bluuebunny/crossref_metadata_2025 huggingface dataset:
Differentiation of human peripheral blood monocytes to macrophages is associated with changes in the cellular respiratory burst activity
Authors: Johansson Agneta , Dahlgren Claes
Published: May 2005

When phagocytic leukocytes, e.g. neutrophils, monocytes and macrophages, interact with soluble or particulate stimuli, the cells respond with an increased production of reactive oxygen metabolites. This production can be measured with the luminol‐amplified chemiluminescence (CL) technique. In the present study, the CL reaction induced in monocyte‐derived macrophages was investigated and compared to the responses of neutrophils and monocytes. In systems without additives the CL response of macrophages to soluble stimuli (FMLP, PMA and ionomycin) was very low. Addition of a peroxidase (HRP) to the reaction mixtures resulted in a pronounced increase in CL activity. The cellular CL response in macrophages is thus limited by the amount of peroxidase available. The macrophage response differs qualitatively from the responses of neutrophils and monocytes, in that the intracellular phase of the response is missing.
The relation between the synergistic effect of cytostatics and metabolism of V 79B cells In Vitro
Authors: Hudáková A. , Horáková K. , Perényi T.
Published: May 2005

A single‐dose simultaneous application of methotrexate (MTX; 0·002/μg ml−1) and cisplatin (cis‐Pt; 0·0002/μg ml−1) had a permanent synergistic effect on both synchronized and asynchronous cell populations of V 79B cells. Successive combination of the drugs was manifested synergistically when MTX was applied first. The synchronized cell population was more sensitive to the cytostatics than the asynchronous population.

Treatment with MTX alone, or the combination of MTX–cis‐Pt, as well as their successive combination with the first drug being cis‐Pt, caused gluconeogenesis.
Neutrophil integrin assay for clinical studies
Authors: Bateman J. , Parida S. K. , Nash G. B.
Published: May 2005

The level of expression of neutrophil adhesion molecules may be a useful marker for neutrophil activation in clinical studies. We therefore determined neutrophil integrin expression under various experimental conditions using a Fluorescence Activated Cell Sorter (FACS) after the cells had been labelled with fluorescent conjugated antibodies to the integrin subunits CD11a, CD11b and CD18. Levels of labelled CD11b and CD18 increased after activation with the chemotactic peptide formyl‐methionyl‐leucyl phenylalanine (fMLP) in a dose‐ and time‐dependent manner, but CD11a did not, indicating that CD11a would not be a useful marker of neutrophil activation. The baseline expression of CD11b and CD18 on unstimulated neutrophils was similar in heparin and EDTA anti‐coagulated blood but the response to activation with fMLP was significantly less for the EDTA anti‐coagulated samples (p < 0·01 in paired t‐test). The labelling of integrins was significantly higher in unfixed whole blood samples compared to samples fixed with 1 per cent paraformaldehyde. However, the increase in labelling induced by fMLP was similar whether or not the samples were fixed after activation. Labelling of CD11b and CD18 was greater for preparations of isolated neutrophils than for neutrophils in whole blood, and the response to fMLP stimulation tended to be lower for the isolated cells. Our results indicate that heparin should be used as anti‐coagulant in clinical studies utilizing whole blood if subsequent activation of neutrophils is planned (e.g. to detect in vivo priming), although EDTA may be used if baseline expression alone is to be measured. Fixation of blood samples should not affect the ability to detect neutrophil activation.
Phospholipase A2 from Trypanosoma congolense: Characterization and haematological properties
Authors: Nok Andrew J. , Esievo King A. N. , Ibrahim Sani , Ukoha Agwu I. , Ikediobi Christopher O.
Published: May 2005

Phospholipase A2 was isolated from Trypanosoma congolense and purified to electrophoretic homogeneity. The enzyme appeared to exist in a dimeric form with subunit molecular weights of 16 500 and 18 000. It had a pH optimum of 6·8. Kinetic analysis with different substrates, showed that the enzyme had exceptional specificity for 1,2,dimyristoyl‐sn‐phosphatidylcholine and 1,2,dioleoyl‐sn‐phosphatidylcholine with Km values of 1·85 × 10−3 M and 2·12 × 10−3 M respectively. The Arrhenius plot was linear with an activation energy of 5·8 kcal mol−1. Inhibition studies with parahydroxymercuribenzoate and tri‐butyltinoxide were positive thus implicating a thiol group at the catalytic site of the enzyme. The enzyme was stable to heat treatment and possessed haemolytic and anticoagulating properties.
Fructose 1,6‐bisphosphate prevents oxidative stress in the isolated and perfused rat heart
Authors: Rigobello Maria Pia , Galzigna Lauro , Bindoli Alberto
Published: May 2005

Rat hearts were perfused with the Langendorff technique at constant flux in the presence of the oxidizing agents hydrogen peroxide and diamide. Fructose 1,6‐bisphosphate strongly prevented the decline of heart contractility due to the infusion of these oxidizing agents. On the other hand, fructose 1,6‐bisphosphate had no effect on the release of total glutathione into the perfusate but prevented the loss of lactate dehydrogenase indicating a protective effect on cell membranes. Comparing the cytosolic and mitochondrial loss of glutathione, fructose 1,6‐bisphosphate exerted a beneficial action only on the mitochondrial fraction. Several mechanisms of action have been considered to explain the protective action of frutose 1,6‐bisphosphate. In our experimental conditions fructose 1,6‐bisphosphate might stimulate its own production giving rise to dihydroxyacetone phosphate, that, after reduction to glycerol 3‐phosphate, can permeate the mitochondrial membrane with the final production of energy.
Reduced 4‐hydroxynonenal degradation in hearts of spontaneously hypertensive rats during normoxia and postischemic reperfusion
Authors: Grune T. , Schönheit K. , Blasig I. , Siems W.
Published: May 2005

4‐Hydroxynonenal (HNE) degradation was investigated in isolated perfused rat hearts of the WKY and SHR strains before and after ischemia. HNE (10 μmoles l−1) were infused and the concentration of HNE in the effluent was determined. The rate of initial consumption was about 50 nmoles min−1 g−1 wet weight in hearts of both the WKY and SHR rats. In the WKY rat hearts, this rate of HNE degradation did not change during several minutes of HNE infusion and also remained constant during postischemic reperfusion. In the hearts of the SHR rats the HNE degradation rate declined within 5 min to 25 nmoles min−1 g−1 wet weight. Also during postischemic reperfusion, there was a lower HNE degradation rate in the SHR rat hearts than in the WKY rat hearts. The influence of hypertrophy on the rate of HNE degradation is discussed. It is suggested that the low degradation of the cytotoxic lipid peroxidation product, HNE, in hypertrophic hearts may contribute to reduced antioxidant defence in those hearts.
Hyaluronan‐mediated protective effect against cell damage caused by enzymatically produced hydroxyl (OH·) radicals is dependent on hyaluronan molecular mass
Authors: Presti Donatella , Scott John E.
Published: May 2005

Hyaluronan (HA) protected tendon fibroblasts against cell damage mediated by hydroxyl radicals (OH·) as demonstrated by release of 51Cr from labelled cells. Protection afforded by high molecular mass (Mr) HA (1218 kDa) was much more effective than that provided by lower (176 kDa and 668 kDa) Mr HA.

OH· was generated by coupling H2O2 produced by glucose oxidase: glucose to [Fe2+‐EDTA] chelate in a Fenton‐type system. The flux of OH· was measured by a spectrofluorimetric assay of salicylate produced by the reaction of benzoate with OH·. Cell damage caused by the OH· generating system was prevented in the presence of catalase, which destroyed H2O2. Damage caused in a standard incubation time increased with increased amounts of glucose oxidase.

Protection against OH·‐mediated cell damage increased with increasing concentration of HA. The presence of HA did not interfere with the enzyme‐Fenton system, as monitored by production of gluconate. On the other hand, HA scavenged OH· produced by the enzyme‐Fenton system, as shown by competition with benzoate, which produced less salicylate in the spectrofluorimetric assay in the presence of HA.

The reaction of OH· with HA was measured directly by a pulse radiolysis technique in which a hydrated electron (e) produced OH· by the reaction with nitrous oxide. Second order rate constants obtained in distilled H2O or in phosphate buffer showed no dependence on HA Mr. Similarly, fluorimetric assay of the flux of in the enzyme‐Fenton system confirmed that HA competed with benzoate, thus lowering salicylate production, and the flux was also independent of the molecular mass of HA.

These results demonstrate that part of the HA‐mediated protection against enzyme‐Fenton produced OH· and other reactive oxygen‐derived toxic species was not a consequence of either the primary or secondary structure of HA, but rather depends on higher order HA organization. Some aspects of the formation of the HA meshwork (tertiary structure) are Mr dependent. We therefore propose that cell‐anchored HA meshworks excluded relatively large enzyme molecules from the immediate environment of the cell, thus reducing the flux of OH· etc. at the cell surface and diminishing cell damage.
Mechanism of lysosome rupture by dipeptides
Authors: Bird Susan J. , Lloyd John B.
Published: May 2005

Low concentrations of some neutral dipeptides, such as L‐Ala‐L‐Ala, rapidly disrupt rat liver lysosmes. The phenomenon has been attributed to an osmotic imbalance generated by the production of amino acids in the lysosme by lysosomal dipeptidase activity. This hypothesis is challenged by testing several pairs of dipeptides available in both D‐ and L‐forms and a range of dipeptides whose susceptibility to lysosomal dipeptidase activity is known. A good correlation was found between the lytic ability of dipeptides and their capacity to cross the lysosome membrane and be hydrolysed by lysosomal dipeptidase. The osmotic‐imbalance hypothesis is critically evaluated in the light of the results and of recent information concerning the carrier‐mediated transport of amino acids and dipeptides across the lysosome membrane. It is concluded that intralysosomal generation of amino acids remains the most plausible explanation of the lytic activity of dipeptides, and that the dipeptide proter(s) in the lysosome membrane must have higher Km than the amino acid porters.
Subpopulations of tau interact with microtubules and actin filaments in various cell types
Authors: Henríquez Juan P. , Cross Daniel , Vial Clarisa , Maccioni Ricardo B.
Published: May 2005

It has been demonstrated that microtubule‐associated proteins (MAPs) interact with tubulin in vitro and in vivo. However, there is no clear evidence on the possible roles of the interactions of MAPs in vivo with other cytoskeletal components in maintaining the integrity of the cell architecture. To address this question we extracted the neuronal cytoskeleton from brain cells and studied the selective dissociation of specific molecular isospecies of tau protein under various experimental conditions. Tau, and in some cases MAP‐2, were analysed by the use of anti‐idiotypic antibodies that recognize epitopes on their tubulin binding sites. Fractions of microtubule‐bound tau isoforms were extracted with 0·35 M NaCl or after the addition of nocodazole to allow microtubule depolymerization. Protein eluted with this inhibitor contained most of the assembled tubulin dimer pool and part of the remaining tau and MAP‐2. When the remaining cytoskeletal pellet was treated with cytochalasin D to allow depolymerization of actin filments, only tau isoforms were extracted. Immunoprecipitation studies along with immunolocalization experiments in cell lines containing tau‐like components supported the findings on the roles of tau isospecies as linkers between tubulin in the microtubular structure with actin filaments. Interestingly, in certain types of cells, antibodyreactive tau isospecies were detected by immunofluorescence with a discrete distribution pattern along actin filaments, which was affected by cytochalasin disruption of the actin filament network. These results suggest the possible in vivo roles of subsets of tau protein in modulating the interactions between microtubules and actin filaments.
Characterization of the effect of peak shifts on the performance of the Kalman filter in multicomponent analyses
Authors: Webster Gail Hartmann , Cecil Todd L. , Rutan Sarah C.
Published: May 2005

The effect of peak shifts on the performance of a Kalman filter multicomponent analysis algorithm has been investigated. A series of Gaussian test systems were employed to characterize the concentration estimation errors and the morphology of the on‐line residuals (the innovations sequence). Both forward and reverse filters were used in the generation of the innovations sequences. It was found that the difference between the forward and reverse innovations sequences gave an accurate indication of the direction and magnitude of the peak shift. A series of overlapped two‐component systems were also investigated. Again, a correlation between the difference innovations and the degree of the response shift was observed. The behavior of the Kalman filter in fitting the shifted fluorescence emission spectrum of benzo[a]pyrene was also examined. The response for benzo[a]pyrene in cyclohexane solution was compared to that obtained on the surface surface of a reversed phase thin layer chromatography plate. A red shift of 4 nm was detected for the surface spectrum by observing the difference innovations sequence. This approach holds promise for correction of response shifts in multicomponent systems.
Three‐way methods for the calibration of chromatographic systems: Comparing PARAFAC and three‐way PLS
Authors: Smilde Age K. , Doornbos Durk A.
Published: May 2005

For the calibration of chromatographic systems, different methods can be used. One class of methods utilizes three‐way approaches. The calibration problem is stated in such a way that the decomposition of a three‐way array can serve for the prediction of retention on new stationary phases.

Two three‐way approaches are presented: the Unfold‐PCA and PARAFAC models. The theory of both methods is presented and the differences are highlighted, the main difference being that PARAFAC is a trilinear decomposition whereas Unfold‐PCA is not. Both three‐way methods are evaluated on a small data set consisting of retention measurements of eight solutes at six mobile phase compositions on six stationary phases. The differences in performance of the two models are minor.

For calibration purposes, two variants of the methods are discussed: three‐way PLS and an extension of PARAFAC. Again the theory and differences between the two methods are explained. The predictive performance of the two methods is compared using the same data set as earlier. The differences in predictive performance, however, are minor. Both methods are capable of predicting 98% of the variation in the test sets. Yet, there are other considerations when comparing methods than predictive performance, e.g. the quality of the predictions.
Comments on the PLS kernel algorithm
Authors: De Jong Sijmen , Ter Braak Cajo J. F.
Published: May 2005

Lindgren et al. (J. Chemometrics, 7, 45–49 (1993)) published a so‐called kernel algorithm for PLS regression of Y against X when the number of objects is very large. The algorithm is based solely on deflation of the cross‐product matrices XTX, YTY and XTY. The algorithm is now described in a shorter and more transparent way and compared with a similar algorithm for the singular value decomposition of XTY.
Metal‐organic vapor phase epitaxial growth of cubic Gallium selenide, Ga2Se3
Authors: Ng Tat Lin , Maung Nicolas , Fan Guanghan , Poole Ian B. , Williams John O. , Wright Andrew C. , Foster Douglas F. , Cole‐Hamilton David J.
Published: May 2005

We present results of a study using metal‐organic vapor phase epitaxy (MOVPE) to synthesize thin films of cubic Ga2Se3 on both nearly lattice matched GaP and mismatched GaAs substrates. We find that trimethylgallium (TMGa) pre‐reacts with the hydride H2Se in the gas phase to yield films which are only partially epitaxial and that the combination of TMGa with ditertiarybutylselenide produces films of the best crystal quality under steady state flow conditions. We also show that GaAs is an unsuitable substrate for MOVPE growth of gallium selenide due to exchange reactions at the interface leading to poorly bonded films.
Enhancer trapping with a red fluorescent protein reporter in Drosophila
Authors: Akimoto Ai , Wada Housei , Hayashi Shigeo
Published: May 2005

Enhancer trapping allows the detection of genomic transcriptional enhancers and provides a rich source of cell‐ and region‐specific markers in Drosophila. We report here the development of a P‐element–based enhancer‐trap vector with a nuclear red fluorescent protein (DsRed) reporter for enhancer detection. We demonstrate that this vector can be used for a standard enhancer‐trap screen as well as for targeted replacement of previously characterized P‐element insertions. We isolated DsRed insertion strains of hedgehog, patched, apterous, teashirt, and dachshund that label specific regions of imaginal discs. The time required for red fluorescence maturation in the eye imaginal disc was estimated to be 22.5 hr. The DsRed markers can be combined with green fluorescent protein markers for double labeling of living Drosophila tissues. Developmental Dynamics 233:993–997, 2005. © 2005 Wiley‐Liss, Inc.
NeuroD1 in the endocrine pancreas: Localization and dual function as an activator and repressor
Authors: Itkin‐Ansari P. , Marcora E. , Geron I. , Tyrberg B. , Demeterco C. , Hao E. , Padilla C. , Ratineau C. , Leiter A. , Lee J.E. , Levine F.
Published: May 2005

The basic helix–loop–helix transcription factor NeuroD1 regulates cell fate in the nervous system but previously has not been considered to function similarly in the endocrine pancreas due to its reported expression in all islet cell types in the newborn mouse. Because we found that NeuroD1 potently represses somatostatin expression in vitro, its pattern of expression was examined in both strains of mice in which lacZ has been introduced into the NeuroD1 locus by homologous recombination. Analysis of adult transgenic mice revealed that NeuroD1 is predominantly expressed in β‐cells and either absent or expressed below the limit of lacZ detection in mature α‐, δ‐, or PP cells. Consistent with a previous report, NeuroD1 colocalizes with glucagon as well as insulin in immature islets of the newborn mouse. However, no colocalization of NeuroD1with somatostatin was detected in the newborn. In vitro, ectopic expression of NeuroD1 in TRM‐6/PDX‐1, a human pancreatic δ‐cell line, resulted in potent repression of somatostatin concomitant with induction of the β‐cell hormones insulin and islet amyloid polypeptide. Additionally, NeuroD1 induced expression of Nkx2.2, a transcription factor expressed in β‐ but not δ‐cells. Transfection studies using insulin and somatostatin promoters confirm the ability of NeuroD1 to act as both a transcriptional repressor and activator in the same cell, suggesting a more complex role for NeuroD1 in the establishment and/or maintenance of mature endocrine cells than has been recognized previously. Developmental Dynamics 233:946–953, 2005. © 2005 Wiley‐Liss, Inc.
Ammonium detection using an ion‐selective electrode array in flow‐injection analysis
Authors: de Viteri F. J. Sáez , Diamond Dermot
Published: May 2005

An ammonium detection system using ion‐selective electrodes (ISEs) in flow‐injection analysis (FIA) is described. Because of the low selectivity of the nonactin ammonium selective electrode toward some common ions, notably potassium, different selectivity enhancement techniques have been examined. An array of ISEs selective for ammonium, sodium, potassium, and calcium was used as the FIA detector, and the array response was modeled via the Nikolskii‐Eisenmann equation and the Projection Pursuit regression model. The interfacing of the system to a PC‐386SX computer using an Analog Devices RTI‐815 I/O card is also described. The results show that the use of an array of ISEs under FIA regimes for the detection of ammonium in the activity range 10 to 0.01 mM gives much improved results for the determination of ammonium in aqueous samples than the use of a single ammonium electrode. The performance of the array under steady‐state and FIA conditions is compared.
Electrocatalytic oxidation and amperometric determination of sulfhydryl compounds at a copper hexacyanoferrate film glassy carbon electrode in liquid chromatography
Authors: Zhou Jianxun , Wang Erkang
Published: May 2005

Electrocatalytic oxidation of sulfhydryl compounds was effective on a copper hexacyanoferrate (CuHCF) film glassy carbon electrode, at a significantly reduced overpotential (0.55 to 0.65 V) and for a broader pH range (2.0 to 7.0). The electrocatalysis was evaluated in terms of several preliminary experimental parameters, such as buffer pH, surface adsorbent concentration, operating potential, mobile‐phase flow rate, and concentration dependence in batch and flow systems. Electrochemical liquid chromatography (LCEC) of sulfhydryl compounds gave a linearity over two orders of magnitude and detection limits at picomole level. Overlaying the CuHCF film electrode surface with a Nafion layer is essential for improving the catalysis stability and offers enhancement of the liquid chromatography electro chemistry (LCEC) response.
Determination of oxidase enzyme substrates using cross‐flow thin‐layer amperometry
Authors: Yang Liu , Kissinger Peter T.
Published: May 2005

Thin‐layer cross‐flow amperometric detectors with enzyme modified working electrodes were developed for the determination of oxidase substrates in flowing streams. The enzyme was covalently immobilized in an osmium redox polymer film on the electrode surface. Electrodes modified with glucose oxidase or lactate oxidase were prepared for the continuous monitoring of glucose or lactate. A peroxidase electrode coupled with oxidase enzyme reactors was also explored for the determination of glucose, lactate, and acetylcholine/choline in continuous monitoring systems or following liquid chromatography. The effects of interferences, oxygen, mass transfer limiting membrane, and perfusate composition on electrode performance are discussed.
Determination of free formaldehyde in nail preparations by differential pulse voltammetry
Authors: Wang Lai‐Hao , Chen Zong‐Shiow
Published: May 2005

Free formaldehyde in nail preparations, extracted with dichloromethane and 0.002 M hydrochloric acid (1:2v./v.), was determined by differential pulse voltammetry(DPV) using a thin‐film mercury electrode (TFME) in a basic solution. The various factors such as deposition time, concentration of mercury (II) and of coexisting organic and inorganic interferences on the precision of the analysis have been explored. Under optimum conditions the peak potential of formaldehyde was −1.652V [vs. the saturated calomel electrode(SCE)], and a linear current vs. concentration relationship was over 2.0–80.0 mg L−1. The results obtained were in good agreement with those of the official European Economic Community(EEC) acetylacetone method.
Voltammetric determination of dopamine in the presence of ascorbic acid at a chemically modified electrode
Authors: Zen Jyh‐Myng , Chen I‐Lang
Published: May 2005

A Nafion/ruthenium oxide pyrochlore chemically modified electrode (CME) was used for the selective determination of dopamine (DA) in the presence of a high concentration of ascorbic acid by square‐wave voltammetry. Compared to a bare glassy carbon electrode, the CME exhibits an apparent shift of the oxidation potentials in cathodic direction and a marked enhancement of the current response. The selective sensing of DA is achieved by combining the electrocatalytic function of the ruthenium oxide pyrochlore catalyst with the charge‐exclusion and preconcentration features of Nation. With a preconcentration time of 60 s at a potential of −0.3 V (vs. Ag/AgCl), linear calibration plots are obtained for dopamine in 0.1 M, phosphate buffer (pH 7.4) over 0–20 μM with a detection limit (3σ) of 0.1 μM.
Lead‐selective membrane electrodes based on dithiophenediazacrown ether derivatives
Authors: Yang Xinhao , Kumar Naresh , Chi Hua , Hibbert D. Brynn , Alexander Peter W.
Published: May 2005

Three double‐armed diazacrown ethers with two thiophene side groups, 7,16‐dithenyl‐1,4,10,13‐tetraoxa‐7,16‐diazacyclooctadecane (DTDC), 7,16‐dithenoyl‐1,4,10,13‐tetraoxa‐7,16‐diazacyclooctadecane (DTODC), and 7,16‐di‐(2‐thiopheneacetyl)‐1,4,10,13‐tetraoxa‐7,16‐diazacyclooctadecane (DTAODC), have been synthesized and used as novel neutral lead(II) ionophores in ion selective electrode applications. The relationship between the molecular structure of these ionophores and electrochemical properties (linear range, response time, selectivity) of the membrane electrode is discussed. The optimum conditions for the preparation of the electrodes are described. The optimized dithenoyldiazacrown had a detection limit of pPb = 5.7, and Nernstian range with slope 29.2 mV decade−1 from pPb = 5.0 to 2.7. Mercury and silver ions are the major interferences. These electrodes are applied to potentiometric titrations of lead(II) ions and show promise for the determination of lead ions in water samples.
Electrochemical and surface characterization of pH sensor based on bisulfate‐doped poly(pyrrole)
Authors: Jovanovic Vladislava M. , Dekanski Aleksander , Vlajnic Goran , Jovanovic Momir S.
Published: May 2005

Pyrrole was electrochemically polymerized on glassy carbon support in NaHSO4 solutions under different conditions (method applied, concentration of monomer, concentration of the main solution, thickness of the film). These variables were used to study the preparation effect on the resulting properties, in particular on the potentiometric response of the PPy‐HSO4 electrodes. The electrodes were characterized by cyclic voltammetry, as well as by STM and AES techniques. Utilities of the polymer application as a sensor were studied by direct potentiometric measurements. The results obtained show that poly(pyrrole) doped with bisulfate ions can serve as pH sensor, with the slope of E/pH curves approaching the theoretical value.
Field Strnegth changes during isoelectric focusing
Authors: Jackiw B. Andrew , Brown Ray K.
Published: May 2005

Conductivity gaps in electrofocusing gels were measured as a function of the duration of isoelectric focusing, using a device for the determination of segmental voltage along the gel length (Jackiw, B. A., et al., Electrophoresis 1980, 1, 102–106 Transient and permanent conductance gaps were found on electrofocusing with both commercial and home‐made carrier ampholytes of a variety of pH‐ranges and chemical compositions. All pH gradients investigated exhibited conductance gaps. The dynamics of conductance gap formation and the relative magnitude of voltage maxima were compared in electrofocusing gels made with the various types of carrier ampholytes, With ampholyte‐ampholyte mixtures, and with ampholyte‐amino acid mixtures.
A continuous‐flow isoelectric focusing system for protein fractionation: Its description and application in the partial purification of choline acetyltransferase
Authors: Basset Paul , Froissart Chantal , Vincendon Guy , Massarelli Raffaele
Published: May 2005

A continuous‐flow isoelectric focusing (IEF) system for protein fractionation, using a non‐commercial separation chamber and carrier ampholytes synthesized in the laboratory, is described. The resolving power and the sample‐loading capacity have been tested by the fractionation of hemoglobin mixtures. Hb A2and HbA (ΔpI 0.4) were separated with a sample loading of 4 g of hemoglobin per day. The maximal temperature in the gel was 5 °C, the average field strength 30 V/cm and the residence time 6 h. The continuous‐flow IEF system was further used as a preliminary step in the purification of choline acetyltransferase (EC. 3.2.1.6) from human placenta. The results obtained with choline acetyltransferase indicated that continuous‐flow IEF can be used for the purification of enzymes when other preparative IEF systems are not effective, particularly when the sample is present in large volume.
Acrylamide gel electrophoresis of hydrophobic proteins: Gas vacuole protein
Authors: Simon Robert D.
Published: May 2005

The hydrophobic protein comprising the gas vacuoles of a variety of procaryotic microorganisms is insoluble in detergents (e.g. sodium dodecyl sulfate) and cannot be electrophoresed in detergent‐containing systems. The protein is solubilized by mixtures of phenol‐acetic acid‐urea (PAU) and the development of a slab gel system containing PAU which successfully separates the gas vacuole protein is described. In the system, phenol is polymerized directly into the gel, and the high background staining which results can be minimized by the proper choice of gel thickness, acrylamide/Bis ratio, and polymerization conditions. There is a direct relationship between the electrophoretic mobility and molecular weight of protein standards in the PAU system; however, several proteins including histones H2, H3, H4 and bovine serum albumin have a faster rate of gration than expected. It has been possible to separate and identify the gas vacuole protein from Halobacterium halobium, Halobacterium salinarium strain 5, and the two species of cyanobacteria, Anabaena flos‐aquae and Microcystis aeruginosa. Mobility of the gas vacuole proteins in the four species are consistent with molecular weights of 16 800, 16 800, 14 700 and 15 700 respectively. Gels produced using the PAU system can be successfully fluorographed.
Amylase1polymorphism of human parotid saliva: Detection of a new allele, AMY by isoelectric focusing and AMY1population data from Germany
Authors: Kühnl Peter , Tischberger Hubert
Published: May 2005

Prolonged isoelectric focusing (IEF) of human saliva in narrow pH range polyacrylamide gels (pH 5–7) gave evidence for the existence of a new allele in the amylase1(AMY1) system. It has tentatively been designated AMY15and, inherited in an autosomal codominant way, as the variant phenotype AMY15–1, was observed in four individuals of a Yugoslavian family through three generations. The isoelectric point (pI) of the AMY15gene product is intermediate to AMY11 and AMY12. The allele frequencies of AMY1in 170 healthy, unrelated individuals from Hesse, Germany, were determined as follows: AMY11= 0.909, AMY12= 0.070, and AMY13= 0.021. With a single exclusion chance of 8.0 % for non‐fathers, AMY1might become a useful genetic marker in cases of disputed paternity as well as for “gene mapping” the fine structure of chromosome 1 in man.
Characterization of mouse lymphocytes by analytical automatic cell electrophoresis
Authors: Shimizu Motomu , Iwaguchi Takao
Published: May 2005

Mouse splenic lymphocytes were examined for electrophoretic mobility by an automatic cell electrophoresis. The whole population of lymphocytes showed a bimodal histogram of electrophoretic mobility. In contrast, adherent cells to a nylon wool column (B cell‐rich) and nonadherent cells treated with anti‐mouse IgG and complement (T cell‐rich) gave a unimodal histogram. A linear relationship existed between the electrophoretic mobility of mixture of T and B cells and their ratio detected by the method of immunofluorescence, suggesting that the ratio of T to B cells can easily be determined from the electrophoretic mobility of lymphocytes.
Horizontal gradient polyacrylamide gel electrophoresis
Authors: Altland Klaus , Hackler Rolf
Published: May 2005

A simple Technique for horizontal gradient polyacrylamide gel electrophoresis (PAGE) in discontinuous buffer systems is described, in which one‐dimensional, two‐dimensional and double one‐dimensional separation patterns can be obtained. The advantages of this technique are: (i) that samples can be applied by overlaying on a variable broad area of the concentrating gel, (ii) that the complete separating gel and the contact with the concentrating gel remain sandwiched between glass plates from the moment of gel polymerization till the end of electrophoretic separation, (iii) that many gels are poured from one gel solution, and (iv) that the gels can be stored, due to an identical buffer composition in the concentrating and separating gel.
pH‐Gradient development and focusing speed in thin‐layer polyacrylamide gel isoelectric focusing: A comparison between PharmalyteR, AmpholineR and ServalytR
Authors: Låås Torgny , Olsson Ingmar
Published: May 2005

The rate of pH gradient development and protein migration towards their isoelectric points(pI) were investigated for thin‐layer polyacrylamide gel isoelectric focusing (PAGIF) using Pharmalyte 3–10, Ampholine 3.5–9.5 and Servalyt 2–11 as carrier ampholytes. Using volt‐hours(Vh) as the controlling parameter, it was found that both pH gradient development and protein focusing proceeded very similarly with all three carrier ampholytes, Only two significant (but minor) differences between the carrier ampholytes were observed: (1) due to a faster voltage rise with Pharmalyte, the Vh needed for focusing are achieved faster than with the other two carrier ampholytes and (2) focusing of protein with alkaline pI's (> pH 8) is slightly slower with Servalyt (both in time and Vh). This is presumably explained by the low field strength at the alkaline end of the pH gradient with Servalyt. Also, it was found that pH gradient development was completely independent of applied voltage when Vh were used as controlling parameter.
Polymerization kinetics of polyacrylamide gels. III. Effect of catalysts
Authors: Righetti Pier Giorgio , Gelfi Cecilia , Bosisio Adriana Bianchi
Published: May 2005

The kinetics of photopolymerization, in presence of riboflavin or riboflavin‐5′‐phosphate (FMN), have been studied either spectrophotometrically, at 283 nm, or chemically, by titrating unreacted double bonds with permanganate. The two sets of data are in good agreement and suggest that at least 8 h of light exposure are needed to ensure 95% conversion of monomers into polymers. Up to now, it was generally accepted that photopolymerization should proceed for 1 h. It is not necessary to prolong light exposure for longer than 8 h, because even a 24 h illuminatin period only improves the conversion from 95% to 96%. Although conditions are described which ensure at least 95% conversion of both riboflavin‐and persulfate‐catalyzed gels, their visco‐elastic properties are widely different, suggesting that the final structure of these two matrices must vary considerably as a function of the catalyst used.
Comparison of rare PGM1 variants by isoelectric focusing and conventional electrophoresis: Identification of five new variants
Authors: Dykes Dale D. , Polesky Herbert F.
Published: May 2005

A comparison of specimens tested in our laboratory for the red cell enzymes sub‐system PGM1 by isoelectic focusing and conventional electrophoriesis demonstrated 11 rare allotypes. In addition to the allotypes PGM, PGM, PGM, , and , we indengified five new variants: , , , , . Of the five new variants only and could be separated from common PGM1 variants on conventional elehoresis. Because of the unique banding patterns for the variants observed, it was found necessary to use both isoelectic focusing and conventional methods for proper identification. Due to the presence for several different momeclatures for naming PGM variants, it is felt that a universal nomenclature should be defined to deal with PGM variants using both methods of analysis.
Synthetic inorganic ion exchangers XXI. Electrochromatographic separations of metal ions on lanthanum antimonite‐impregnated paper
Authors: De Anil K. , Chakraborty Punyabrata
Published: May 2005

Electrochromatographic studies of several metal ions on lanthanum antimonateimpregnated papers have been carried out. Various background electrolytes were used for these studies at different voltage and time intervals. On the basis of the differential mobilities of metal ions, which depend on the ion exchange properties of lanthanum antimonite and the nature of complex formation with the electrolytes, some important binary and ternary separations have been achieved.
A technique for the detection of variable electrophoretic patterns of hair proteins
Authors: Budowle Bruce , Acton Ronald T.
Published: May 2005

A reproducible isoelectic focusing method has been developed which displays the structural proteins of hair. By staining with the photochemically derived stain, hair protein variability was observed. On a limited number of individuals, five different isoelectric focusing patterns were detected. In addition, polychromatic differences of proteins with similar isoelectric points were found.
Silver staining of DNA in polyacrylamide gels: Linearity and effect of fragment size
Authors: Goldman David , Merril Carl R.
Published: May 2005

A simplified, inexpensive silver stain for proteins in polyacrylamide gels also stains DNA with a sensitivity of 1 ng (< 0.03 ng/mm2). Sensitivity of detection is superior to ethidium bromide and, for 11 double‐stranded fragments of ϕX174 DNA, staining is linear with respect to concentration. Surprisingly, the slope of staining is similar for pieces of DNA larger than 300 base pairs, but is greater for fragments of smaller size. Staining of DNA with silver is a practical alternative to visualization with ethidium bromide and may serve as a tool for elucidating the basis of silver staining.
Rapid staining of proteins in ultrathin‐layer isoelectric focusing in polyacrylamide gels
Authors: Frey Manuela D. , Radola Bertold J.
Published: May 2005

A method for protein staining after isoelectric focusing (IEF) in polyacrylamide gel is described in which the total time for fixation, staining and complete gel background destaining is reduced to 3–5 min. This is achieved by (i) staining dry gels on silanized supports instead of hydrated gels, (ii) reducing gel thickness to 50 μm, (iii) using Servalyt carrier ampholytes with good destaining properties, and (iv) using dyes with low affinity for carrier ampholytes, namely Serva Violet 17 or 49, facilitating destaining of gel background. The sensitivity of staining is 100–200 ng protein/ mm2over a 10 cm separation distance and 20–30 ng protein/ mm2over 3 cm gels (miniature focusing). Addition of 5–20 % methyl acetate to the staining or destaining solution reduces the sensitivity of staining significantly. Most effective fixation of proteins in 50 μm gels is achieved with 20% trichloroacetic acid. In solutions of alcohol and acetic acid, appreciable amounts of proteins are leached after incubation for 5–20 min. Paraformaldehyde, malondialdehyde or glutaraldehyde (5–10 %) exhibit no fixative effect on proteins in 50 μm gels when used directly or after precipitation of the proteins with trichloroacetic acid. The selective leaching of proteins under a variety of experimental conditions shows that adequate fixation is particularly important in work with basic, low molecular weight and/or hydrophobic proteins.
Gel electrofocusing in a natural pH gradient of pH 3–10 generated by a 47‐component buffer mixture
Authors: Cuono Charles B. , Chapo Giselle A.
Published: May 2005

A natural pH gradient with a pH range of 3–10 was generated from a mixture of 47 buffers utilizing a polyacrylamide gel slab format. The gradient is stable for at least 18 h at 100 V/cm of gel, with the attainment of the steady state at 1 h and of isoelectric protein positions within 2.5 h. Gels containing buffer constituents at a final concentration of 10–20 mM exhibit a conductivity which is approximately one order of magnitude less than that exhibited by any synthetic carrier ampholyte mixture (SCAM), thereby permitting a high degree of resolution in analytic applications. Excessive Joule heating and artefactual protein‐protein interaction, both potential drawbacks to electrofocusing at lower ionic strengths, were not observed in this study. The pH gradient generated by the buffer mixture makes possible for the first time electrofocusing of proteins under constant conditions in longitudinal studies, independently of the source and batch of carrier constituents. Furthermore, this system completely circumvents the physical, chemical and biologic problems frequently encountered with SCAMs.
Specific antiserum staining of two‐dimensional electrophoretic patterns of human plasma proteins immobilized on nitrocellulose
Authors: Anderson N. Leigh , Nance Sharron L. , Pearson Terry W. , Anderson Norman G.
Published: May 2005

Human plasma proteins separated by high‐resolution two‐dimensional electrophoresis have been electrophoretically transferred to sheets of nitrocellulose using a modification of the method of Towbin, Staehelin, and Gordon [8]. Although the proteins have been denatured in sodium dodecyl sulfate and separated into subunits, the nitrocellulose‐bound molecules still react with appropriate specific antisera even after storage of the transfer in air at room temperature for 5 months. Of 25 proteins whose location in the pattern had been previously determined, 24 are specifically revealed on transfers of whole plasma patterns by appropriate antiserum. In addition, 6 previously unidentified proteins (prothrombin, C1s, C4γ, C1s inhibitor, Ig J‐chain, and a1AP glycoprotein) have been identified in the pattern for the first time using the transfer technique. It therefore seems likely that a large majority of proteins (> 96 % in this study) retain sufficient conformation throughout the analytical procedure (or can regain it easily afterwards) to be recognized immunologically. The transfer technique thus constitutes a generally useful immunological “third‐dimension” in the high‐resolution separation of proteins. Of three monoclonal antibodies similarly tested, none could detect antigen transferred to nitrocellulose from a two‐dimensional gel, while each bound specifically to the appropriate antigen absorbed in native form to the nitrocellulose.
Improvements of isoelectric focusing in agarose for direct tissue isoelectric focusing
Authors: Thompson Basil J. , Dunn Michael J. , Burghes Arthur H. M. , Dubowitz V.
Published: May 2005

Improvements of isoelectric focusing (IEF) in agarose are described that result in consistently obtaining broad, linear pH gradients in the range pH 4 to 9.2 at equilibrium. Cathodic drift has been reduced by the use of a blend of commercial carrier ampholytes and by incorporating lysine and arginine in the catholyte as spacers. Resolution has also been enhanced by the use of thin gels, supplemented with sorbitol and glycerol. In addition, a comparison of two agarose preparations for isoelectric focusing was performed using two commercial carrier ampholytes. To minimise background staining, Triton complexes were removed by modification of the fixing and clearing procedures. These modifications to IEF in agarose are being applied to direct tissue isoelectric focusing (DTIF) of normal and diseased human skeletal muscle.
Detection of bacteriophage‐antibody complexes by agarose gel electrophoresis
Authors: Serwer Philip , Hayes Shirley J.
Published: May 2005

To detect bacteriophage‐antibody complexes, specific antiserum has been incubated with bacteriophages and antiserum‐induced aggregation has been detected by agarose gel electrophoresis, using differential sieving of the gel to fractionate aggregates by size. Agarose gel electrophoresis should also be of use in monitoring reaction of other virus‐sized antigens with antibodies.
Design types and performances of preparative polyacrylamide gel electrophoresis apparatus
Authors: Akaiwa Shigeo
Published: May 2005

The designs of both gel and elution chambers in preparative electrophoresis apparatuses are key elements for their performance. The present paper describes four types of designs which were developed by the author to alleviate the shortcomings found in the preceding designs. Performance tests, using human hemoglobin (Hb), partially purified haptoglobin 2‐1 type (Hp 2‐1), and bis‐albumin‐containing serum, show that insufficient dissipation of Joule heat and a heterogeneous electric field cause curvature and slant of the sample zones, and that only a chamber of optimal shape and size leads to minimal dilution, giving a good resolution. Moreover, notch, angle, and rim (Fig. 5), designed to support the gel, and tapering of the elution chamber to minimize its volume, are also important ministructures for the performance of the instrument. Unoptimal sets of these structures also cause concave sample bands and result in bad resolution and excessive dilution. These findings emphasize that in scaling up the apparatus, the structure must be designed on the basis of the detailed fundamental experiments from macrostructure to microstructure by using models of actual size.
Implementation and application of a method to quantitate 2‐D gel electrophoresis patterns
Authors: Jansson Peter A. , Grim Larry B. , Elias John G. , Bagley Elisabeth A. , Lonberg‐Holm Karl K.
Published: May 2005

We describe a system for accurate photometry and analysis of two‐dimensional (2‐D) gels, and its application to both high and low resolution gels. Image acquisition is video‐based and includes methods of data correction necessary for accurate photometry. The analysis is based upon a method described previously by Lutin et al. [8], which we have modified for improved stability and adapted to run on our PDP 11/60 computer. We illustrate analysis of a typical gel with a sequence of images showing the data while Gaussian components are estimated, removed, added and fitted as the iterative algorithm proceeds. We conclude by demonstrating the use of a mathematical curve‐of‐growth model for calibrating peak volumes vs. protein loading.
An improved electroelution method for the recovery of DNA fragments from agarose and acrylamide gels
Authors: Ho Nancy W. Y.
Published: May 2005

A simple elution tube has been constructed which allows reliable electroelution of DNA restriction fragments from agarose and acrylamide gels. This method enables the recovery of DNA fragments of 50 to over 50 000 base pairs from cylindrical or slab gels with yields between 70 % to 90 %. This method has several distinct features which make it outstanding. (1) It concentrates DNA from a large sample of gels into a small volume of buffer (less than 500 μl) during elution oso that the recovered DNA can be directly precipitated out with alcohol for subsequent use. (2) The recovered DNA is contaminated with only a minimal amount of agarose or acrylamide gel so that it may be subsequently digested with most restriction endonucleases with normal efficiencies. (3) It is particularly useful for the recovery of DNA fragments that have been subjected to end labeling with [γ‐32P] ATP and polynucleotide kinase, because it not only recovers the DNA from the gel, but also purfies the fragments from the contaminating background [γ‐32P] ATP. (4) Many different sizes of DNA fragments can be recovered at the same time. In addition, the elution tubes are easy to construct with not much more cost than that of a piece of plexiglas tubing.