bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of hypo‐ and hyperthyroidism on the function and metabolism of macrophages in rats
Authors: Rosa L. F. B. P. Costa , Safi D. A. , Curi R.
Published: May 2005

The effect of thyroid hormones on monocyte migration, phagocytic capacity and hydrogen peroxide production by macrophages and the effect of these hormones on glutamine and glucose metabolism was investigated. The experiments were performed on resident, thioglycollate‐ and BCG‐stimulated cells from hypo‐ and hyperthyroid rats. High plasma levels of thyroid hormones suppressed the migration of monocytes and hydrogen peroxide production, whereas hypothyuroidism did not affect cell migration but rasied the phagocytic capacity and the hydrogen peroxide production. Hyperthyroidism increased the activities of glutaminase and hexokinase and the rates of decarboxylation of [U‐14C]‐glutamine and [U‐14C]‐glucose in inflammatory and activated cells. Hypothyroidism stimulated glucose metabolism and had only a slight effect on glutaminolysis. The activity of the TCA cycle was, however, diminished in the presence of high plasma levels of thyroid hormones and enhanced by the hypothyroid state. These findings suggest that the functional changes observed are more likely to be related to the activity of the TCA cycle rather than to glutaminolysis and glycolysis.
Bivascular liver perfusion in the anterograde and retrograde modes: Zonation of the response to inhibitors of oxidative phosphorylation
Authors: Constantin Jorgete , Ishii‐Iwamoto Emy , Suzuki‐Kemmelmeier Fumie , Yamamoto Nair S. , Bracht Adelar
Published: May 2005

The action of cyanide (500 μM), 2,4‐dinitrophenol (50 μM) and atractyloside (100 μM) on glycogen catabolism and oxygen uptake was investigated in the bivascularly perfused liver of fed rats. Cyanide, 2,4‐dinitrophenol and atractyloside were infused at identical rates into the hepatic artery in either the anterograde or retrograde perfusion. The accessible aqueous cell spaces were determined by means of the multiple‐indicator dilution technique. Glucose release, oxygen uptake and glycolysis were measured as metabolic parameters.

Oxygen uptake changes per unit cell space caused by atractyloside (inhibition) and 2,4‐dinitrophenol (stimulation) were equal in the retrograde perfusion (periportal cells) and the anterograde perfusion (space enriched in perivenous cells); the decreases caused by cyanide were higher in the retrograde perfusion. Glucose release from periportal cells was not increased upon inhibition of oxidative phosphorylation, a phenomenon which was independent of the mechanism of action of the inhibitor. There were nearly identical changes in glycolysis in the periportal and perivenous cells.

It was concluded that: (1) oxygen concentration in the perfused rat liver, if maintained above 100 μM, had little influence on the zonation of the respiratory activity; (2) in spite of the lower activities of the key enzymes of glycolysis in the periportal hepatocytes, as assayed under standard conditions, these cells were as effective as the perivenous ones in generating ATP in the cytosol when oxidative phosphorylation was impaired; (3) the key enzymes of glycogenolysis and glycolysis in periportal and perivenous cells responded differently to changes in the energy charge.
Citrinin affects the oxidative metabolism of BHK‐21 cells
Authors: Chagas Generoso M. , Campello Annibal P. , Kluppel Ma. Lúcia W. , Oliveira Ma. Benigna M.
Published: May 2005

The effects of citrinin on energy production along the respiratory chain and on glycolytic lactate production were examined in BHK‐21 cultured cells. Citrinin inhibited the oxygen consumption rate by about 45 per cent. The respiratory rate of digitonin‐treated cells energized with succinate, in the presence of ADP, was reduced by about 39 per cent. The mycotoxin inhibited the glucose utilization of BHK‐21 cells by about 86 per cent. Cells treated with citrinin produced a small quantity of pyruvate, but were unable to produce lactate. It is concluded that BHK‐21 cells cannot generate lactate when oxidative metabolism is inhibited by citrinin. The perturbations in BHK‐21 cells caused by citrinin are due to alterations in mitochondrial function and in the glycolytic anaerobic pathway.
Numerical deconvolution of gas chromatograph peaks using Jansson's method
Authors: Crilly Paul Benjamin
Published: May 2005

Severely overlapped gas chromatographic peaks can be resolved using Jansson's method. Jansson's method is an iterative nonlinear deconvolution technique and consists of Van Cittert's algorithm with the relaxation factor a function of the kth estimate of the peak shape. The maximum peak amplitude of the instrument and peak non‐negativity serve as constraints to improve the peak estimate. Super‐resolution is achieved without significantly degrading the chromatogram signal‐to‐noise ratio. The method only requires a knowledge of the instrument impulse response function and maximum peak height.
Extraction of near infra‐red spectral information by fast fourier transform and principal component analysis. Application to the discrimination of baking quality of wheat flours
Authors: Devaux Marie‐Françoise , Bertrand Dominique , Robert Paul , Morat Jean‐Luc
Published: May 2005

Digitalized continuous near infra‐red reflectance (NIR) spectra are composed of a great number of data which must be reduced for microcomputer mathematical treatment. The sequence ‘fast Fourier transform preceding principal component analysis’ was tested to perform data size reduction without a large loss of information. The method was applied on a collection of wheat spectra composed of 351 data. Ten resulting data, which described 99.5% of the total variance, were kept. The relevance of the method was estimated by the ability of the resulting data (i) to regenerate the original signal, and (ii) to discriminate the baking quality of the wheat by stepwise multiple discriminant analysis. The average difference between initial and regenerated spectra was −2.4 × 10−3 log (1/R) units and the standard deviation was 1.16 × 10−3 log (1/R) units. The discrimination treatments gave 89.9% of well classified samples for the calibration test and 90.5% for the prediction test. The application of these mathematical treatments to other continuous signals is discussed.
Mechanism of the hydrolysis of adenosine 5′‐triphosphate: A regression analysis of kinetic data
Authors: Hibbert D. Brynn , Sandall John P. B.
Published: May 2005

Data of the hydrolysis of adenosine 5′‐triphosphate have been re‐evaluated using a computer program that numerically integrates the differential rate equations within a routine that optimizes rate coefficients given a suitable model and concentration versus time data.

The model is tested by calculation of Hamilton R‐values, the Fisher F‐statistic, a sensitivity analysis, the standard errors on the rate coefficients and by constructing contour maps of the objective function versus two rate coefficients.

An optimization using only phosphate concentration data cannot distinguish between a model in which adenosine 5′‐monophosphate is formed predominantly directly with a molecule of pyrophosphate, and one in which it is formed via adenosine 5′‐diphosphate. A more accurate set of rate coefficients is calculated from existing data and the relative importance of the two paths determined.
A bootstrap resampling scheme for using the canonical correlation technique in rank estimation
Authors: Tu Xin M.
Published: May 2005

Rank estimation by canonical correlation analysis in multivariate statistics has been proposed as an alternative approach for estimating the number of components in a multicomponent mixture. A methodological turning point of this new approach is that it focuses on the difference in structure rather than in magnitude in characterizing the difference between the signal and the noise. This structural difference is quantified through the analysis of canonical correlation, which is a well‐established data reduction technique in multivariate statistics. Unfortunately, there is a price to be paid for having this structural difference: at least two replicate data matrices are needed to carry out the analysis.

In this paper we continue to explore the potential and to extend the scope of the canonical correlation technique. In particular, we propose a bootstrap resampling method which makes it possible to perform the canonical correlation analysis on a single data matrix. Since a robust estimator is introduced to make inference about the rank, the procedure may be applied to a wide range of data without any restriction on the noise distribution. Results from real as well as simulated mixture samples indicate that when used in conjunction with this resampling method, canonical correlation analysis of a single data matrix is equally efficient as of replicate data matrices.
The Present and future of CVD studies in Korea
Authors: Choi S. S.
Published: May 2005

South Korea is one of the most rapidly growing countries in terms of technological developments. Advanced materials in general and CVD in particular will undoubtedly play an important role in those developments. In this essay Professor Choi outlines the short history of CVD in Korea, high‐lights some of the key features of present day Korean CVD, and points out future trends. He pays particular attention to the range of results presented at the first Korean Chemical Vapor Deposition Symposium, which was held last year.
Chemical vapor deposition of Gallium selenide and indium selenide nanoparticles
Authors: Stoll Sarah L. , Gillan Edward G. , Barron Andrew R.
Published: May 2005

Nanometer‐sized structures can exhibit quite different properties from those of their macroscopic forms. In this paper is is reported that, under suitable conditions, well‐defined nanoparticles of InSe and GaSe may be obtained from the vapor phase thermolysis of heterocubane molecules. For example, InSe grown from [(EtMe2C)InSe]4 consists of spheres with a mean diameter of 88 nm (standard deviation s.d. = 30 nm), while GaSe grown from [(tBu)GaSe]4 produces pseudo‐spherical nanoparticles with a mean diameter of 42 nm (s.d. = 13 nm). Possible controlling mechanisms are discussed.
Thin film growth and microstructure analysis of CeO2 prepared by MOCVD
Authors: Becht Michael , Morishita Tadataka
Published: May 2005

CeO2 Thin films were prepared by metal‐organic chemical vapor deposition (MOCVD) using tetrakis(2,2,6,6‐tetramethyl‐3,5‐heptanedionato)cerium(iv), [Ce(thd)4], as the precursor. The films were grown in a horizontal cold‐wall reactor at low pressure and temperatures of 400–900°C in an atmosphere of argon/oxygen. SrTiO3, yttria stabilized zirconia (YSZ), and MgO were used as substrates. Films were prepared on single crystalline substrates with a (100) orientation as well as on polycrystalline YSZ. The film microstructure was investigated by X‐ray diffraction and the surface morphology was studied by scanning electron microscopy (SEM). The growth rate was determined using data obtained by profilometry and cross‐section SEM. The films are polycrystalline with a (100) preferential orientation with respect to the substrate orientation. Low oxygen partial pressures deteriorated the crystalline quality. The SEM images of the surface and the cross sections demonstrated a columnar microstructure. The growth rates in the range of 25–250 nm/h were strongly dependent on the precursor feeding rate but less affected by the deposition temperature and substrate material.
Amperometric glucose sensors based on glucose oxidase immobilized in nafion
Authors: Rishpon Judith , Gottesfeld Shimshon , Campbell Carmen , Davey John , Zawodzinski Thomas A.
Published: May 2005

A glucose‐sensitive electrode composed of glucose oxidase (GOX) embedded in a layer of the polymer Nafion is described. Electrodes of various configurations on which the layer was cast were tested to determine their response to glucose additions to phosphate buffer and human whole serum samples. These electrodes have good response characteristics and several desirable features. Glucose sensors based on Nafion/GOX layers cast onto platinum electrodes respond to glucose additions with a rapid response time (2 to 4 seconds), have a long lifetime when stored in a phosphate buffer (55 days before termination of testing), have a reduced sensitivity to local oxygen tension, respond linearly to additions of glucose to the serum, and are not fouled by exposure to blood. The Nafion confers some permselectivity on the layer, thus providing a degree of discrimination against anioni interferents.
Dual wall‐tube electrode cell for use in clay‐modified electrode studies
Authors: Stein Jennifer A. , Fitch Alanah
Published: May 2005

A dual tube‐electrode cell has been constructed for use in clay‐modified electrode (CME) studies. The optimization and characterization of the wall‐tube cell through both visual and electrochemical measurements is described. Its use in some CME studies is then described. The system is found to give excellent insight into the behavior of a CME.
Ion‐exchange properties of poly(L‐lysine) coatings on pyrolytic graphite electrodes
Authors: Bianco Pierre , Haladjian Jean , Giannhndrea Sophie
Published: May 2005

Pyrolytic graphite electrodes are coated with films of poly(L‐lysine) (PLL). The polyelectrolyte films bind multiply charged counterions. Cyclic voltammetry is used to investigate the incorporation of Fe(CN) anions as a function of time, concentration, pH, and ionic strength. The stability of the film is studied after transfer of the PLL coatings into an electrolyte solution. The PLL‐modified pyrolytic graphite (PG) electrode proves to be very effective in facilitating electron transfers of negatively charged proteins, such as ferredoxin and hydrogenase.
Copper‐sensitive modified poly(pyrrole) electrode
Authors: Arca Meral , Yildiz Attila
Published: May 2005

A copper ion sensitive poly(pyrrole) electrode, containing diethyldithio‐carbamate as a ligand is described. The ligand was incorporated into the electrochemically‐prepared polymer coating via anion exchange. Cyclic voltammetry and infrared spectroscopy were used to characterize the modified electrode surface. The uptake of copper ions was achieved by electroreduction at −0.65 V (vs. SCE), and the cyclic‐voltametric anodic peak current of the complex ion at +0.55 V (vs. Ag/AgCl) was used for analysis. A detection limit for copper ions was estimated to be 1 ppm.
Electrocatalytic reduction of hydrogen peroxide on a palladium‐modified carbon paste electrode
Authors: Cai Xiaohua , Kalcher Kurt , Kölbl Gottfried , Neuhold Christian , Diewald Wolfgang , Ogorevc Bozidar
Published: May 2005

A palladium‐dispersed carbon paste electrode was used for the electrocatalytic reduction of hydrogen peroxide. After cycling the potential between 0.4 and −0.8V (vs. SCE) in pure dilute alkaline solution (10−3M NaOH), the resulting electrode surface exhibited stable and sensitive electrocatalytic response for hydrogen peroxide. The involved catalytic mechanism was thoroughly investigated. The electrocatalytic effect was attributed to electrogenerated elemental palladium at the electrode surface. The catalytic reduction of hydrogen peroxide proceeds via the hydroxyl radical, OH·. Dissolved oxygen can also be catalytically reduced in a similar way to hydrogen peroxide. The height of the cathodic catalytic current peak, as obtained by linear sweep voltammetry (LSV) was directly proportional to the hydrogen peroxide concentration over a very wide concentration range (0.1–600mgL−1). The detection limit (3σ) was calculated as 50 μgL−1 H2O2.
Detection of new features associated with the oxidation of microcrystalline tetrathiafulvalene attached to gold electrodes by the simultaneous application of electrochemical and quartz crystal microbalance techniques
Authors: Shaw Shannon J. , Marken Frank , Bond Alan M.
Published: May 2005

Simultaneous voltammetric or double potential step and quartz crystal microbalance experiments combined with the results obtained from optical and surface analysis measurements have allowed new mechanistic aspects of the electrochemical oxidation of microcrystalline particles of tetrathiafulvalene (TTF) attached to a gold substrate, which is placed in aqueous electrolyte media, to be unravelled. The use of the microcrystalline form of the solid rather than thin films makes it possible to obtain short time domain data which enables features for the reduction and the oxidation process consistent with a nucleation process at the solid electrode‐aqueous electrolyte interface to be detected. Electrochemical quartz crystal microbalance (EQCM) and electron microprobe analysis experiments confirm that the overall process in the oxidation of microcrystals of TTF in aqueous KBr, KI, KClO4, NaBr and CsBr electrolyte media involves the uptake of the appropriate nonsolvated anion (X‐) in order to maintain charge neutrality. Reduction of the oxidized TTF leads to the expulsion of the anion. The rates of uptake and expulsion are both very rapid for the bromide ion, but involve slower reaction steps for iodide and perchlorate anions. The oxidation of TTF particles attached to a gold electrode can be described by the equation

The number of electrons transferred to the solid, n, may theoretically vary over a wide range, but in the case of X− =Br− is found to be fixed with only one product formed (n =0.7). The peak potential and shape of voltammograms have been studied as a function of scan rate, electrolyte concentration and the nature of the anion and cation. A Nernstian change in peak potential is observed when the electrolyte anion concentration is varied. The wave shape, but not the charge, also varies with electrolyte anion and concentration. In contrast, no dependence on the electrolyte cation is found. All the data imply that incorporation of the anion into the solid provides the charge neutralization mechanism. This study confirms that use of microcrystalline forms of a solid enables a wide range of time domains (and techniques) to be applied to electrochemical studies of solids.
Cyclic voltammetry of a unique quinone–quinol equilibrium
Authors: Maali Nagwa Abo El , Berg Hermann
Published: May 2005

The thermochromic quinone‐quinol equilibrium of more than 20 derivatives was studied by classical DC‐polarography. This tautomery causes one reversible two‐electron wave and one irreversible (about 1 V) more negative wave. This double‐wave current‐voltage curve occurs only in the case of one asymmetric oxyethylamino substitution in the 2‐position for benzoquinones and for naphthoquinones. Our present investigations characterize further this unique slow equilibrium by the influence of the scan rate ν on cyclic voltammograms. The peak potentials are shifted to more negative values with increasing ν. Both the heights of the second wave and of the positive anodic step increase linearly with ν, whereas the height of the positive kinetic reduction wave behaves similarly only when ν < 25mVs−1. However, at ν > 50mVs−1the decyclization rate of quinol to quinone has no further influence on the first peak height andk2was determined directly and independently of former DC‐polarographic and spectrometric results.
The effect of zinc(II) and some chromium complexes on the constant current chronopotentiometry of insulin
Authors: Honeychurch Michael J. , Ridd Michael J.
Published: May 2005

The constant current chronopotentiometry (CCCP) of zinc‐free insulin was examined and the effect of additions of zinc(II) or either of two chromium complexes to the zinc‐free zinc was studied. The zinc free insulin gave a constant current chronopotentiometric (CCCP) response which indicated that the freeze drying process, which was part of its preparation, may have denatured the insulin. The addition of zinc(II) appeared to reverse this since the response following zinc additions was similar to that of native insulin. Of the two chromium complexes investigated for their influence on insulin electrochemistry tripicolinato chromium(III) had no effect on the CCCP response, trans‐Diaquoethylenebis‐(salicylideneiminato) chromium(III) had an indirect effect by appearing to restore the native conformation of the insulin following the apparent denaturation caused by the removal of zinc.
Staircase voltammetry with finite diffusion space
Authors: Lovrić Milivoj , Komorsky‐Lovrić Šbojka , Scholz Fritz
Published: May 2005

A theory of the staircase voltammetry in a thin‐layer cell is developed. The influence of the cell thickness and the redox reaction rate constant on the current and the peak potentials is analyzed. For moderately thick cells, the kinetic maximum is predicted. The relationship between peak currents and the square root of the scan rate is not linear and the response may entirely vanish if the redox reaction is fast, the cell is very thin and the scan rate is slow.
Modification of pH gradients in isolelectric focusing by the pH of the anolyte
Authors: An der Lan Birgit , Chrambach Andreas
Published: May 2005

In isoelectric focusing on polyacrylamide gel, pH gradients exhibited a systematic shift along the pH‐axis in proportion to the pH of the anolyte. Thus, more acidic anolytes produced more acidic pH gradients, and more basic ones gave rise to more basic pH gradients. In all cases the pH's of the anolyte and catholyte were kept outside the pI‐range of the carrier ampholytes used. Increasing the pH of the anolyte stabilized pH gradients, while the pH of the catholyte, within the range tested, had no systematic effect on pH gradient stabilization.

From a practical viewpoint, modification of pH gradients by the anolyte provides increased flexibility in pH gradient design for electrofocusing. From a theoretical viewpoint, the selective anolyte effect on pH gradients in isoelectric focusing on polyacrylamide gels supports the hypothesis previously advanced by Murelet al., which relates pH gradient decay in electrofocusing to progressive protonation from the anodic pH gradient terminus.
Molecular weight determinations of RNA by gel electrophoresis
Authors: Carmichael Gordon G.
Published: May 2005

There are a number of methods‐currently available for determining the molecular weights of RNA molecules by gel electrophoresis. This article reviews these techniques briefly and appraises the major advantages and disadvantages of each.
pH Gradient flattening in isoelectric focusing in long polyacrylamide gels
Authors: Rapaport Ray N. , Jackiw Andrew , Brown Ray K.
Published: May 2005

Isoelectric focusing in 60 cm long gels improves resolution in multiple‐component systems by providing very shallow, linear pH gradients. Broad range ampholytes available commercially or synthesized in our laboratory develop pH gradients of 0.1 pH/cm at voltages of 1500 to 4500 V, with running times of 25–30 h. Shallower gradients result with narrow range ampholytes or separator isoelectric focusing. Hemoglobin, human serum albumin, and an α‐N‐acetylgalactosaminidase have been used for defining this system.
Distorted zones in isoelectric‐focusing systems
Authors: Jonsson Mats
Published: May 2005

A maximum utilization of the high resolving power of isoelectric focusing requires that the focused zones are both planar and perpendicular to the direction of focusing. This paper reports on extensive investigations carried out with a rectangular quartz column with provisions for direct photography of zonal shapes. The experiments have been performed in density gradients, but the conclusions may be generalized to other stabilizing media. It is demonstrated that distorted zones are formed if a second pH gradient, perpendicular to the direction of focusing, originates at one end of the column. The generation of such a pH gradient may have different causes: erroneous loading of the column, too high an initial power input, presence of salts of weak electrolytes in the sample, or unsuitable positioning of the electrodes. The effects of the second pH gradient on the shapes of the focused carrier‐ampholyte and protein zones are interpreted on the basis of the mechanism creating the natural axial pH gradient. The stability of the disturbed system is discussed in terms of the diffusional, electrical, and convective mass flows of its components.
An investigation into the mechanism of electrophoretic desorption of immunoglobulin G from protein A‐Sepharose
Authors: Haff Lawrence A.
Published: May 2005

The effect of voltage on the rate of desorption of radiolabeled rabbit immunoglobulin G (IgG) from protein A‐Sepharose was studied. In the absence of voltage, the rate of desorption was measured using a vast excess of unlabeled IgG to prevent re‐adsorption. Electrophoretic desorption was measured using a column equipped with electrodes and with eluent flow occurring in the direction of electrophoresis. The rate of desorption was very sensitive to voltage, and, furthermore, desorption could occur at a rate 25 times faster than in the absence of a voltage. The results are interpreted to indicate that the presence of voltage not only altered the equilibrium of the system but altered the equilibrium or rate constants of the system.
Cellulose acetate electrophoresis of glycosaminoglycans: Determination of concentration and specific activity by solid‐phase spectrophotomertry and fluorography
Authors: Stack Michael T. , Golichowski Alan M.
Published: May 2005

A simple method for the quantitative determination of glycosaminoglycan concentration and radioactive precursor incorporation into glycosaminoglycans is describe. Tissue glycosaminoglycans are separated by electrophoresis on cellulose acetate sheets. Sheets are then impregnated with diphenyloxazole in toluene and tritium activity quantitated by fluorography. Absolute absorbance measurement of X‐ray film fluorograms exposed for 80 h demonstrate a sensitivity of 50 dpm per samle (< 5 dpm/mm2) and reproducibility of 7% Diphenyloxazole is removed with toluene and the sheets are stained with Alcian Blue. Absolute absorbance determination allow quantity measurement of mass with a sensitivity of 0.05 nmol of disaccharide subunit and a reproducibility of 6%. Beer's law is followed to 3nmol. Example of the utilization of this technique in studying radionuclide incorporation into glycosaminoglycans in these tissues‐ articuular cartilage, uterine cervix and skin‐are presented.
Simplified silver protein detection and image enhancement methods in polyacrylamide gels
Authors: Merril Carl R. , Goldman David , Van Keuren Margaret L.
Published: May 2005

The use of silver for detection of protein in polyacrylamide gels is becoming widespread. An effort has been made to develop a silver stain which minimizes the time to perform the stain, the amount of silver used, and the complexity of the procedures, and which maximizes the sensitivity. This stain is almost 200‐fold more sensitive than the Coomassie Brilliant Blue R‐250 stain. This silver stain procedure has been shown, with eight purified proteins, to generally be linear over a 40‐fold range in concentration, between 0.005 ng/mm2 to 2.0 ng/mm2. For greater sensitivity, a recycling procedure has been developed. This procedure is capable of amplifying trace proteins which could not be visualized with previous silver stain techniques. Analysis of the kinetics of image development revealed that the silver ion was the limiting factor in image formation. By recycling the gel through silver nitrate solutions, silver ions are replenished in the gel, permitting amplification of further details when the gel is treated with a developing solution. A number of problems inherent in silver stain detection of proteins in polyacrylamide gels are discussed with suggested remedies.
A procedure for stabilization of cytopherometers for immunological applications
Authors: Koenig Ulf D. , Bongartz Lothar , Kozan A. Burak
Published: May 2005

A device commonly used for cell electrophoresis is the cytopherometer. Its application in an immunological tumor test, however, pointed out many shortcomings and instabilities. In order to use cytopherometers in cellular immunology, a procedure was worked out to stabilize this instrument. By enlarging the electrode chambers, using a simple and efficient buffer in all compartments, coating the electrophoresis chamber, and by following a set of electrophoretic parameters (current, temperature, etc.), an intraassay variance of 1.8 % and an interassay variance of approximately 1 % were reached.
Improved isoelectric focusing of normal and variant forms of erythrocyte galactose‐1‐phosphate uridyl transferase
Authors: Vaccaro Anna M. , Muscillo Michele , Mandara Ida , Salvioli Rosa
Published: May 2005

An improved isoelectric focusing (IEF) technique has been developed and used to detect the erythrocyte galactose‐1‐phosphate uridyl transferase isoenzymes. The normal enzyme was resolved by IEF in four bands with isoelectric points (pI) between 5.80 and 6.08. Numerous samples, classified according to electrophoresis as Duarte or Los Angeles homozygotes, showed the same IEF pattern composed of four bands with pI's between 5.60 and 5.90. The Duarte‐normal and Los Angelesnormal heterozygotes gave an IEF pattern of 4‐5 bands (pI 5.60‐6.08), with relative intensities that varied according to their activity.
Characterization of intracellular deoxyribonucleases using polynucleotide‐polyacrylamide gel electrophoresis
Authors: Brown Glenn E. , Karpetsky Timothy P. , Rahman Aneesa , McFarland Edward , Haroth Mary B.
Published: May 2005

Intracellular deoxyribonucleases (DNases) in human peripheral blood leukocytes have been identified and characterized by polynucleotide‐polyacrylamide gel electrophoresis (PPAGE). This technique combines the protein separating powers of polyacrylamide gel electrophoresis with the sensitivity of detection characteristic of an enzyme assay. Visualization of nuclease activity is made possible by the inclusion of high molecular weight DNA substrate within the sieving portion of a narrow polyacrylamide gel. No sodium dodecyl sulfate, urea, or other protein denaturants are used in this procedure. However, electrophoresis of the contents of up to 106 cells/gel is accomplished using conditions under which the enzymes are inactive. The positions of DNases are then revealed by incubation of the gel in an appropriate buffer followed by staining for intact DNA. Colorless regions correspond to the presence of enzyme. Used as a pattern recognition tool, PPAGE revealed that leukocytes from normal donors contained lower levels of certain activities than did cells from donors with chronic myelogenous leukemia. The rapid simultaneous characterization of multiple activities is made possible by simply using different buffer compositions in the incubation step. For example, ionic strength and pH optima may be found directly for activities present in unpurified human intracellular contents. The reproducibility of this method permits comparison of individual activities by using Ferguson plot analysis to detect differences or similarities among enzymes. These analyses revealed that DNase activities in human peripheral blood leukocytes fell into a minimum of two families. Modifications of overall effective charge probably account for microheterogeneity.
Photometric evaluation of slab gels II: Delimitation and integration of peaks in one‐dimensional electropherograms
Authors: Yakin Habib M. , Kronberg Harald , Zimmer Hans‐G. , Neuhoff Volker
Published: May 2005

In order to increase accuracy and precision of quantifying one‐dimensional electropherograms by digital signal processing, a set of algorithms is presented that operate on the optical density values acquired by a linear scanning densitometer. These operations include smoothing of the measuring values, background correction by an optimized piecewise linear baseline, and integration of the peak areas after two‐sided fit of half‐Gaussian profiles to each stained protein band. The algorithms are implemented as BASIC‐programs and are readily adaptable to a variety of electrophoretic separations. The procedure has been applied successfully in research [1] as well as in clinical evaluations [2].
Crude‐tissue‐extract analysis by micro‐electrophoresis in cylindrical continuous polyacrylamide gradient gels
Authors: Murach Karl‐Friedrich , Beltle Wolfgang , Bopp Martin
Published: May 2005

Continuous gradient gels in capillaries with acrylamide concentrations ranging from 2–20 % or alternatively from 2–40 % are equally suited for the fractionation of complex mixtures of macromolecules in aqueous solutions within a range of molecular weights from 103‐106. A time‐saving method for the simultaneous preparation of up to 48 identical linear micro‐gradient polyacrylamide gels is described. With this method it was possible to compare the patterns of aqueous extracts and to determine tissue‐specific bands of the mossFunaria hygrometrica L. Sibth.in the low molecular weight range for the first time.
Formation of natural pH gradients in sequential moving boundary systems with solvent counterions II. Predicted and experimental properties
Authors: Buzás Zsuzsanna , Hjelmeland Leonard M. , Chrambach Andreas
Published: May 2005

Sequential moving boundary electrophoresis with protons and hydroxyl ions as the sole counterions, consisting solely of six acids (system A) and of six bases (system B), gives rise to natural steady‐state pH gradients similar to those predicted by theory (pH 2.4–5.5 for system A, pH 10.3–12.2 for system B). Mixtures of the constituents in system A and system B gave rise to a natural pH gradient spanning the pH range between the terminal electrolytes (pH 2.4 and 12.2) predicted for systems A and B. The predicted boundary displacement is negligibly small. Experimentally the boundary displacement could not be determined in view of the difficulty of separating it from the initial transient state in which the pH gradient is formed, and a final state in which the pH gradient decays.
Anomalous conductivity zones in electrophoresis I. Basic theory for two‐ion systems
Authors: Spencer Michael
Published: May 2005

The progressive increase in circuit resistance observed at times in polyacrylamide gel electrophoresis is attributable in part to the formation of zones of altered salt concentration at the interfaces between gel and solution. Similar changes have been reported for electrophoresis through sucrose gradients. Such zones must develop wherever migrant ions suffer a change in transport number. The paper develops a basic theory for such concentration changes. It is shown that a moving boundary will tend to arise at any interface between two media, leaving behind it a zone of altered concentration. The effect will in general be most noticeable at low ionic strengths and with electrolytes whose ions differ considerably in size, a condition applying to many common buffers. Where both ions are relatively small, diffusion is likely to obscure the boundary movement, but this will not affect the integrated change in concentration. The effect could have various deleterious consequences in gel electrophoresis, particularly in preparative apparatus. There are also implications for techniques such as isoelectric focusing and for the analysis of membrane phenomena.
Anomalous conductivity zones in electrophoresis. II. Theory for three‐ion systems and for changes in pH
Authors: Spencer Michael
Published: May 2005

The theory developed in the previous paper is extended to cover a system containing three ions. If an “inactive” electrolyte whose ions are equally affected by a retarding medium is added to a “zone‐former” for which this is not the case, the main effect is to reduce the integrated concentration changes at the interfaces. Where a buffer contains two ions carrying charges of the same sign, the theory predicts a progressive change of pH within a zone of altered concentration. Other sources of pH change are also considered. Where a buffer includes an uncharged weak acid or base, changes in ionic concentration at an interface will lead to changes in pH. Other changes may arise from the migration of water ions, whose contributions are normally neglected. This is not justified where there are large discontinuities in conductivity. Expressions are derived for the changes in pH under different circumstances. In some cases no steady‐state is possible and there are accelerating shifts of pH at the boundaries of a zone. This could have important consequences in practical electrophoresis.
Detection of subnanogram quantities of DNA and RNA on native and denaturing polyacrylamide and agarose gels by silver staining
Authors: Guillemette Joseph G. , Lewis Peter N.
Published: May 2005

A silver staining method is described for detecting subnanogram amounts of DNA and RNA on polyacrylamide and agarose gels. The key step for increased sensitivity and linearity is an initial soaking of the gel in a 0.1% solution of cetyltrimethylammonium bromide (CTAB). Agarose‐containing gels can be silver stained after treatment with formaldehyde. The methods described are comparatively rapid, extremely sensitive (≅ 150 pg of DNA) and inexpensive (0.4 g of AgNo per 25 ml of slab volume).
Two‐dimensional electrophoresis in the development of clinical laboratory tests
Authors: Young Donald S. , Tracy Russell P.
Published: May 2005

Before any new test should be introduced into clinical laboratory practice the need for the test should be carefully assessed. Generally, new tests should only be made available when they have distinct advantages over other tests to assess the same organ function or disease process. The appropriateness of the test must be considered in relation to its intended application and it must be examined in the proper clinical context when it is advocated as a diagnostic test. The potential applications of two‐dimensional electrophoresis in the clinical laboratory have been examined. These include its actual use as a diagnostic test with serum and urine specimens, if the simultaneous examination of several proteins can be justified, and if an important role in the identification of disease related changes in protein concentrations can be established which could subsequently be exploited to develop specific diagnostic tests; then the method has a valid application in the search for new clinical procedures.
Isolation by free‐flow electrophoresis and immunological detection of troponin T from turkey muscle: An application in food chemistry
Authors: Schweiger Anton , Baudner Siegfried , Günther Herbert O.
Published: May 2005

The identification of turkey meat in food products has been accomplished using specific antisera against turkey troponin T. This protein (Mr 37 000) was extracted from fresh muscle tissue and isolated by a procedure that included free‐flow electrophoresis as final fractionation step. Preparations of troponin T with purities of more than 95 % were applied as antigen in the immunization of rabbits. Several standard immunological techniques were used to determine additions of turkey meat in food products heated to different temperatures (including roasting temperature). That way, it was possible to also detect additions of chicken meat on the basis of a partial identity between troponins T from turkey and chicken muscle.
Nonspecific binding of p‐phenylenediamine‐pyrocatechol peroxidase substrate to electrofocused proteins
Authors: Cantarow Walter D. , Saravis Calvin A. , Sampson Christian E.
Published: May 2005

Nonspecific staining of electrofocused protein bands was found with the peroxidase product of Hanker‐Yates reagent (a mixture of p‐phenylenediamine and pyrocatechol). No difficulty was encountered with diaminobenzidine as peroxidase substrate.
Pseudo‐ligand affinity chromatography and high‐voltage isoelectric focusing as a multiparameter method for separation and identification of plasma proteins
Authors: Allen Robert C. , Arnaud Philippe
Published: May 2005

A system is described that utilizes physical‐chemical characteristics of macromolecules, not restricted to charge‐size variations, for the separation of the plasma proteins. The first step, using pseudo‐ligand affinity chromatography, separates the plasma proteins into fractions which were characterized by fused rocket immuno‐electrophoresis. Then electrophoresis in the form of high voltage gradient isoelectric focusing on ultrathin‐layer polyacrylamide gels is employed to further separate each of the fractions by charge differences. The advantages of this technique are that the proteins are maintained without noticeable denaturation, allowing a variety of direct functional tests. One is not restricted to only charge‐size variations for separation and the technique is a preparative‐to‐analytical procedure. Furthermore, the higher voltage gradients employed increase resolution of proteins significantly and require separation times of only 30–35 min. The limit of detectability is 50 μg/dl of protein, using silver diamine staining. The feasibility of this and related techniques as applied to the study of genetic polymorphisms and as an aid in the correlation and identification of spots obtained in dissociating‐denaturing two‐dimensional electrophoresis methods is emphasized.
Two‐dimensional polyacrylamide gel electrophoresis of nuclear proteins in human meningiomas
Authors: Unteregger Gerhard , Zang Klaus D. , Issinger Olaf‐Georg
Published: May 2005

The nuclear protein fraction from human meningioma cells was compared with the nuclear protein fraction of fibroblasts, derived from the same patients. The protein pattern obtained was visualized either with the silver stain technique or from labelled cells by fluorography. Whereas no specific polypeptides corresponding to tumor cells or fibroblasts could be detected in one‐dimensional polyacrylamide gel electrophoresis (1‐DE), small differences occur in the two‐dimensional gel patterns (2‐DE), using the silver protein detection. Cell cultures which were labelled during phase G 1 show twice as many polypeptides on the 2‐DE electropherogram with respect to the silver‐stained gels. Besides several proteins detectable in tumor cells and fibroblasts with both methods, a set of 5 polypeptides with an apparent molecular weight (Mr) of 30 000 and an isoelectric point (pI) near 6.1 could be detected in the 2‐DE pattern from labelled tumor cells. Furthermore, the tumor cells showed a remarkably enhanced incorporation of label in proteins in the pH range 5.5–7.0 and with Mr from 14 000–40 000. The fluorograms from both cell types reveal more than 40 polypeptides with a pI of 4.5–5.5 and an Mr from 12 000–45 000, which had never been detected on the silver‐stained electropherograms. The results indicate that cells derived from meningiomas exhibit a pattern of nuclear proteins quite similar to that obtained from fibroblasts with the same diploid karyotype. Furthermore, it seems necessary to combine the high‐resolution separation techniques with other analytical methods, e. g. specific labelling procedures, to enlighten the role of nuclear proteins during the onset of tumorgenesis.
Approximate mobility calculation of composed ions
Authors: Michov Budin M.
Published: May 2005

Equations are proposed for calculating the approximate mobility of ions on condition that they are considered to be composed of ions with known mobilities.
Avidin biotin amplified immunoperoxidase staining of angiotensin‐1‐converting enzyme transferred to nitrocellulose after agarose isoelectric focusing
Authors: Lanzillo Joseph J. , Stevens Joanne , Tumas John , Fanburg Barry L.
Published: May 2005

Angiotensin‐1‐converting enzyme (ACE) subjected to isoelectric focusing in agarose gel can be transferred to nitrocellulose in a reliable and reproducible manner. Specific detection of ACE is enhanced 2‐fold by replacing enzyme‐conjugated reagents with avidin biotin‐conjugated reagents. Sensitivity is not diminished when the carcinogenic substrate 3,3′‐diaminobenzidine is replaced with the non‐carcinogenic substrate 3‐amino‐9‐ethylcarbazole. This improved procedure was used to monitor the spontaneous change in the isoelectric point of ACE which occurred during purification of ACE from outdated human plasma.
An improved and simplified apparatus for protein extraction and concentration from gel slices, using moving boundary electrophoresis
Authors: Lan Birgit An Der , Horuk Richard , Sullivan James V. , Chrambach Andreas
Published: May 2005

A previous design of an apparatus for extraction and concentration of proteins from gel slices by moving boundary electrophoresis has been simplified, making it into an upper buffer reservoir accessory for the multifunctional gel tube apparatus commonly used for gel electrophoresis. The design of the collection cup has been improved to facilitate assembly and thereby reduce the risk of leakage. The performance of the apparatus was evaluated by the isolation from sodium dodecyl sulfate ‐ polyacrylamide gel electrophoresis of radiolabeled bovine serum albumin and glucagon receptor.
High resolution electrophoresis of extracts of a baby hamster kidney cell line before and after transformation by polyoma virus
Authors: Marshall Thomas , Latner Albert L.
Published: May 2005

The polypeptide patterns obtained following denaturation and high resolution two‐dimensional electrophoresis of proteins from baby hamster kidney cells before and after transformation by Polyoma virus have been compared by silver staining incorporating methylamine. The polypeptides were resolved either by non‐equilibrium pH‐gradient electrophoresis or isoelectric focusing in the first dimension. Two different approaches to isoelectric focusing were employed in order to achieve satisfactory resolution over a wide range of isoelectric point. Both cell lines gave remarkably similar two‐dimensional patterns of spot distribution which allowed changes to be detected in the relative abundance of a number of polypeptides. None of the polypeptides detected were specific to either of the two cell lines.
Effects of chemical exposure on rat serum proteins revealed by a modified technique of two‐dimensional gel electrophoresis
Authors: Marshall Thomas , Vesterberg Olof
Published: May 2005

Rat sera were analysed by high resolution two‐dimensional electrophoresis to detect polypeptides sensitive to chemical exposure. Exposure to carbon tetrachloride, trichloroethylene or dimethylformamide increased the intensity of a polypeptide with electrophoretic characteristics (pI 4.3, isoelectric point; molecular weight, MW 45 000) similar to those of human orosomucoid. The two‐dimensional procedure was modified by using only selected gel lengths (pH range 4.5–6.5) from the first‐dimension isoelectric focusing gels. Three such gel sections were positioned side by side on the top of a second‐dimension gel slab prior to electrophoresis. Using this approach, heavily stained regions of the two‐dimensional pattern were clarified by allowing simultaneous analysis in one gel slab of the same sample at three different dilutions. With this modification other potential marker proteins for chemical exposure were detected. The advantages and application of this modified technique are discussed.
Enzymatic amplification of electroimmunoprecipitates in agarose gels with horseradish peroxidase‐labeled protein A
Authors: Taketa Kazuhisa
Published: May 2005

Electroimmunoprecipitates in agarose gels were amplified with protein A‐horseradish peroxidase (HRP) conjugate. The method is simple and detected quantitatively as low as 0.5 ng of human α‐fetoprotein (AFP) in electroimmunodiffusion. The alternative methods employing the second antibody, labeled initially or afterwards with HRP, failed to stain AFP immunoprecipitates. Direct application of anti‐AFP immunoglobulins‐HRP in electroimmunodiffusion produced less contrasted colored precipitates. Immunoprecipitates of other proteins were similarly visualized by the protein A‐HRP method, avoiding the use of specifically labeled antibody.