bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of neurotransmitter receptors following unilateral L1–S2 deafferentation: NK1, NK3, NMDA, and 5HT1a receptor binding autoradiography
Authors: Croul S. , Sverstiuk A. , Radzievsky A. , Murray M.
Published: May 2005

Following surgical deafferentation of the spinal cord, cut dorsal roots degenerate, and spared projections compensate for this loss by collateral sprouting (reactive reinnervation). Light microscopic immunocytochemistry has shown sprouting by selected undamaged intraspinal projections, including those that express the transmitters substance P and serotonin. Quantitative immunoelectron microscopy supports these results by demonstrating loss and subsequent recovery of substance P‐containing terminals and an increase in serotonin‐containing terminals. To test the hypothesis that changes in afferent innervation modulate neurotransmitter receptors on second‐order neurons, we used receptor binding autoradiography in this model. Adult rats were subjected to L1–S2 unilateral dorsal rhizotomy and killed at 1, 2, 6, or > 20 weeks after surgery. Receptor binding densities of tachykinin (neurokinins‐1 and ‐3), glutamate (N‐methyl‐D‐aspartate), and serotonin (serotonin‐1a) receptors were assayed in the lumbar dorsal horn. Neurokinin‐1 binding density was increased in lamina II of the deafferented side by 1 week after surgery, remained elevated at 2 weeks, and returned to control values by 6 weeks. Neurokinin 3 binding density was elevated at 2 weeks and then returned to control levels. N‐methyl‐D‐aspartate receptor binding showed slight but not statistically significant increased binding density at 6 and at > 20 weeks. No significant changes were found in serotonin‐1a receptor binding density. The elevations in tachykinin receptor binding density occur when afferents in the dorsal horn are degenerating and suggest reactive up‐regulation of the receptor. The return to normal levels coincides with reactive reinnervation in the spinal cord, which restores synaptic numbers. Changes in N‐methyl‐D‐aspartate binding occur much later than the restitution of synaptic numbers but may indicate a role for this receptor in synaptic stabilization following reactive reinnervation. © 1995 Wiley‐Liss, Inc.
Projections of ankle joint afferents to the spinal cord and brainstem of the chicken (Gallus G. domesticus)
Authors: Gentle Michael J. , Hunter Louise N. , Sterling Robert J.
Published: May 2005

The projections of the ankle joint capsule afferents were studied by transganglionic transport of horseradish peroxidase injected directly into the ankle joint. The number and size of the labelled dorsal root ganglion cells were measured from synsacral nerves 2–9. In the dorsal root ganglia, all sizes of sensory neurones were labelled, and the largest number of labelled cells was in ganglia 5–7. The extensive sympathetic innervation of the ankle joint was identified by the large number of cell bodies labelled in the sympathetic ganglia of the paravertebral chain. Labelled afferent fibres projected to the spinal cord from the 2nd to the 8th synsacral nerves, with the rostral projection mainly via Lissauer's tract and the dorsal funiculus. Terminal labelling in the dorsal horn was identified in laminae I–III and VI, with a slight projection to V. Two areas of dense labelling, which did not correspond with the largest number of labelled dorsal root ganglion cells, were identified. A rostral area with the highest density of label was observed at the level of synsacral nerves 3–4 and a second slightly less dense area between synsacral nerves 7–8. In the caudal medulla, diffuse terminal labelling was observed in the nucleus gracilis et cuneatus, nucleus of the tractus solitarius, and the nucleus cuneatus externus.

These results are discussed in a comparative context to identify similarities and differences between different primary afferent projections in birds and mammals and to highlight the possible functional significance of the avian articular afferent projection. © 1995 Wiley‐Liss, Inc.
Comparative ultrastructural localization of the NMDAR1 glutamate receptor in the rat basolateral amygdala and bed nucleus of the stria terminalis
Authors: Gracy K. Noelle , Pickel Virginia M.
Published: May 2005

The N‐methyl‐D‐aspartate (NMDA)‐type glutamate receptor in the basolateral amygdala (BLA) has been implicated in activity‐dependent plasticity important for cortically evoked acquisition of fear‐potentiated startle response. We examined the ultrastructural immunoperoxidase labeling of the R1 subunit of the NMDA receptor in the BLA of adult rats to determine the potential cellular and subcellular sites mediating the effects generated by NMDA activation. The localization was compared with that seen in the bed nucleus of the stria terminalis (BNST), the major efferent pathway from the central nucleus of the amygdala, which has a more pronounced involvement in autonomic function. Electron microscopy established that in the BLA, 68. 4% (n=177) of the profiles showing NMDAR1–like immunoreactivity (NMDAR1–LI) were dendrites, and 19. 8% were distal tips of astrocytic processes. In contrast, profiles containing NMDAR1–LI (n=262) in the BNST were more equally distributed between dendrites (37. 4%) and axons (38. 2%). The subcellular localization of NMDAR1 immunoreactivity was, however, similar in both regions. Our findings provide the first ultrastructural evidence that glutamate may prominently act through NMDAR1 receptors to elicit postsynaptic actions on intrinsic neurons in the BLA and BNST. The results also indicate that, in the BLA, the NMDAR1 receptor plays an important role in astrocytic function, whereas the receptor is more preferentially a presynaptic modulator in axons which terminate in or pass through the BNST. © Wiley‐Liss, Inc.
Metabotropic glutamate receptor mGluR5 subcellular distribution and developmental expression in hypothalamus
Authors: Van Den Pol Anthony N. , Romano Carmelo , Ghosh Prabhat
Published: May 2005

The metabotropic glutamate receptor mGluR5 is a G‐protein coupled receptor that plays a key role in release of Ca2+ from internal stores via inositol triphosphate mobilization. Western and Northern blot analyses revealed a greatly enhanced expression of mGluR5 in rats during early stages of hypothalamic development compared with the adult. This enhanced developmental expression provides an explanation for the dramatic physiological response of developing neurons to metabotropic glutamate receptor activation and supports the argument that metabotropic glutamate receptors may play an important role in hypothalamic development. During development, expression of the mGluR5 gene was reduced, not only in the hypothalamus but also in other regions of the brain. A differential decrease in mGluR5 protein was found in different brain regions with Western blot analysis. The hypothalamus showed a sixfold decrease in mGluR5 with dovelopment, whereas the cortex showed only a threefold decrease.

Immunocytochemistzy with an afflnity‐purified antibody against a peptide deduced from the cloned mGluR5 gene revealed selective expression in some regions in the adult hypothalamus. In the adult and developing (postnatal day 10) brain, immunoreactive neurons were found in the suprachiasmatic nucleus, preoptic area, lateral hypothalamus, and mammillary region, areas where the related metabotropic glutamate receptor mGluR1 is also found. In contrast, the ventromedial nucleus, an area critically involved in the regulation of food intake and metabolic balances, showed strong mGluR5 immunoreactivity but no mGluR1 immunoreactivity. Little or no mGluR5 staining was found in the neurosecretory neurons of the paraventricular, supraoptic, and arcuate nuclei. Ultrastructurally, mGluR5 was associated with the cytoplasmic face of the plasmalemma on hypothalamic dendrites, dendritic spines, and neuronal perikarya in the adult. The strongest immunoreactivity was found in patches on the membrane, sometimes associated with the postsynaptic side of synapses and sometimes associated with nonsynaptic dendritic or perikaryal membrane. Intense immunostaining was found on some astrocyte processes surrounding synaptic complexes containing asymmetrical synapses. These astrocytes would be in an ideal position to receive excitatory signals from glutamatergic axons. Unlike the punctate appearance of immunolabeling on neuronal membranes, astrocytes showed continuous staining along the plasma membrane. © Wiley‐Liss, Inc.
Reduced gravity evaluation of potential spaceflight‐compatible flow cytometer technology
Authors: Crucian Brian , Sams Clarence
Published: May 2005

Background

The presence of a spaceflight‐compatible flow cytometer onboard the International Space Station would be extremely beneficial for in‐flight medicine and reduced gravity research. In the past, commercially available flow cytometers were not suitable for use during spaceflight due to their size, weight, power, and sheath fluid requirements. Recent advances in cytometer technology have enabled features that now make a spaceflight‐compatible cytometer possible. We evaluated a small, robust novel cytometer with design features that minimize or eliminate many incompatibilities with spaceflight. This cytometer is highly miniaturized and lightweight, does not require sheath fluid, and uses a low‐energy diode laser. The ability to achieve laminar particle flow without sheath fluid is important because an instrument using large liquid volumes and producing an equivalent amount of hazardous liquid waste would not meet spaceflight constraints.

Methods

For this study the cytometer was modified so that stained liquid cell samples could be delivered during reduced gravity. The cytometer was then evaluated onboard the NASA KC‐135 reduced gravity research aircraft. The KC‐135 uses a parabolic flight path to generate essentially zero gravity conditions for 30‐s increments. Typically 40 parabolas are flown per mission, resulting in approximately 20 min of reduced gravity during which research may be performed. During this evaluation, bead‐based cytometer precision, photomultiplier tube linearity, and leukocyte immunophenotype analysis were performed during reduced gravity. The flight data were then compared with ground‐based control data and data generated using a reference cytometer (Beckman‐Coulter XL).

Results

This novel cytometer functioned well during reduced gravity and produced data comparable to those of ground‐based controls with only minor caveats. The reduced gravity cell immunophenotype data were indistinguishable from ground control data and reference cytometric data. Bead‐based instrument precision (coefficient of variation) was slightly increased during reduced gravity operation, but not to a degree that would affect most common flow cytometric applications. The ability of the instrument to collect absolute cell counts was validated.

Conclusions

This study represents the first generation of real‐time flow cytometry data during zero gravity. With modifications, the evaluated cytometer technology could be the basis from which an operational spaceflight‐compatible flow cytometer is developed. Published 2005 Wiley‐Liss, Inc.
Piracetam in the treatment of cognitive impairment in the elderly
Authors: Reisberg Barry , Ferris Steven H. , Schneck Michael K. , Corwin Jene , Mir Pervez , Friedman Eitan , Sherman Kathleen A. , McCarthy Martin , Bartus Raymond T.
Published: May 2005

Reisberg, B., S.H. Ferris, M.K. Schneck, J. Corwin, P. Mir, E. Friedman, K.A. Sherman, M. McCarthy, and R.T. Bartus: Piracetam in the treatment of cognitive impairment in the elderly. Drug Dev. Res. 2: 475–480, 1982.

Piracetam (Nootropil, 2‐oxopyrrolidone acetamide) has been extensively investigated for the treatment of cognitive impairment. Initial studies on normal subjects and patients with mild or moderate cognitive decline have been somewhat encoruaging. Accordingly, we conducted a further evaluation of the effects of piracetam in the treatment of elderly outpatients 60 to 85 years of age with mild to moderate memory impairment consistent with a diagnosis of Primary Degenerative Dementia (PDD). In our first study, we examined the effects of piracetam in 20 patients. All patients received 7.2 g of piracetam and placebo for 4 weeks in accordance with a double‐blind, randomized treatment order, crossover design with 1‐week washout periods prior to each crossover period. Hence, the total study period for each patient was 10 weeks (1‐4‐1‐4). An analysis of 43 psychometric measures revealed significant improvement (P < 0.05) in only three measures, all favoring the treatment condition. Recent findings support a rationale for examining the effects of piracetam in conjunction with cholinergic precursors in patients with cognitive decline. In our second study we conducted a 1‐week open trial of 1.6 g of piracetam t.i.d. in conjunction with 3 g of choline cholride t.i.d. in 15 patients. Four patients were rated as clinically improved. These “responders” were all subjects with moderate cognitive impairment. The responders showed much higher RBC choline levels than the nonresponders, both at baseline and during treatment. We conclude that the present evidence indicates that the effects of piracetam treatment alone in elderly outpatients with mild to moderate congnitive decline are subtle and not of proven clinical significance. However, studies of longer duration and of piracetam in combination with other agents may eventually show genuine clinical utility.
New methods to determine the CNS effects of antigeriatric compounds: EEG topography and glucose use
Authors: Buchsbaum Montes S. , Cappelletti John , Coppola Richard , Regal Francois , King Anna C. , van Kammen D. P.
Published: May 2005

Buchsbaum, M.S., J. Cappelletti, R. Coppola, F. Regal, A. C. King, and D. P. van Kammen: New methods to determine the CNS effects of antigeriatric compounds: EEG topography and glucose use. Drug Dev. Res. 2:489–496, 1982.

It has previously been possible to measure regional glucose use in the cerebral cortex in animals only with autoradiographic techniques. With the advent of position emission tomography (PET) using (18F‐2DG) 18F‐2‐deoxyglucose, it is now possible to assess local glucose uptake in μmol/100 g tissue/min in normal volunteers or patients. The PET technique is complex and costly and cannot be repeated frequently because of the radiation dosage. However, local glucose use is closely tied to cerebral blood flow, and studies have related blood flow to EEG frequency measures. In this study, we have simultaneously investigated local glucose metabolism using 18F‐2DG with PET and EEG frequency with 16‐lead topographic mapping in six normal controls. Subjects sat in an acoustically treated darkened room with eyes closed for 10 min prior to, and 30 min following injection of 3–5 mCi 18F‐2DG. Following uptake, seven to eight horizontal scans parallel to the canthomeatal line were made. EEG recordings are made beginning 1 min after injection of the isotope and continuing for 30 min with 12 standard 10/20 system points on the left hemisphere and midline, and 4 additional points between existing posterior leads. Ten‐second EEG epochs are edited for artifacts and then analyzed by fast Fourier transform techniques. Using a cross‐sectional whole‐head atlas, a standardized, approximately equal‐area two‐dimensional representation of a lateral view of the brain was developed and 10/20 system scalp coordinates were projected onto it. EEG power estimates are interpolated for all points on this brain map. Using digital techniques, a 1‐cm‐thick cortical strip is peeled off each PET, slice and conformed to the lateral brain view, and values between strips are interpolated. The result is two simultaneously obtained electrophysiologic and metabolic lateral views of brain function displayed in gray‐scale values represented by dot density. In some subjects with eyes closed, alpha power is high in low glucose‐use regions, such as the occiput. Temporal regions appear low both in glucose use and in most power bands. Parallels between alpha distribution and glucose use are illustrated.
Sonochemical Synthesis and Characterization of Ni(C4H6N2)6(PF6)2, Fe(C4H6N2)6(BF4)2, and Ni(C4H6N2)6(BF4)2 in 1‐Butyl‐3‐methylimidazole with Hexafluorophosphate and Tetrafluoroborate
Authors: Jacob David S. , Makhluf Shirli , Brukental Ishay , Lavi Ronit , Solovyov Leonid A. , Felner Israel , Nowik Israel , Persky Rachel , Gottlieb Hugo E. , Gedanken Aharon
Published: May 2005

This paper reports on the inorganic sonochemical synthesis of crystalline Ni(C4H6N2)6(PF6)2, Fe(C4H6N2)6(BF4)2, and Ni(C4H6N2)6(BF4)2 in the ionic liquids, 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMI)+(PF6)– and 1‐butyl‐3‐methylimidazolium tetrafluoroborate (BMI)+(BF4)–. The structures of the compounds were determined and refined by using powder X‐ray diffraction methods. The salts crystallize in the trigonal P$\tilde {3}$ space group. IR, Raman and 19F NMR spectroscopic studies confirm that the PF6– and BF4– anions are not bonded to the metal cations. Magnetic measurements reveal the paramagnetic nature of the complexes. Mössbauer investigations show that the iron atom in Fe(C4H6N2)6(BF4)2 is in the oxidation +2 state. The broad EPR signals, which reflect the octahedral symmetry of the nickel complexes, were measured for Ni(C4H6N2)6(PF6)2 and Ni(C4H6N2)6(BF4)2. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)
1‐D Open‐Channeled 3‐D Supramolecular Self‐Assembled Frameworks Encapsulating Unprecedented Cyclic (H2O)8 Clusters or Solvent Molecules
Authors: Li Dong‐sheng , Wang Yao‐yu , Luan Xin‐jun , Liu Ping , Zhou Cai‐hua , Ma Hai‐rui , Shi Qiz‐hen
Published: May 2005

Two novel 1‐D coordination polymers, {[Cu(Hhbd)(bpy)]·3H2O}n (1) and {[Cu(Hhbd)(phen)]·H2O·MeOH}n (2) [Hhbd = α‐hydroxybutanedioicate dianion, bpy = 2,2'‐bipyridine, phen = 1,10‐phenanthroline], were rationally designed and synthesized with an identical construction strategy, using Hhbd ligands as bridges and chelating aromatic ligands as terminal ligands. Interestingly, the chelating aromatic ligand and the –OH groups of the Hhbd ligand play an important role in the self‐assembly of the polymeric coordination chain by providing potential supramolecular recognition sites for π–π aromatic stacking and hydrogen‐bond interactions, resulting in the self‐assembly of the molecular double chains to give a 3‐D supramolecular framework with 1‐D open channels. More significantly, in 1 the host framework encapsulates unprecedented centrosymmetric cyclic (H2O)8 clusters that have a chair conformation and are connected into 1‐D arrays by supramolecular association along the 1‐D open channels. In contrast, the channels of 2 contain lattice water and disordered methanol molecules. TGA and XRPD show that the host frameworks of 1 and 2 are stable when the guest solvent molecules are removed from the channels. Variable‐temperature magnetic susceptibility measurements for both complexes indicate the presence of weak antiferromagnetic exchange interactions between adjacent copper(II) ions. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)
Enzymatic and chemical synthesis of phosphatidylcholine regioisomers containing eicosapentaenoic acid or docosahexaenoic acid
Authors: Lyberg Ann‐Marie , Adlercreutz Dietlind , Adlercreutz Patrick
Published: May 2005

Regioselective incorporation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into phosphatidylcholine (PC) was carried out using enzymatic and chemical synthesis. Incorporation at the sn‐1 position was successfully achieved by lipase‐catalysed esterification of 2‐palmitoyl‐lysophosphatidylcholine (LPC), although in most cases, the enzymes incorporated EPA and DHA at lower rates than other fatty acids. For the incorporation of DHA, Candida antarctica lipase B was the only useful enzyme, while incorporation of EPA was efficiently carried out using either this enzyme or Rhizopus arrhizus lipase. The highest yields in the lipase‐catalysed reactions were obtained at the lowest water activity (close to 0). However, by carrying out the reactions at a higher water activity of 0.22, more EPA and DHA were incorporated. Esterification of 2‐palmitoyl‐LPC with pure EPA at this water activity converted 66 mol‐% of LPC to PC using Rhizopus arrhizus lipase as catalyst. When the fatty acid was DHA and the catalyst Candida antarctica lipase B, 45 mol‐% of PC was obtained. For incorporation of EPA and DHA at the sn‐2 position, phospholipase A2 was used, but the reaction was very slow. Chemical coupling of 1‐palmitoyl‐LPC and EPA or DHA was more efficient, resulting in complete conversion of LPC.
Book Review: Rapeseed and Canola Oil – Production, Properties and Uses. By Frank D. Gunstone
Authors: Pokorný Jan
Published: May 2005

No abstracts.
Ionized and total magnesium level in blood serum and plasma of healthy and III adults
Authors: Maj‐Zurawska Magdalena , Hulanicki Adam , Drygieniec Danuta , Pertkiewicz Marek , Krokowski Marcin , Zebrowski Andrzej , Lewenstam Andrzej
Published: May 2005

Ionized magnesium was measured potentiometrically using a clinical analyzer MICROLYTE 6 (KONE, Finland). Total magnesium was measured colorimetrically. Ionized magnesium (iMg) concentration changes with pH were made to observe the accuracy of the results if they were not recalculated to the actual pH of blood or recalculated with some uncertainty in parameters. The dependence of iMg and iCa on heparin (used as anticoagulant in plasma samples) was made. Less than 40 units per 1 mL of blood decreased the concentration of iMg and iCa less than 1.5%. The range of iMg and total Mg in serum and plasma of healthy and ill adults (short bowel syndrome, myocardial infarct) was investigated and compared. In both illnesses, the concentration of ionized and/or total magnesium is significantly different from the range of healthy people. The range of iMg and total Mg as well as the percentage of iMg against total Mg seem to be specific for a given illness.
Benzoic acid determination through competitive inhibition of mediated bioelectrocatalysis
Authors: Smit Mark H. , Rechnitz Garry A.
Published: May 2005

A bioelectrochemical system for the sensing of benzoic acid is described. An enzyme‐based oxygen electrode is constructed where the enzymatic consumption of oxygen by tyrosinase is monitored electrochemically through the mediator 1,2 naphthoquinone, 4‐sulfonate (NQS). The bioelectrocatalysis of oxygen can be electrochemically initiated and terminated and is sensitive to inhibition by benzoic acid in a concentration‐dependent manner. Inhibition of catalytic current results from a competitive interaction of benzoic acid at the enzymatic docking site of NQS. This inhibition of catalytic activity is reversible, and full catalytic activity can be reinstated following the removal of benzoic acid from solution. The sensing of benzoic acid is dependent on the nature of the mediator used, occurring with NQS but not with ferricyanide. These studies provide support for the possibility that analytes can be targeted for detection in such inhibition‐based sensing through the proper selection of mediator.
Analytical performances of lipophilic diamides based alkaline earth ion‐selective electrodes
Authors: Tóth Klára , Lindner Ernö , Horváth Mária , Jeney Judit , Pungor Ernö , Bitter István , Ágai Béla , Töke László
Published: May 2005

Based on complex chemical considerations, lipophilic diamide derivatives have been designed and synthesized as ionophores for liquid membrane electrodes. The structure‐selectivity studies revealed that the majority of the ligands exhibit calcium ion selectivity, except the compound bis‐N,N‐dicyclohexyl‐malonamide. The latter has an almost equal selectivity for Ca2+ and Mg2+ and sufficiently rejects sodium ions, thus it lends itself to water hardness measurements.
Oligosiloxane‐modified calix[4]arene ionophores for silicone‐rubber‐membrane sodium ion‐sensitive field‐effect transistors
Authors: Tsujimura Yutaka , Yokoyama Masaaki , Kimura Keiichi
Published: May 2005

Unsymmetrical and symmetrical calix[4]arene esters carrying oligosiloxane moiety were designed for neutral carriers of silicone‐rubber‐membrane sodium ion‐sensitive field‐effect transistors (ISFETs). The incorporation of oligosiloxane moiety to calix[4]arene ionophores promotes their dispersibility in silicone rubber, thus bringing about high stability and low impedance of the ion‐sensing membranes and thereby high sensitivity and fast response of the resulting sodium ISFETs.
Membraneless immobilization of xanthine oxidase on organic conducting salt/silicone oil electrodes
Authors: Korell Ulrich , Spichiger Ursula E.
Published: May 2005

Xanthine oxidase (XOD, E.C. 1.2.3.2) has been immobilized on amperometric electrodes made from the organic conducting salt tetrathiafulvalene‐p‐tetracyanoquinodimethane (TTF‐TCNQ) and silicone oil. Xanthine oxidase was immobilized by various techniques; immobilization by means of hydrophobic interaction was found to yield optimum analytical performance. Except for buffer, no coreactants are required for sensor operation. The response of the XOD electrode to its substrate hypoxanthine can be described by the enzyme‐kinetic Michaelis‐Menten formalism with excellent correlation (in most cases, Γ2 > 0.995). Steady‐state currents are usually measured within 10 seconds. Apparent KM (typically 3 to 10 μM) and saturation current values (0.2 to 3.7 μA, measured with new electrodes) strongly depend on the preparation procedure chosen as did the useful lifetime of the XOD sensors which was up to several weeks at room temperature. The described immobilization methodologies circumvent diffusion problems associated with well‐established biosensor preparation techniques (e.g., physical entrapment in gel or between dialysis membranes) and thus yield biosensors of superior analytical performance.
Development of a regenerable amperometric immunosensor for 7‐hydroxycoumarin
Authors: Lu Bin , Smyth Malcolm R. , Quinn John , Bogan Declan , O'Kennedy Richard
Published: May 2005

A regenerable immunosensor, based on a competitive assay principle and utilizing electrochemical detection, was developed for the determination of 7‐hydroxycoumarin. Horseradish peroxidase (HRP) labeled anti‐7‐hydroxycoumarin antibody was employed in the determination, and the enzyme activity was measured as a function of current due to the reduction of hydrogen peroxide in the presence of a mediator (potassium ferrocyanide). Sensor preparation involved chemical immobilization of 7‐hydroxy‐coumarin‐BSA conjugate on a glassy carbon electrode surface using a water soluble carbodiimide, 1 ‐ethyl‐3(3‐dimethyl‐aminopropyl) carbodiimide, as coupling reagent in the presence of N‐hydroxysuccinimide. The competition between the free and immobilized forms of the hapten for their HRP labeled antibody in solution allows for the quantitation of free 7‐hydroxycoumarin. The sensor can be regenerated by simply immersing the electrode tip in acidic media. Under these conditions the hapten‐antibody complex was selectively disrupted without adversely affecting the sensor performance. The conditions for removing the non‐specific adsorption of the enzyme conjugate were also studied. The results showed that the detection range obtained for the hapten was physiologically relevant.
Development of a conducting polymer‐based microelectrode array detection system
Authors: John Richard , Ongarato Daniela M. , Wallace Gordon G.
Published: May 2005

An individually addressable microelectrode array system has been developed in the course of this work. The performance of this system was investigated using conventional electrochemistry (oxidation/reduction or ferrocene carboxylic acid) to demonstrate that independent potential control and current measurement at each of the array components was possible. The practical utility of this system has been demonstrated using a conducting polymer based microarray detection system subsequent to Ion Chromatography. The detection of simple electroinactive ions (e.g., Cl−, SO42−, K+, NH4+) using the array system was achieved and the response at each of the array components was studied as a function of polymer composition.
Disposable nickel screen‐printed sensor based on dimethylglyoxime‐containing carbon ink
Authors: Wang Joseph , Nascimento Valberes B. , Lu Jianmin , Park Deog Su , Angnes Lucio
Published: May 2005

Disposable complexing (preconcentrating) sensor strips for trace nickel have been fabricated by doping the screen‐printed carbon ink with ligand dimethylglyoxime (DMG). The carbon ink is shown to be a useful matrix for incorporating the DMG, with the ligand maintaining its attractive coordination chemistry following the printing and firing processes. The response is characterized with respect to the ink composition, preconcentration period and potential, solution conditions and other variables. A short (30 s) accumulation period yields a detection limit of 5 μg/L nickel. The highly stable response indicates great promise for use as reusable devices. Measurement of nickel in a river water sample is demonstrated. Such incorporation of ligands into screen‐printed carbon inks provides a promising route for thick‐film sensors based on the preconcentration/voltammetric strategy.
The potentiometric stripping analysis of proteins
Authors: Honeychurch Michael J. , Ridd Michael J.
Published: May 2005

Ten proteins were analyzed by potentiometric stripping analysis (PSA) following reductive accumulation on a HMDE and oxidation with dissolved oxygen. During the reductive accumulation period the surface disulfide bonds are reduced to sulfhydryl pairs. The rate of oxidation of the sulfhydryl pairs is determined by the transport of dissolved oxygen to the electrode surface. Comparison of the PSA response to that obtained by constant current chronopotentiometry (CCCP) of the reduced protein in a deoxygenated solution showed that the presence of dissolved oxygen, in the PSA experiments stabilized the proteins from further conformational changes following the reduction of the surface disulfide bonds.
Investigation into electrochemical pretreatment of carbon fiber electrodes for the analysis of bamipine and imipramine
Authors: Surmann Peter , Peter Bernhard
Published: May 2005

The influence of the parameters of electrochemical pretreatment of carbon fiber electrodes on the differential pulse voltammetric response of bamipine and imipramine is described. Upon increasing the pretreatment potential a slight activation is followed by a deep suppression of peak current in the analysis of both substances. After a turning point the currents rise again and exceed the initial values many times over. The peak potential is affected as well. A slight deactivation is followed by a greater activation. The surfaces were characterized by chronoamperometry, chronopotentiometry and inverse voltammetry of the electrode material. At the carbon fiber epoxy matrix electrode it is possible to distinguish three different stages, which are interpreted as a phase of slightly activating functional groups in a submonolayer, a phase of passivating multilayer oxidation and finally a strongly activating phase of burn‐off and consumption of the electrode surface with change in its structure. These are thought to be responsible for the effects at the surface. It was shown that the effects within the third phase are diametrically opposed for aromatic and aliphatic amines.
Photo‐electroanalysis: Indirect determination of anthracene using the oxygen reduction in aqueous acetonitrile
Authors: Eklund John C. , Hallick Alan , Kumbhat Sunita , Compton Richard G.
Published: May 2005

A novel method for the photoelectroanalysis of anthracene, as a model compound, is proposed. This technique utilizes a gold electrode in a flowing system of electrolyte, air saturated with oxygen. On irradiation of the electrode with UV‐vis light there is a reduction in the level of the oxygen four‐electron reductive limiting current due to a reaction of the excited state anthracene and oxygen in acetonitrile/water solution. A linear response over four orders of magnitude (10−2 to 10−1 mol dm−3 was observed between this reduction of current and the concentration of anthracene suggesting the application of this type of technique to the detection of trace levels of aromatic molecules.
Analysis of skin fibroblast proteins in Duchenne muscular dystrophy: 1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Authors: Burghes Arthur H. M. , Dunn Michael J. , Statham Helen E. , Dubowitz Victor
Published: May 2005

Protein profiles of skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) and normal individuals have been compared using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS) combined with various detection systems. Protein patterns of normal and DMD fibroblasts obtained usin 10 % polyacrylamide gels showed no qualitative or quantitative differences. The use of acrylamide gels results in increased resolution. No qualitative or quantitative differences were observed using Coomassie Brilliant Blue R‐250 staining. However, Coomassie Brilliant Blue lacks the sensitivity to detect many minor components. Therefore a silver staining procedure was used, which resulted in greatly increased detection sensitivity. Background staining did not increase with gel concentration, but stain development proceeded more slowly at higher acrylamide concentrations. No qualitative differences were observed between normal and DMD profiles. Fibroblasts radiolabelled with [35S]‐methionine were analysed using gradient gels. No qualitative differences between DMD and normal patterns were observed by autoradiography of dried gels. However, quantitative analysis of slicing of gel rods revealed differences between normal and DMD fibroblasts. Two methods of statistics were used to analyse this data, firstly a method that accounts for inter‐experimental variation (termed “batch statistics”) and secondly the standard t‐test. Regions of 73 000 to 68 000 and 48 000 molecular weight were decreased in DMD samples and this finding was significant by both methods of statistical analysis. Regions of 175 000 and 53 000 molecular weight were significantly elevated in DMD preparations only by t‐test, whereas a regions of 32 × 103 molecular weight was only significantly elevated when tested by batch statistics.
Analysis of skin fibroblast proteins in Duchenne muscular dystrophy: 2. Isoelectric focusing under dissociating conditions
Authors: Burghes Arthur H. M. , Dunn Michael J. , Statham Helen E. , Dubowitz Victor
Published: May 2005

Protein profiles of skin fibroblasts labelled with [35S]‐methionine from patients with Duchenne muscular dystrophy (DMD) have been compared with those of cells from normal individuals by isoelectric focusing on thin polyacrylamide gels containing denaturing and solubilising agents cast on silanized supports. The routine method of sample solubilisation used a mixture of 8 M urea and 2 % NP‐40. The inclusion of sodium dodecyl sulphate (SDS) in the sample was found to result in removal of non‐ionic detergent from the gel even if a competition step had been used. In the presence of SDS less material remained at the point of sample application with a concomitant increase in bands in the middle region of the gels. Gels containing a mixture of sulfobetaine and NP‐40 were found to be inferior to those with 8 M urea and 2 % NP‐40, and high levels of urea were found to precipitate the zwitterionic detergent. No consistent qualitative differences between normal and DMD patterns were observed using any of the solubilisation methods on pH 3 to 10 or pH 8 to 10.5 gels. In order to facilitate quantitative analysis by gel slicing techniques, a plastic sheet was developed which would reliably bind acrylamide in the presence of 8 M urea and 2 % NP‐40. The quantitative analysis revealed significant differences between normal and DMD profiles, but the relevance of these changes to the disease state awaits further investigation.
Quantitative aspects of silver deposition in proteins resolved in complex polyacrylamide gels
Authors: Guevara Juan , Johnston Dennis A. , Ramagali Louis S. , Martin Barbara A. , Capetillo Sylvia , Rodriguez Lewis V.
Published: May 2005

Deposition of silver or silver compounds in proteins separated in linear gradient polyacrylamide electrophoretic gels is influenced by protein concentration and location, polyacrylamide concentration, extent of washing after incubation in ammoniacal silver hydroxide solution, and development time in a citric acid: formaldehyde solution. Our modification of existing silver staining methods provided routine resolution of polypeptides separated in 1.0 to 1.5‐mm‐thick gels without high background staining or surface staining and with picogram detection sensitivity. Removing electrophoretic components, such as sodium dodecyl sulfate (SDS), sulfhydryl reagents (2‐mercaptoethanol, dithiothreitol and dithioerythreitol), glycerol, tris‐glycine, carrier ampholytes, urea, and acetic acid, was essential in order to attain silver staining of proteins with a negligible background. Ammoniacal silver ions or silver ions, associated and unassociated with proteins separated in polyacrylamide gels, were removed during the washing step following incubation in ammoniacal silver hydroxide solution. Loss of stain intensity at discrete protein clusters was shown to correlate with polyacrylamide concentration in gradient gels. Standardizing the washing step enhanced reproducibility of staining. Using this silver staining method and computer‐assisted image processing, it was possible to detect quantitatively as little as 27 picograms of protein per mm in 10 %‐20 % linear gradient polyacrylamide gels (1.2 mm thick). Based on the lowest detection limits for Coomassie Brilliant Blue R‐250 staining of protein (10 nanograms), this method for silver staining is at least 370‐fold more sensitive.
Silver stain after isoelectric focusing of unconcentrated cerebrospinal fluid: Visualization of total protein and direct immunofixation of immunoglobulin G
Authors: Confavreux Christian , Gianazza Elisabetta , Chazot Guy , Lasne Yves , Arnaud Philippe
Published: May 2005

A silver stain technique has been developed to study proteins in unconcentrated cerebrospinal fluid (CSF) after isoelectric focusing. This method is highly sensitive, and bands containing 25 to 50 ng protein can be clearly distinguished, so that small volumes (40 μl maximum) of native CSF can be used. Individual proteins (e. g., immunoglobulin G) can be detected easily by specific immunofixation. It is also possible to perform direct precipitation and direct specific immunofixation on a single gel in order to compare side by side the patterns of the whole and of specific proteins from different samples of CSF. The technique is simple and highly reproducible, and results are obtained 6 h after sample deposition (if immunofixation is used, a further 24 h washing is necessary). The sensitivity and versatility of this technique (immunofixation can be applied to the detection of any antigen) should permit its extension to other biological fluids with a low protein content.
A fast flat‐bed laser scanner for the digital acquisition of two‐dimensional electrophoresis patterns
Authors: Marsman Herman , van Resandt Roelof Wijnaendts
Published: May 2005

Analysis of two‐dimensional electrophoresis patterns demands computer assistance. Specialized machines to read and store the pattern of an autoradiograph or of a “wetgel” into a digital memory are rare. A fast and flexible opto‐mechanical picture reading and storage machine, specially designed for the computer‐aided analysis of two‐dimensional electrophoresis patterns, has been constructed. It is now possible to digitize, for example, a picture of 250 × 400 mm into pixels of ca. 250 × 250 μm with a grey level resolution of 8‐bit within 70 s. Digitizing at significant pixel sizes down to 64 μm is possible at correspondingly longer acquisition times for the areas to be digitized. The design is a flat‐bed scanner. One axis is scanned optomechanically (Galvanometer scanned laser beam), the other axis is scanned mechanically. The area covered by the scanning laser beam has a size of 250 × 400 mm or smaller. The control of the scanner, including pixel density and sampling synchronization is executed by a microprocessor system.
Design of a vertical gel slab electrophoresis apparatus containing a permanent salt bridge
Authors: Schafer‐Nielsen Claus
Published: May 2005

An electrophoresis apparatus of the vertical slab gel type was designed to obtain a unit in which the number of spare parts and manual operations necessary for its assembly is significantly reduced as compared to the designs that are commonly used at present. The new apparatus is assembled in less than 60 s, and the gels are moulded in situ., i. e. without the use of a separate casting stand. With all electrode reservoirs placed within the base unit, the apparatus obviates the need for bulky external electrode reservoirs. The compact design is made possible by use of a permanently mounted porous plastic bridge that acts as a seal during moulding of the gel and as a conducting salt bridge during electrophoresis. The apparatus permits simultaneous running of two gels, and runs on single gels may be performed independently without filling the electrode reservoirs for the other gel.
An evaluation of DNA fingerprinting for forensic purposes
Authors: Gill Peter , Lygo Joan E. , Fowler Susan J. , Werrett David J.
Published: May 2005

Many highly polymorphic minisatellite loci can be detected simultaneously in the human genome by hybridisation to probes consisting of tandem repeats of the ‘core’ sequence. The resulting DNA fingerprints produced by Southern blot hybridisation are comprised of multiple hypervariable DNA fragments, show somatic and germline stability and are completely specific to an individual. DNA of high molecular weight can be isolated from blood and semen stains up to 4 years old. Sperm nuclei can be separated from vaginal debris and the sperm DNA examined in isolation, allowing the positive identification of rapists. Correspondingly, vaginal DNA can be isolated from extracts of penile swabs. DNA can also be isolated from extracts of buccal swabs. A blind trial involving 43 blood samples, 11 bloodstains and 11 semen stains has been successfully carried out. It is envisaged that genetic fingerprinting will shortly form an important part of casework procedure in forensic science laboratories.
Effect of ligand‐affinity differences of human hemoglobin variants on electrophoretic behavior and their isolation and functional characterization
Authors: Rochette Jacques , Deburgrave Nathalie , Bohn Brigitte , Dodé Catherine , Poyart Claude , Krishnamoorthy Rajagopal
Published: May 2005

A natural sulfated polysaccharide (agaropectin), contained in crude agar, can be used as a medium for electrophoretic separation of hemoglobin mutants, constituting a particular class of protein‐ligand interactions. Mutations which either modify the electrostatic charge at the surface of the hemoglobin molecule or not, have been studied according to their putative interaction with the medium. Using conformational specificities of the hemoglobin molecule, we have also demonstrated that isoelectric focusing on a polyacrylamide gel in the absence of heme ligands represents a useful, convenient and rapid procedure for isolating silent Hb variants in their native form, provided that they exibit an abnormal Bohr effect.
Characterization of human catalase by isoelectric focusing in presence of urea
Authors: Alonso Antonio , Visedo Guillemo , Sancho Manuel , Fernández‐Piqueras José
Published: May 2005

Human catalase from erythrocytes and liver were analyzed by polyacrylamide gel isoelectric focusing in presence and absence of urea using two different pH gradients, namely pH 6–8 and pH 6.7–7.7. In presence of urea, human catalase focused in the pH range 6.75–7.0, slightly anodal to that of hemoglobin A. In narrow pH gradients, human erythrocyte catalase was microheterogeneous. Neuraminidase from different sources and peptide‐N‐glycosidase F were applied to investigate the presence of sialic acid and/or carbohydrate chains in human catalase. A shift in the focusing pattern of both erythrocyte and liver catalase towards the anode was observed after treatment with one of the commercially available neuraminidase preparations. This unusual result could be related to a contaminating protease since no effect was observed when the catalases were treated in presence of a serine protease inhibitor. In contrast, bovine liver and Maccaca erythrocyte catalase did not display any detectable change in their focusing patterns after treatment with any of the neuraminidase preparations.
Two‐dimensional electrophoresis of membrane proteins: Separation of myelin proteins
Authors: Person Håkan , Överholm Torsten
Published: May 2005

A method for two‐dimensional electrophoretic separation of myelin proteins is presented. The first dimension consists of isoelectric focusing of lyophilized and delipidated membrane proteins, solubilized in a mixture of the nonionic detergent Triton X‐100, the zwitterionic detergent CHAPS, 9 M urea and carrier ampholytes, and incorporated into a slab before separation. Subsequent discontinouous sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was performed ny moulding the isoelectric focusing slab gel with its supporting glass plate into the stacking gel. This method proved to give highly reproducible results since mechanical forces and thus the risk of stretching, folding or rupture of the isoelectric focusing slab gel is minimized. Furthermore, by immunoblotting. the positions of myelin‐associated glycoprotein and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase were established with specific antibodies.
High‐performance human myocardial two‐dimensional electrophoresis database: Edition 1996
Authors: Müller Eva‐Christina , Thiede Bernd , Zimny‐Arndt Ursula , Scheler Christian , Prehm Joachim , Müller‐Werdan Ursula , Wittmann‐Liebold Brigitte , Otto Albrecht , Jungblut Peter
Published: May 2005

The master gel of the human myocardial two‐dimensional electrophoresis (2‐DE) gel database contains about 3300 protein spots characterized in terms of isoelectric point (pI) and molecular mass. A high‐performance technique was applied, using large gels (23 × 30 cm). Isoelectric focusing with anodic sample preparation and nonequilibrium running conditions (NEPHGE) was combined with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in 15% acrylamide gels in the second dimension. The range of pI extends from pH 4.5 to 9.6. Seventy proteins were identified by combinations of amino acid analysis, N‐terminal and internal sequencing, immunostaining, matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) peptide mass fingerprinting, post‐source decay MALDI‐MS and ladder sequencing by carboxypeptidase P. The identification of additional proteins, not found in the master gel, was achieved by immunoblotting. Unequivocal identification with high sensitivity and good yield was obtained by combining internal sequencing and MALDI‐MS. In‐gel digestion, the concentration and purification of peptides in a peptide collecting device, and the improved FRAGMOD program for peptide mass fingerprinting have added to the security and sensitivity of identification. The high‐performance human myocardial 2‐DE database was built up with proteins detected by the TOPSPOT program. Spots within six sections of the whole pattern are clickable. Protein description includes detailed information about identification, characterization, and links to the related SWISS‐PROT, other 2‐DE databases and Medline entries. The database is constructed in accordance with four of the rules for a federated database.