cancers.
A systematic investigation into the recovery of radioactively labeled proteins from sodium dodecyl sulfate‐polyacrylamide gels
Authors: Zhou Shaobo , Bailey Matthew J. , Dunn Michael J. , Preedy Victor R. , Emery Peter W.
Published: January 2004

We report the results of a systematic investigation designed to optimize a method for quantifying radioactivity in proteins in sodium dodecyl sulfate‐polyacrylamide gels. The method involves dissolving appropriately sized pieces of gel in hydrogen peroxide and heating to 70°C overnight followed by liquid scintillation counting. H2O2 had no effect on the count rates of [14C]bovine serum albumin (BSA) when counted in a conventional liquid scintillation system, and the count rates remained stable for several days. Temperatures below 70°C resulted in incomplete extraction of radioactivity from gels containing [14C]BSA, but there was also a significant reduction in count rates in samples incubated at 80°C. At 70°C recovery was not affected by the amount of sample loaded onto the gel or by the staining procedure (Coomassie Brilliant Blue or SYPRO Ruby). Recoveries were in the range of 89–94%, and the coefficient of variation for five replicate samples was 5–10%. This method offers a reliable way of measuring the amount of radioactivity in proteins that have been separated by electrophoresis. It may be useful, for example, in quantitative metabolic labeling experiments when it is necessary to know precisely how much tracer has been incorporated into a particular protein.
Direct ampholyte‐free liquid‐phase isoelectric peptide focusing: Application to the human serum proteome
Authors: Xiao Zhen , Conrads Thomas P. , Lucas David A. , Janini George M. , Schaefer Carl F. , Buetow Kenneth H. , Issaq Haleem J. , Veenstra Timothy D.
Published: January 2004

In this study, we utilized a multidimensional peptide separation strategy combined with tandem mass spectrometry (MS/MS) for the identification of proteins in human serum. After enzymatically digesting serum with trypsin, the peptides were fractionated using liquid‐phase isoelectric focusing (IEF) in a novel ampholyte‐free format. Twenty IEF fractions were collected and analyzed by reversed‐phase microcapillary liquid chromatography (μLC)‐MS/MS. Bioinformatic analysis of the raw MS/MS spectra resulted in the identification of 844 unique peptides, corresponding to 437 proteins. This study demonstrates the efficacy of ampholyte‐free peptide autofocusing, which alleviates peptide losses in ampholyte removal strategies. The results show that the separation strategy is effective for high‐throughput characterization of proteins from complex proteomic mixtures.
Pyrosequencing™ technology at elevated temperature
Authors: Eriksson Jonas , Gharizadeh Baback , Nordström Tommy , Nyrén Pål
Published: January 2004

To date, the Pyrosequencing™ technology has been performed at 28°C due to the low thermostability of the firefly luciferase. In this study, firefly luciferase was stabilized in the presence of glycine betaine, allowing DNA sequencing at 37°C. By increasing the temperature to 37°C, false signals due to primer‐dimers and loop‐structures were decreased significantly. In addition, a combination of (i) replacing the natural dGTP with 7'deaza‐dGTP in the polymerase chain reaction (PCR), (ii) 1.6 M glycine betaine, and (iii) an increase of the temperature to 37°C enabled us to sequence a DNA template with the initial sequence 3'‐ATGGCCCGGGGGGGAGCTCCA … 5'. Furthermore, we describe a method to analyze if a primer forms a primer‐dimer with extendable 3'‐ends.
Vapor and liquid phase detection of cyanide on a microchip
Authors: Lu Qin , Collins Greg E. , Evans Thomas , Hammond Mark , Wang Joseph , Mulchandani Ashok
Published: January 2004

A capillary electrophoresis microchip is used to selectively and sensitively monitor cyanide levels in both vapor (HCN(g)) and aqueous (NaCN in drinking water) phases. Laser‐induced fluorescence detection is applied using a violet diode laser to monitor the fluorescent isoindole derivative formed by the reaction of cyanide with 2,3‐naphthalenedicarboxaldehyde (NDA) and taurine. Air sampling of hydrogen cyanide is achieved using a miniature impinger (2 mL), giving collection efficiencies as high as 79% for a sampling rate of 1.0 L/min and a 10 s sampling time (relative standard deviation RSD: 2.7% for n = 5). Following the addition of NDA and taurine to either the vapor phase impinger sample or an aqueous drinking water sample, the NDA/cyanide derivative can be detected in just over 40 s on the microchip, giving a detection limit of 0.56 μg/L and a linear dynamic range from 0.56 μg/L–2.4 mg/L. The detection limit for hydrogen cyanide in air was determined to be 2.3 ppb (mole%). On‐chip derivatization of cyanide by NDA was successful, although a 50% decrease in signal intensity was observed due to insufficient time for completion of the reaction on the microchip. A number of different interferents were examined, and only iron(II) and chlorine showed any interference due to their capability for masking the presence of cyanide by reacting with free cyanide in solution.
Modelling and simulation of fires in vehicle tunnels
Authors: Gasser I. , Struckmeier J. , Teleaga I.
Published: January 2004

Applying a low‐Mach asymptotic for the compressible Navier–Stokes equations, we derive a new fluid dynamics model,which should be capable to model large temperature differences in combination with the low‐Mach number limit. The model is used to simulate fires in vehicle tunnels, where the standard Boussinesq‐approximation for the incompressible Navier–Stokes seems to be inappropriate due to the high temperatures developing in the tunnel. The model is implemented using a modified finite‐difference approach for the incompressible Navier–Stokes equations and tested in some realistic fire events. Copyright © 2004 John Wiley & Sons, Ltd.
Newton linearization of the incompressible Navier–Stokes equations
Authors: Sheu Tony W. H. , Lin R. K.
Published: January 2004

The present study aims to accelerate the convergence to incompressible Navier–Stokes solution. For the sake of computational efficiency, Newton linearization of equations is invoked on non‐staggered grids to shorten the sequence to the final solution of the non‐linear differential system of equations. For the sake of accuracy, the resulting convection–diffusion–reaction finite‐difference equation is solved line‐by‐line using the proposed nodally exact one‐dimensional scheme. The matrix size is reduced and, at the same time, the CPU time is considerably saved due to the decrease of stencil points. The effectiveness of the implemented Newton linearization is demonstrated through computational exercises. Copyright © 2004 John Wiley & Sons, Ltd.
Neurotrophin 3 promotes purification and proliferation of olfactory ensheathing cells from human nose
Authors: Bianco John I. , Perry Chris , Harkin Damien G. , Mackay‐Sim Alan , Féron François
Published: January 2004

Several studies have demonstrated the potential of olfactory ensheathing cells for the repair of central and peripheral nerve injury. However, the majority of these studies have been performed with olfactory ensheathing cells derived from the olfactory bulbs, situated inside the skull. A more clinically relevant source of olfactory ensheathing cells is the olfactory mucosa, located in the nose. To be successful, an autologous transplant of nasal ensheathing glia would require a large number of purified cells. To address this issue, we have focused our research on three neurotrophic factors, namely nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), and neurotrophin 3 (NT3). We show here that their respective receptors, TrkA, TrkB, TrkC, as well as p75NTR (the low affinity NGF receptor), are expressed in vitro by the nasal ensheathing cells; the three neurotrophins promote purification and proliferation of these glial cells, with an optimal concentration of 50 ng/ml; and human ensheathing cells can be easily biopsied and highly purified using a serum‐free medium supplemented with NT3. This technique opens the door for clinical trials in which nasal ensheathing cells will be autotransplanted in humans suffering from nerve injury. © 2003 Wiley‐Liss, Inc.
Association of the ecto‐ATPase NTPDase2 with glial cells of the peripheral nervous system
Authors: Braun Norbert , Sévigny Jean , Robson Simon C. , Hammer Klaus , Hanani Menachem , Zimmermann Herbert
Published: January 2004

Cellular signaling via extracellular nucleotides appears to play a major role in the functioning of the peripheral nervous system. Information regarding the functional characterization of nucleotide P2 receptors or their expression pattern has been accumulating rapidly; however, very little is known regarding the distribution of ecto‐nucleotidases in the periphery. The extracellular level of nucleotides is controlled by ecto‐nucleotidases, whereby the three membrane‐bound members of the ecto‐nucleoside triphosphate diphosphohydrolase (E‐NTPDase) family are of special functional importance. Using enzyme histochemistry and immunostaining, we demonstrate that NTPDase2 is associated with nonmyelinating Schwann cells of the rat sciatic nerve, whereas NTPDase1 is restricted to blood vessel walls. NTPDase2 immunoreactivity was detected from embryonic day E18 onward, suggesting that immature Schwann cells express the enzyme. With the onset of myelination, NTPDase2 immunoreactivity remained associated solely with nonmyelinating Schwann cells. NTPDase2 was absent from perisynaptic Schwann cells but was associated with fibroblasts covering the endplate at some distance. In addition, NTPDase2 immunoreactivity was associated with the satellite glial cells in dorsal root ganglia and sympathetic ganglia, and with the enteric glia surrounding the cell bodies of ganglionic neurons of the myenteric and the submucous plexus. In contrast to NTPDase1, NTPDase2 preferentially hydrolyzes nucleoside triphosphates over nucleoside diphosphates and thus can act either in inactivating or in producing P2 receptor ligands. Our results suggest that NTPDase2 plays an important role in the control of nucleotide‐mediated activation of peripheral neurons or glia and in the dialogue between these two cell types. © 2003 Wiley‐Liss, Inc.
Activated glia induce neuron death via MAP kinase signaling pathways involving JNK and p38
Authors: Xie Zhong , Smith Carolyn J. , Van Eldik Linda J.
Published: January 2004

Chronic glial activation in neurodegenerative diseases contributes to neuronal dysfunction and neuron loss through production of neuroinflammatory molecules. However, the molecular mechanisms, particularly the signal transduction pathways involved in glia‐dependent neuron death, are poorly understood. As a first step to address this question, we used a neuron‐glia co‐culture system that allows diffusion of soluble molecules between glia and neurons to test the potential importance of mitogen‐activated protein kinase (MAPK) signaling pathways in the glia‐induced neuron death. Activation of glia in co‐culture by lipopolysaccharide (LPS) induced apoptotic‐like neuron death. The MAPKs tested (p38, JNK, ERK1/2) were activated in both glia and neurons following LPS treatment, suggesting their involvement in both glial activation and neuronal response to diffusible, glia‐derived neurotoxic molecules. Inhibitors of p38 and JNK partially blocked neuron death in the LPS‐treated co‐culture, whereas an ERK1/2 pathway inhibitor did not protect neurons. These results show that p38 and JNK MAPKs, but not ERK1/2 MAPK, are important signal transduction pathways contributing to glia‐induced neuron death. © 2003 Wiley‐Liss, Inc.
Cloning, transport properties, and differential localization of two splice variants of GLT‐1 in the rat CNS: Implications for CNS glutamate homeostasis
Authors: Sullivan Robert , Rauen Thomas , Fischer Frauke , Wießner Michael , Grewer Christof , Bicho Ana , Pow David V.
Published: January 2004

At least two splice variants of GLT‐1 are expressed by rat brain astrocytes, albeit in different membrane domains. There is at present only limited data available as to the spatial relationship of such variants relative to the location of synapses and their functional properties. We have characterized the transport properties of GLT‐1v in a heterologous expression system and conclude that its transport properties are similar to those of the originally described form of GLT‐1, namely GLT‐1α. We demonstrate that GLT‐1α is localized to glial processes, some of which are interposed between multiple synapse types, including GABAergic synapses, whereas GLT‐1v is expressed by astrocytic processes, at sites not interposed between synapses. Both splice variants can be expressed by a single astrocyte, but such expression is not uniform over the surface of the astrocytes. Neither splice variant of GLT‐1 is evident in brain neurons, but both are abundantly expressed in some retinal neurons. We conclude that GLT‐1v may not be involved in shaping the kinetics of synaptic signaling in the brain, but may be critical in preventing spillover of glutamate between adjacent synapses, thereby regulating intersynaptic glutamatergic and GABAergic transmission. Furthermore, GLT‐1v may be crucial in ensuring that low levels of glutamate are maintained at extrasynaptic locations, especially in pathological conditions such as ischemia, motor neurone disease, and epilepsy. © 2003 Wiley‐Liss, Inc.
Downregulation of steroidogenic acute regulatory protein (StAR) gene expression by cyclic AMP in cultured Schwann cells
Authors: Benmessahel Yasmina , Troadec Jean‐Denis , Cadepond Françoise , Guennoun Rachida , Hales Dale Buchanan , Schumacher Michael , Groyer Ghislaine
Published: January 2004

Steroidogenic acute regulatory protein (StAR) plays a key role in the availability of cholesterol to the inner mitochondrial membrane, where the first step of steroidogenesis, its conversion to pregnenolone, takes place. Here, we demonstrate for the first time that the StAR gene is also expressed in the rat sciatic nerve and in cultured Schwann cells. The addition to the culture medium of the cAMP‐elevating agent forskolin or of the cAMP analogue 8Br‐cAMP produced a time‐course extinction of StAR gene expression. An inverse relationship was demonstrated between StAR gene expression and the intracellular cAMP content. Accordingly, pharmacological inhibition of the activities of Schwann cell adenylyl cyclase or of phosphodiesterase IV resulted in modifications of StAR gene expression. Since StAR gene expression is stimulated by cAMP in classical steroidogenic cells, our work is the first demonstration of a negative regulation of StAR gene by cAMP. © 2003 Wiley‐Liss, Inc.
Sulfatide is a negative regulator of oligodendrocyte differentiation: Development in sulfatide‐null mice
Authors: Hirahara Yukie , Bansal Rashmi , Honke Koichi , Ikenaka Kazuhiro , Wada Yoshinao
Published: January 2004

Galactosylceramide (GalC) and its sulfated analogue, sulfatide, are major galactosphingolipid components of myelin and oligodendrocyte plasma membranes in the nervous system. We previously hypothesized that these galactolipids play functional roles in the regulation of oligodendrocyte terminal differentiation by acting as sensors/transmitters of environmental information. Evidence strongly supports this idea. First, these molecules are initially expressed on the cell surface at the interface at which oligodendrocyte progenitors first enter terminal differentiation. Second, exposure of oligodendrocyte progenitors to anti‐GalC/‐sulfatide (RmAb) or antisulfatide (O4), but not anti‐GalC (O1), antibodies leads to the reversible arrest of oligodendrocyte lineage progression at this interface. Third, in cerebroside galactosyl transferase‐null mice (Cgt−/−) that are unable to synthesize either GalC or sulfatide, terminal differentiation and morphological maturation of oligodendrocytes are enhanced. In the present study, we examined oligodendrocytes differentiation in cerebroside sulfotransferase‐null mice (Cst−/−) that lack sulfatide but express GalC. We show that cerebroside sulfotransferase mRNA expression begins already in the embryonic spinal cord and progressively increases with age, that the late progenitor marker POA is not synthesized in the absence of this enzyme, and that, most notably, there is a two‐ to threefold enhancement in the number of terminally differentiated oligodendrocytes both in culture and in vivo, similar to that in mice lacking both GalC and sulfatide. We conclude that primarily sulfatide, rather than GalC, is a key molecule for the negative regulation of oligodendrocyte terminal differentiation. © 2003 Wiley‐Liss, Inc.
Glial activation modulates glutamate neurotoxicity in cerebellar granule cell cultures
Authors: Pérez‐Capote Kamil , Serratosa Joan , Solà Carme
Published: January 2004

We studied the influence of glial cells on the neuronal response to glutamate toxicity in cerebellar granule cell cultures. We compared the effect of glutamate on neuronal viability in neuronal vs. neuronal‐glial cultures and determined this effect after pretreating the cultures with the lipopolysaccharide (LPS) of Escherichia coli, agent widely used to induce glial activation. Morphological changes in glial cells and nitric oxide (NO) production were evaluated as indicators of glial activation. We observed that glutamate neurotoxicity in neuronal‐glial cultures was attenuated in a certain range of glutamate concentration when compared to neuronal cultures, but it was enhanced at higher glutamate concentrations. This enhanced neurotoxicity was associated with morphological changes in astrocytes and microglial cells in the absence of NO production. LPS treatment induced morphological changes in glial cells in neuronal‐glial cultures as well as NO production. These effects occurred in the absence of significant neuronal death. However, when LPS‐pretreated cultures were treated with glutamate, the sensitivity of neuronal‐glial cultures to glutamate neurotoxicity was increased. This was accompanied by additional morphological changes in glial cells in the absence of a further increase in NO production. These results suggest that quiescent glial cells protect neuronal cells from glutamate neurotoxicity, but reactive glial cells increase glutamate neurotoxicity. Therefore, glial cells play a key role in the neuronal response to a negative stimulus, suggesting that this response can be modified through an action on glial cells. © 2003 Wiley‐Liss, Inc.
Cost‐effectiveness analysis and health care resource allocation: decision rules under variable returns to scale
Authors: Elbasha Elamin H. , Messonnier Mark L.
Published: January 2004

We argue that health programs are administered in settings that often violate the frequently stated assumption of constant returns to scale in the provision of health services. Three types of returns to scale are identified from the general economic literature: returns to scale with respect to population, effectiveness, and quality. We show that decision rules based on incremental cost‐effectiveness ratios or cost–benefit tests are not optimal if returns to scale are not constant. We derive the optimal decision rules under variable returns to scale using optimization techniques, and employ several examples to illustrate the concepts and methods. Published in 2003 by John Wiley & Sons, Ltd.
The value of risk‐free cigarettes – do smokers underestimate the risk?
Authors: Hammar Henrik , Johansson‐Stenman Olof
Published: January 2004

The health risk of smoking is valued using the contingent valuation method, applied to a Swedish sample of smokers. The respondents were asked to put a value on newly developed cigarettes with no associated health risks. The average additional willingness to pay for the new cigarettes is estimated to be between 10 and 41 SEK per pack, where the variation is due to statistical method, discounting, and whether the open‐ended or closed‐ended question format is used. Using medical data on life shortening effects of smoking, the results indicate rather low values put on a lost life‐year, compared to most existing estimates based on other methods. This may indicate that smokers do underestimate the health risk of smoking. There is also initial optimism‐bias regarding people's own ability to quit smoking at will. However, there are remaining methodological questions and we found little or no sensitivity to scope. Copyright © 2003 John Wiley & Sons, Ltd.
Responses to standard gambles: are preferences ‘well constructed’?
Authors: Baker Rachel , Robinson Angela
Published: January 2004

Standard gamble (SG) is commonly used to elicit preferences in order to assess health related quality of life. There has been little qualitative research exploring how respondents answer such questions.

An SG study was designed to elicit values for the health states associated with anti‐hypertensive medication, stroke and cardiovascular disease. This paper describes a qualitative study that was carried out alongside the SG exercise in order to document the thought processes respondents bring to bear in formulating their responses. Data were generated using ‘think aloud’ techniques and semi‐structured interviews. Values were generally well‐constructed: responses were thoroughly considered, and respondents made complex trade‐offs and arrived at a point of indifference. However, some respondents incorporated inappropriate information into their choices, redefining the hypothetical ‘Option B’ resulting in problems interpreting the probabilistic information. Consideration of non‐health factors was commonplace, in particular the impact of choices on others. We discuss these findings in terms of the use of qualitative methods in health economics and the wider discourse surrounding the theoretical underpinnings of health state valuation. Copyright © 2003 John Wiley & Sons, Ltd.
Cost‐effectiveness analysis based on the number‐needed‐to‐treat: common sense or non‐sense?
Authors: Sønbø Kristiansen Ivar , Gyrd‐Hansen Dorte
Published: January 2004

This paper explores and critically discusses some of the methodological limitations of using the number‐needed‐to‐treat (NNT) in economic evaluation. We argue that NNT may be a straightforward measure of benefit when the effect of an intervention is immediate, but that serious problems arise when the effect is delay rather than avoidance of an adverse event. In this case, NNT is not a robust or accurate measure of effect, but will vary considerably and inconsistently over time. This weakness will naturally spill over onto any CEA based on NNT. A literature review demonstrated that CEAs based on NNT were all published within the last five years, and that all studies suffered from important limitations. A major weakness of using NNT is the imposed restrictions on the outcome measure, which can only be strictly uni‐dimensional and non‐generic. Using NNT in economic evaluations is obtained at a cost in terms of both methodological shortcomings, and a reduced ability for such evaluations to serve as a useful tool in decision making processes. The use of NNT in economic evaluations might be better avoided.

To every complicated question, there is a simple, straightforward, easy – and probably wrong answer (Occam's Sledgehammer). Copyright © 2003 John Wiley & Sons, Ltd.
Insomnia, biological clock, and the bedtime decision: an economic perspective
Authors: Yaniv Gideon
Published: January 2004

While insomnia can be a symptom of numerous mental and physical illnesses, it is frequently diagnosed as a sleep disorder in its own right, caused mainly by stressful life events or by non‐synchronization of individuals' biological sleep–wake cycle with the one they choose to practice. Because of irregular work schedules, late‐night entertainment, or rapid crossing of several time zones, individuals might retire to bed earlier or later than their biological bedtime, experiencing difficulties in falling asleep. The present paper develops a simple economic model of the bedtime decision, viewing the individual as a rational decision‐maker who determines her insomnia level through consciously weighing the cost and benefit of deviating from her biological bedtime. The model is then used to examine the individual's response to stress, yielding a prediction which is consistent with observed behavior, although not with sleep therapists' recommendations. Finally, the model is applied to the case of transmeridian flights, explaining jet lag as a rational adjustment to a misalignment between the individual's slow‐to‐adapt internal clock and her external environment. Copyright © 2003 John Wiley & Sons, Ltd.
Simulation of a Hirschman–Herfindahl index without complete market share information
Authors: Nauenberg Eric , Alkhamisi Mahdi , Andrijuk Yuri
Published: January 2004

This paper utilizes maximum likelihood methods to simulate a Hirschman–Herfindahl index (HHI) for markets in which complete market share information is unavailable or delayed. Many jurisdictions either may be unable to administratively collect data or experience delays in collection that make data regarding turbulent markets of limited use. With the development of this method, regulatory authorities monitoring health‐care competition or health‐care firms can now use market surveys – in which reliable recall is often limited to the largest three or four firms – to produce an on‐the‐spot measure of market concentration. Copyright © 2003 John Wiley & Sons, Ltd.
Increasing patient choice in primary care: the management of minor ailments
Authors: Bojke Chris , Gravelle Hugh , Hassell Karen , Whittington Zoe
Published: January 2004

We examine the effects of an intervention to provide easier access to pharmacists for patients with minor ailments. The intervention allowed pharmacists to prescribe and dispense medicines currently limited to general practitioners (GPs) without patients losing their right to free prescriptions. We show that the total number of GP consultations was unaffected by the intervention but that the proportion which were for minor ailments decreased. We also use estimate multinomial models of patient choices between GP and pharmacies and find that the main determinant is the type of minor ailment. Distance appears to have no effect on patient choice. Copyright © 2003 John Wiley & Sons, Ltd.
The effects of higher cigarette prices on tar and nicotine consumption in a cohort of adult smokers
Authors: Farrelly M.C. , Nimsch C.T. , Hyland A. , Cummings M.
Published: January 2004

The objective of this paper is to estimate the demand for tar and nicotine in cigarettes as a function of cigarette prices in a cohort of cigarette 11 966 smokers followed for 5 years. Data for the analysis come from a longitudinal telephone survey of 11 966 smokers who were interviewed in 1988 and 1993 as part of the Community Intervention Trial for Smoking Cessation (COMMIT). Separate models are estimated for three age groups to account for differences in levels of addiction and brand loyalty across age. We found that smokers respond to higher cigarette prices by reducing the number of cigarettes smoked per day but also by switching to cigarettes that are higher in tar and nicotine per cigarette. Copyright © 2003 John Wiley & Sons, Ltd.
Oral mucositis: A challenging complication of radiotherapy, chemotherapy, and radiochemotherapy. Part 2: Diagnosis and management of mucositis
Authors: Scully Crispian , Epstein Joel , Sonis Stephen
Published: January 2004

Background.

Oral mucositis is a common sequel of radiotherapy, chemotherapy, and radiochemotherapy in patients with cancer or patients requiring hemopoietic stem cell transplants. Mucositis has a direct and significant impact on the duration of disease remission and cure rates, because it is a treatment‐limiting toxicity. Mucositis also affects survival because of the risk of infection and has a significant impact on quality of life and cost of care.

Methods.

This article reviews publications on the diagnosis and management of oral mucositis accessible from a MEDLINE search using as key words mucositis, radiotherapy, chemotherapy, hemopoietic stem cell transplant, and oral.

Conclusions.

Conventional care of patients with mucositis is currently essentially palliative, with good oral hygiene, narcotic analgesics, and topical palliative mouth rinses. © 2004 Wiley Periodicals, Inc. Head Neck 26: 77–84, 2004
The expression of key cell cycle markers and presence of human papillomavirus in squamous cell carcinoma of the tonsil
Authors: Li Wei , Thompson Carol H. , Cossart Yvonne E. , O'Brien Christopher J. , McNeil Edward B. , Scolyer Richard A. , Rose Barbara R.
Published: January 2004

Background.

Chemical carcinogens induce squamous cell carcinoma (SCC) of the head and neck by targeting the p53 and the retinoblastoma (pRb) pathways. Human papillomavirus (HPV) might have an etiologic role in these cancers at particular sites. Few studies have compared cell cycle protein expression in HPV‐positive and HPV‐negative tumors in this region.

Methods.

Fifty tonsil SCCs were analyzed for HPV by PCR and for expression of cell cycle proteins (p53, pRb, p16INK4A, p21CIP1/WAF1, p27KIP1, and cyclinD1) by immunohistochemistry.

Results.

HPV was present in 42%; almost all were type 16. There were statistical associations between HPV positivity and reduced expression of pRb and cyclinD1, overexpression of p16, and younger patient age. Tumor with down‐regulated p27 tended to have down‐regulated pRb and p21.

Conclusions.

HPV‐positive tonsil SCCs have distinct molecular pathways. Their association with younger patient age suggests that they are biologically distinct from HPV‐negative tumors. © 2004 Wiley Periodicals, Inc. Head and Neck 26: 1–9, 2004
Enhancing the outcome of free latissimus dorsi muscle flap reconstruction of scalp defects
Authors: Lipa Joan E. , Butler Charles E.
Published: January 2004

Background. Reconstruction of scalp and calvarial defects after tumor ablation frequently requires prosthetic cranioplasty and cutaneous coverage. Furthermore, patients often have advanced disease and receive perioperative radiotherapy. We evaluated the complications of scalp reconstruction with a free latissimus dorsi muscle flap in this setting.

Methods. The complications and the oncologic and aesthetic outcomes of six consecutive scalp reconstructions with a free latissimus dorsi muscle flap and skin graft in five patients with advanced cancer were retrospectively evaluated. Patient, tumor, defect, reconstructive, and other treatment characteristics were reviewed. Reconstructive and perioperative techniques intended to improve flap survival and aesthetic outcome and reduce complications in these patients.

Results. All patients (52–76 years old) had recurrent tumors (sarcoma, melanoma, or squamous cell carcinoma) and received postoperative radiotherapy. The mean scalp defect size was 367 cm2, and partial‐thickness or full‐thickness calvarial resection was required in all six cases. No vein grafts were needed. The mean follow‐up period and disease‐free survival time were 18 and 13 months, respectively. Three patients died of their disease, and two survived disease free. There were no flap failures or dehiscences. Complications consisted of donor site seroma in two patients; partial skin graft loss in one patient; and radiation burns to the flap, face, and ears in one patient. Scalp contour and aesthetic outcome were very good in all cases except for the one case with radiation burns.

Conclusions. Good outcomes were achieved using a free latissimus dorsi muscle flap with a skin graft for flap reconstruction in elderly patients with advanced recurrent cancers who received perioperative radiotherapy. Several technical aspects of the reconstruction technique intended to enhance the functional and aesthetic outcome and/or reduce complications were believed to have contributed to the good results. © 2004 Wiley Periodicals, Inc. Head Neck 26: 46–53, 2004