bluuebunny/crossref_metadata_2025 huggingface dataset:
High glucose and hyperosmolarity increase heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) production in cultured human aortic endothelial cells
Authors: Asakawa Hideki , Miyagawa Jun‐Ichiro , Higashiyama Shigeki , Goishi Katsutoshi , Hanafusa Toshiaki , Kuwajima Masamichi , Taniguchi Naoyuki , Matsuzawa Yuji
Published: May 2005

Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) has been shown to be a potent smooth muscle cell (SMC) mitogen and chemoattractant, and might be a candidate factor for the progression of atherosclerosis. We have investigated the effects of high glucose and hyperosmolarity on HB‐EGF production in cultured human aortic endothelial cells. Following the culture of the cells for 2 days with high concentrations of glucose or in the hyperosmolar conditions, we measured the content of HB‐EGF and the rate of production in the cells using a semi‐quantitative immunofluorescent technique and a metabolic radiolabelling method. With high glucose (16.6 mmol) and hyperosmolar conditions (glucose 5.5 mmol + mannitol 11.1 mmol or glucose 5.5 mmol + raffinose 11.1 mmol), the content of HB‐EGF was significantly increased and the metabolic rate was also significantly increased (more than a twofold increase, compared to that of 5.5 mmol glucose). In conclusion, conditions of high glucose or hyperosmolarity increase HB‐EGF production in human aortic endothelial cells. These results suggest that diabetic macroangiopathy might be attributed at least in part to HB‐EGF‐related vascular changes which may be induced by glucose.
Insulin treatment can abolish changes in glucose and glutamine metabolism of lymphocytes and macrophages caused by the implantation of the walker 256 tumour
Authors: Fernandes Luiz Claudio , Mattozo Carlos Alberto , Machado Ubiratan Fabres , Rosa Luis Fernando B. P. Costa , Curi Rui
Published: May 2005

Activation of lymphocytes and macrophages by the implantation of tumour cells (107 cells per rat) into the left flank of rats increased the conversion of glucose to lactate and of glutamine to glutamate and aspartate and the decarboxylation of [U‐14C]‐glucose and [U‐14C]‐glutamine in incubated cells. In addition, the amount of GLUT1 was increased in macrophages. The effect of insulin treatment on glucose and glutamine metabolism of lymphocytes and macrophages activated by Walker 256 tumour implantation was also examined. For this purpose, insulin was injected subcutaneously (4 U/100 g b.w. daily) after the fourth day of tumour implantation and the rats were killed 10 days afterwards. Insulin treatment fully reverted the changes due to tumour implantation in the metabolism of glucose and glutamine in lymphocytes and of glucose in macrophages.
Palmitoyl‐CoA synthetase on the external surface of isolated rat hepatocytes
Authors: de Groote Katrien , Velghe Griet , Stals Hedwig K. , Baes Myriam I. , Declercq Peter E.
Published: May 2005

After incubating isolated rat hepatocytes with [1‐14C]palmitic acid, CoA and ATP (+MgCl2), a significant amount of [1‐14C]palmitoyl‐CoA was found in the incubation medium. There was no correlation between its rate of synthesis and the degree of intactness of the cells. The results indicate that there is a long‐chain fatty acyl‐CoA synthetase active on the external surface of the hepatocyte plasma membrane. The activity of this enzyme was negligible in primary cultures of rat hepatocytes, suggesting that the exofacial long‐chain acyl‐CoA synthetase is an artifact of the collagenase perfusion technique used to prepare the hepatocytes.
Determination of the dimensionality of spectroscopic data by submatrix analysis
Authors: Cartwright Hugh
Published: May 2005

In the treatment of spectroscopic data by principal component analysis, correct determination of the dimensionality of the data is essential. Submatrix analysis, which involves preliminary analysis of subsets of the data matrix, is described, and shown to be valuable in determining both the size of the factor space and estimates of equilibrium constants in the system, even for data containing abnormally large experimental error.
Relationships between higher‐order data array configurations and problem formulations in multivariate data analysis
Authors: Esbensen Kim. H. , Wold Svante , Geladi Paul
Published: May 2005

A scaffold for detailed understanding of the concept ‘dimensionality’ in data analysis is furnished by a systematic classification of higher‐order data array configurations. Three major types of problem formulation in multivariate data analysis can be characterized for relevant data classes:

data description (intra‐class data structure modelling of inter‐object and inter‐variable relationships)

classification (inter‐class discrimination)

correlation, regression (inter‐variable relationships).

The relationship between these three categories of data analytical problem formulation and the fundamental data array classification is exposed. These relations are augmented to include the general case of data arrays of order R, and R‐way data analysis with the use of bilinear projections is presented. Based upon this, some possible directions for the future development of data analysis may be imagined.
A test of three fitting criteria for multiresponse non‐linear modeling
Authors: Pell Randy J. , Kowalski Bruce R.
Published: May 2005

This work evaluates objective functions for multiresponse non‐linear modeling using computer simulations. Tests are performed under a variety of signal‐to‐noise ratios and noise variance–covariance structures. The standard error of prediction for the model parameters, computed from 50 trials, is used for performance comparisons. The full rank and rank‐deficient problems are considered. For the full rank problem one model was investigated, a first‐order two‐step consecutive reaction model, and two objective functions were considered, the total sum of squares and the determinant criterion. No distinction could be made between the two objective functions for this model.

For the rank‐deficient case two models were investigated, a first‐order two‐step consecutive reaction as in the full rank case, and a pH titration model described by the Henderson–Hasselbalch equation. Three objective functions were investigated for the rank‐deficient case, the total sum of squares, a weighted total sum of squares and the determinant criterion. The total sum of squares was found to perform poorly under all conditions tested compared to the weighted total sum of squares and the determinant criterion. The determinant criterion was found to perform much better than the other two criteria when the data have a combination of a low signal‐to‐noise ratio and high variance–covariance noise structure.
Comparison of pattern recognition descriptors for chemical acoustic emission analysis
Authors: Wentzell Peter D. , Lee Oliver , Wade Adrian P.
Published: May 2005

Features used to characterize acoustic emission signals from chemical systems are evaluated with regard to their potential for pattern recognition. Eight chemical systems involving phase transitions, hydration, dissolution and effervescence are employed and treated as separate signal classes. These are compared pairwise and the discriminatory capabilities of about 50 features are investigated by computing Fisher weights. Time domain and frequency domain descriptors are examined. Correlations among the features evaluated are also reported. Recommended descriptors are the mean and median frequencies, frequency bandwidth, number of level crossings (0% and 25%), crest factor (time and frequency domains), half‐life, kurtosis and normalized percentiles of the signal and its power spectrum. The effectiveness of the recommended descriptors is demonstrated through the separation of signal classes in two different systems (melting ice and an enzyme‐catalyzed gas formation reaction) by principal components analysis.
Computer‐generated multicomponent calibration designs for optimal analysis sample predictions
Authors: Hitchcock Kimberly , Kalivas John H. , Sutter Jon M.
Published: May 2005

This paper utilizes variable step size generalized simulated annealing (VSGSA) to design multicomponent calibration samples for spectroscopic data. VSGSA is an optimization procedure which is capable of converging to exact positions of global optima located on multidimensional continuous functions. On the basis of analysis sample response vectors, optimally designed calibration concentration matrices are obtained assuming knowledge of components present. The complexity of response surfaces established by the optimization criteria is described.
The ectal relations of the right and left parietal and paroccipital fissures
Authors: Wilder B. G.
Published: May 2005

The parietal and paroccipital fissures may be either completely separated by an isthmus, or apparently continuous. When so continuous ectally there may still be an ental and concealed vadum or shallow. Disregarding the vadum on the present occasion, the ectal relations of the two fissures may be designated as either continuity or separation. That continuity occurs more frequently on the left side has been noted by Ecker, Cunningham and the writer. Hitherto, however, statistics have included unmated hemicerebrums as well as mates from the same individuals. The following statement is based upon the cerebrums of 58 adults of both sexes and various nationalities and characters. The speaker has examined 48; the other 10 have been accurately recorded by Bischoff, Dana, Jensen and Mills.

The four possible combinations of right and left continuity and separation occurred as follows:

Left continuity and right separation in 27; 46.5%.

Right and left continuity in 22; 38%.

Right and left separation in 8; I 3.8%.

Left separation and right continuity in I; I.7%.

When 5 groups of persons are recognized the combinations are as follows:

In 8 moral and educated persons, combination I, 62.5; II, 25; III, 12.5.

In 23 ignorant or unknown, I, 56.5; II, 34.8; III, 8.7.

In 20 insane, I, 40; II, 35; III, 20; IV, 5.

In 4 murderers, I, o; II, 75, III, 25.

In 3 negroes, I, 33; II, 67.

So far as these 58 individuals are concerned, the most common combination, viz., left continuity and right separation, is decidedly the rule with the moral and educated, less frequent with the ignorant and unknown, the insane and negroes, and does not occur at all in the murderers. The only instance of the reverse combination (left separation and right continuity) is an insane Swiss woman. The only two known to be left‐handed presented the more frequent combination I.

These statistics suggest many special queries and problems, some of which were briefly indicated. But the speaker wished this to be regarded as a preliminary communication and asked the coöperation of other members in the effort to obtain satisfactory records of larger numbers, particularly of brains of wellborn, moral and educated persons. For this purpose a blank form was outlined and will be sent upon application.
The influence of Fe‐group metals on the CVD of titanium carbide
Authors: Konyashin Igor Y.
Published: May 2005

A number of experiments and computer simulations involving chemical vapor deposition (CVD) of TiC or TiN on various substrates containing Fe, Co or Ni allow the following mechanism of the influence of these metals on the CVD process to be assumed: These metals, being transferred from the substrate into the coating, evidently accelerate the decomposition of methane and enhance its reactivity at the substrate surface. Carburization of titanium, formed due to hydrogen reduction of TiCl4, which is a limiting stage of the TiC CVD, is activated in this way. The deposition rate, structure and morphology of the TiC coatings are affected by the presence of Fe, Co or Ni at the deposition zone, due to their catalytic effect on the process of methane activation and decomposition.
Absolute (calibrationless) chronoamperometric determination with a mercury hemispherical semimicroelectrode
Authors: Bustin Dušan , Mesároš Štefan , Rievaj Miroslav , Tomčík Peter
Published: May 2005

Amperometric determination of electroactive species not requiring calibration described in this work is based on separation of linear and spherical parts of limiting diffusion current of a determined substance to the hemispherical Hg‐semimicroelectrode (radius 37.5 μm). Charges transfered by these two components of current during experiment are comparable for semimicroelectrodes and the determination error is acceptably low (< 10%) for concentration interval 50–500 μmol dm−3. The calibrationless amperometric determination was verified with cadmium and lead. A new way of calculating optimum value of electrode radius to minimize the determination error is also presented.
A simple electropolymerization method for the production of microarray electrodes
Authors: Imisides Mark D. , Wallace Gordon G.
Published: May 2005

The preparation of a polymer based microarray system is described. The technique utilizes electropolymerization to form a nonconductive polymeric layer on a conductive substrate. Provided appropriate electrochemical conditions are used, microarray behavior can be obtained for the resultant modified electrode substrate.
Determination of formation constants of metal complexes by potentiometric stripping analysis
Authors: Tutunji Maha F.
Published: May 2005

A method based on the data obtained from complexometric titration followed by potentiometric stripping analysis is developed. Optimization of the experimental conditions, namely: choice of the proper oxidant, deposition potential, plating time, concentration, and pH, considerably reduced the errors contributed by kinetic dissociation. This method was applied to ethylenediaminetetraacetic acid (EDTA) titration with lead and gave a formation constant for Pb‐EDTA that agreed well with literature values. The method may be applied to the determination of formation constants of 1 : 1 complexes with Kf values about 1020. The feasibility of the metal ion depends mainly on the solubility of the metal in the mercury in addition to its reversible behavior at the thin mercury film electrode.
Highly sensitive detection of L‐lactate and pyruvate by amperometric flow‐injection analysis based on enzymatic substrate recycling and selective detection of hydrogen peroxide
Authors: Yao Toshio , Satomura Masahiro , Nakahara Tuketoshi
Published: May 2005

The poly(1,2‐diaminobenzene)film‐coated platinum electrode was used as an amperometric detector in a flow‐injection system with the lactate dehydrogenase/lactate oxidase coimmobilized reactor involving amplification by substrate recycling. Both L‐lactate and pyruvate are recycled enzymatically during their passage through the enzyme reactor in the presence of reduced nicotinamide adenine dinuceotide (NADH) and oxygen in the carrier stream. As a result, a large amount of hydrogen peroxide is generated in the enzyme reactor. The poly(1,2‐diaminobenzene)‐coated electrode could only selectively detect the hydrogen peroxide generated in the enzyme reactor, without an increase in the base‐line current owing to the direct oxidation of NADH, because the poly(1,2‐diaminobenzene) film effectively prevented NADH in the carrier stream from reaching the electrode surface and allowed only the hydrogen peroxide to penetrate into the polymerized film. In the present flow‐injection analysis (FIA) system, both L‐lactate and pyruvate were determined with a 400‐fold increase in sensitivity compared with the unamplified responses. The detection limit was 2 × 10−9 M (20 fmol) for 10 μL sample injection.
A conductometric detector for capillary separations
Authors: Müller D. , Jelínek I. , Opekar F. , Štulik K.
Published: May 2005

A simple electrochemical cell has been constructed for conductometric detection in capillary electrophoresis or HPLC. A platinum ring electrode is chemically deposited at the capillary end and the other electrode, a platinum wire, is immersed, together with the capillary, in a vessel containing the running buffer or the mobile phase used. The cell has been tested on a model system and its operational parameters have been discussed. The measurement is very sensitive (a limit of determination of ca. 3 × 10−9 M for potassium ions) and the detector response is rapid, comparable with that of the UV photometric detector. The dynamic range is wide (at least five concentration decades) but is nonlinear at high concentrations.
Polarographic maxima revisited
Authors: Noufi Mouna , Yarnitzky Chaim , Ariel Magda
Published: May 2005

The relation between maxima (of the first kind) and drop lifetime‐potential (TE) curves was investigated by recording both the DC polarogram and the TE curve and comparing the latter to that obtained with the blank solution, under identical conditions. The advanced instrumentation for measuring drop life‐time allowed the detection of minute variations occurring in various regions of the applied potential range. Positive maxima were studied with CUII/ClO, CuII/Cl−, PbII/Cl−, FeIII/C2O and UO/C2O systems; negative maxima, with solutions of NiII, ZnII and PbII.

Hypotheses explaining the variations in maximum shape and height and in drop lifetime, taking into account the nature of the electroactive species (mainly, the location of the reduction step vs. the pzc), or the supporting electrolyte concentration are submitted, including the differing behavior observed with positive and negative maxima.
An amperometric lactate sensor based on a NAD+‐analog and lactate dehydrogenase coimmobilized on reticulated vitreous carbon
Authors: Khayyami Masoud , Garcia Nuria Peña , Larsson Per‐Olof , Danielsson Bengt , Johansson Gillis
Published: May 2005

A NAD+‐analog was coimmobilized with lactate dehydrogenase (LDH) on reticulated vitreous carbon (RVC) to give an amperometric lactate biosensor. Both LDH and the NAD+ ‐analog were bound covalently with carbodiimide to the surface of the porous RVC‐material. The electrode was operated in a FIA‐arrangement in the presence or absence of a soluble mediator. Meldola Blue. The stability was poor when the electrode was operated at +400 mV (vs. Ag/AgCl) in the absence of mediator but improved most significantly in the presence of 5 μM mediator, so that 65% of the original activity remained after 16 days. The amperometric currents were smaller with regeneration by mediator at −100 mV than with direct electrochemical oxidation at +400 mV, indicating that the additional steps slow down the reaction rate. Linear calibration plots were obtained from the detection limit, 1 μM, to 500 μM lactate with 5 μM mediator, reoxidized at −100 mV. The sample throughput was about 60 h−1.
Metal analysis in real matrices. Simultaneous voltammetric determination of copper and antimony in alloys
Authors: Locatelli Clinio
Published: May 2005

This work addresses the simultaneous determination of copper(II) and antimony(III) in real matrices by differential pulse (DPASV) and fundamental harmonic alternating current anodic stripping voltammetry (ACASV). The voltammetric measurements were carried out using as supporting electrolyte the same acidic mixture (nitric, hydrochloric and perchloric acids) used in the dissolution of the real matrices with proper dilution. The procedure of the sample preparation is thus reduced to one step hence avoiding errors from long and complex sample handlings prior to the instrumental measurement. The results were verified by the analysis of the standard reference materials NBS‐SRM 631 Spectrographic Zinc Spelter D‐2 and BCS 207/2 Gunmetal. The precision, expressed as relative standard deviation, and the accuracy, expressed as relative error, were, in all cases, less than 5%; the detection limit, for each element and in the experimental conditions employed, was around 10−7 M. The standard addition technique improved the resolution of the voltammetric method, even in the case of very high metal concentration ratios.
Rapid isoelectric focusing and detection of nanogram amounts of proteins from body tissues and fluids
Authors: Allen Robert C.
Published: May 2005

Polyacrylamide gel isoelectric focusing was carried out using 250 μm thick gels backed with polyester films at voltage gradients up to 460 V/cm. Peltier cooling was employed with berrylium oxide heat transfer plates to achieve adequate cooling. Visualization of low protein concentrations was obtained by the use of a highly sensitive silver staining technique. Results indicated that over separating distances of 5.4 cm 150 to 200 bands should be resolved and that considerable differences between two commercial carrier ampholytes exist. Pharmalyte carrier ampholytes from Pharmacia have better continuity and conductance over the range of pH 3–10 but require much higher field strengths to achieve maximal separation than do LKB Ampholines over a similar pH range.
Microheterogeneity of serum myeloma immunoglobulins revealed by a technique of high resolution two‐dimensional electrophoresis
Authors: Latner Albert L. , Marshall Thomas , Gambie Moira
Published: May 2005

A simplified technique of high resolution two‐dimensional electrophoresis has been used to investigate heterogeneity of human serum myeloma proteins. Three types of microheterogeneity have been detected. The heavy chains of IgA, IgM and IgD myeloma proteins demonstrate charge heterogeneity which is far less extensive following neuraminidase treatment and is therefore largely due to sialic acid. The underlying charge heterogeneity, which is apparently resistant to neuraminidase, resembles the charge heterogeneity demonstrated by the heavy chains of IgG myeloma proteins which show no response to neuraminidase treatment. The third type of microheterogeneity is most clearly demonstrated by the heavy chains of IgD myeloma proteins which exhibit heterogeneity with respect to small variations (∼︁3000) in molecular weight. This type of heterogeneity has also been observed, to a lesser extent, in the heavy chain of IgM and IgA myeloma proteins.
One‐ and two‐dimensional electrophoresis in micro‐slab gels
Authors: Poehling Hans‐Michael , Neurhoff Volker
Published: May 2005

All acrylamide slab gel electrophoresis widely used for various separation problems at the marcro‐level can be converted readily to micro‐versions. This is demonstrated in isoelectric focusing, homogeneous or gradient micro‐slab gels, and two‐dimensional separations. The different types of micro‐slab gel electrophoresis have the same resolving capability as the corresponding macro‐method, which is demonstrated with different biological samples. Enzyme staining after two‐dimension separations is also possible. The apparatus, preparation and handling of the different micro‐slab gels is described in full detail.
True and false glycoprotein microheterogeneity observed with lectin crossed affinoimmunoelectrophoresis of inter‐alpha‐trypsin‐inhibitor
Authors: Salier Jean‐Philippe , Faye Loîc , Vergaine Dominique , Martin Jean‐Pierre
Published: May 2005

Crossed affinoimmunoelectrophoresis (CAIE) with free lectin in the first dimension was used to investigate sugar heterogeneity of inter‐α‐trypsin‐inhibitor (ITI) in normal human serum and after purification. Such a heterogeneity was assessed with Lens culinaris agglutinin and Concanavalin‐A, but not with wheat germ agglutinin. The use of a mixture of purified ITI and ITI‐free serum demonstrated that a distortion of the immunoprecipitation patterns for ITI in normal serum was observed with Con‐A and WGA and was due to presence of other glycoproteins. Thus, prior protein purification is sometimes required before obtaining a true pattern of glycoprotein microheterogeneity by lectin CAIE. Furthermore, loading a sugar specific for the lectin in the second dimension gel was shown to be necessary in order to observe all heterogeneous forms of the glycoprotein tested.
Two‐dimensional electrophoresis of membrane and cytosol proteins of mouse liver and brain
Authors: Klose Joachim , Feller Marianne
Published: May 2005

Membrane proteins and cytosol proteins from organs (liver, brain) of inbred mice were separated by two‐dimensional electrophoresis (2DE). The 2DE technique revealed high resolution of complex protein solutions by staining the proteins. The 2DE patterns of membrane proteins showed about 660 protein spots (polypeptides) for both liver and brain. The protein patterns of the cytosols revealed about 800 polypeptides for the liver and about 680 for the brain. The membrane proteins and cytosol proteins of the 2DE patterns represented two protein populations specific for these two cell components. They matched in 7 % (liver) or 23 % (brain) of the total number of membrane protein spots. The matched spots represented dissociable membrane proteins rather than contamination of the isolated membrane fractions by cytosol proteins. Protein patterns of the same cell fraction but from the two different organs investigated contained more than 50 % organ‐unspecific polypeptides.

Several conditions of our 2DE standard procedure were reinvestigated to test the usefulness for our technique of some methodical steps employed in 2DE by other authors. We found that many steps often used in sample preparations impaired rather than improved the 2DE pattern. In particular, addition of SDS to the protein sample resulted in a considerable loss of proteins during isoelectric foucing. The 2DE itself could be simplified by omitting several steps such as prefocusing, equilibration of focusing gels, forming gel gradients, autoradiography. This has some implications for the development of 2DE to a routine method.
A method for continuous preparative electrophoresis using a supporting medium
Authors: Lammel Bernd
Published: May 2005

A method for continuous preparative electrophoresis is presented. It is based on the principle that a supporting medium, e.g. a polyacrylamide gel, is moved continuously relative to an electrical field. At one fixed point, the sample is applied to the gel. The charged particles migrate with different velocities in the supporting medium. Simultaneously, they are dragged along with the supporting medium. This procedure leads to a two‐dimensional separation of charged particles, which is necessary for a continuous operation of an electrophoresis apparatus. Three different designs of apparatuses, using this method, are presented in this paper. The operation of one construction was tested by separating three protein mixtures.
Two‐dimensional immunoelectrophoresis of rat serum apolipoproteins
Authors: Dolphin Peter J.
Published: May 2005

A two‐dimensional crossed immunoelectrophoretic method for the determination of the specificity of antibodies raised against rat serum lipoproteins and apolipoproteins (Apo) is described. The method may also be used for the detection of as yet undesignated apoproteins, associated with the serum lipoprotein fractions, and exploits the superior resolving capacity of 7m urea 10% polyacrylamide gel electrophoresis for the first dimension separation and minimizes physical separation of polyacrylamide and agarose in the second dimension electrophoresis by use of low electroendosmotic flow agarose. The use of polyacrylamide instead of cellulose acetate in the first dimension permits the analysis of larger amounts of antigen protein, crossed imunoelectrophoresis of Apo E which binds to cellulose acetate, and the crossed immunoelectrophoresis of antigens previously focused to their isoelectric points in polyacrylamide gels containing ampholytes.
Isotachophoretic analysis of the end‐product and intermediates in the synthesis of the C‐terminal pentapeptide of bombinin
Authors: Friedel Klaus , Holloway Christopher J.
Published: May 2005

Capillary isotachophoresis has been applied as an analytical monitor during the synthesis of the C‐terminal pentapeptide, Gln‐His‐Phe‐Ala‐Asn, of bombinin. The samples were separated as cations since the C‐terminal carboxyl group was modified to an uncharged amide. Potassium and beeta‐alanine were employed as leading and terminating ions and acetate was the common counter‐ion. The analyses demonstrated that the stages of synthesis up to the tripeptide, Phe‐Ala‐Asn, gave quite pure products, whereas a considerable amount of impurity was detectable after the introduction of His into the molecule. From the peptide chemist's point of view, this amino acid presents special problems. However, the impurities found in the isotachopherograms were not detectable by conventional methods such as thin‐lyer chromatography. The rapid and sensitive method of isotachophoresis should prove useful to the peptide chemist for assessment of his products, and for redesign or modification of non‐optimal synthetic strategies.
Protein concentration and recovery from gel slabs by displacement electrophoresis (isotachophoresis) and the effects of electroosmosis and counter flow
Authors: Öfverstedt Lars‐Göran , Johansson Gunnar , Froman Gunnar , Hjerten Stellan
Published: May 2005

Alternative technical solutions of the concentration of protein samples or the recovery of proteins from gel slabs by displacement electrophoresis are demonstrated. The samples are collected at a boundary between high‐mobility leading ion and a low‐mobility terminating ion either in a small Sephadex G‐25 columns or in free solution in a rotating‐tube free electrophoresis apparatus. The boundary is kept stationary during electrophoresis by a counter flow of leading solution. Spacers and colored markers can be used to facilitate the collection of the proteins. Investigations showed that electroomosis is a very local phenomenon and that, as expected, the magnitude of the effects depends on the conductivity (field strength) of the solution. This implies that electroomosis will vary among different zones in a discontinuous system (as in displacement electrophoresis). Therefore, in most experiments om free solution it is very advantageous to arrange the system such that electrophoresis does not occur.
The use of bacteriophage T4 as a set of molecular weight and isoelectric point markers for two‐dimensional electrophoresis
Authors: Kurian Ponnamma , Gersten Douglas M. , Suhocki Paul V. , Ledley Gary
Published: May 2005

We have studied the use of bacteriophage T4 coat proteins as a complete set of molecular weight and isoelectric point markers for two‐dimensional electrophoresis. We present data which support the feasibility of this approach. The potential utility and advantages of this concept are discussed.
Polymerization kinetics of polyacrylamide gels II. Effect of temperature
Authors: Gelfi Cecilia , Righetti Pier Giorgio
Published: May 2005

The temperature at which polyacrylamide gels are polymerized strongly affects the structure and physical properties of this matrix. The suggested temperature of 0–4 °C has been found to produce highly turbid, highly porous and unelastic gels. At temperature of 25 °C of higher, the gels became progressively transparent, less porous and more elastic. This phenomenon is strongly pronounced in N,N′‐methylenebisacrylamide (Bis) gels and progressively decreases for N,N′‐(1,2‐dihydroxyethylene)bisacrylamide (DHEBA) and N,N′‐bisacrylylcystamine (BAC) gels. It has been attributed to the formation of hydrogen bonds among cross‐linker molecules, which are presumably stabilized by low temperature and progressively broken at higher temperatures. The most extensively H‐bonded compounds seem to be Bis molecules, since they can form four H‐bonds/molecule, followed by DHEBA (in which inter‐molecular H‐bonds have to compete with intra‐molecular H‐bonds) and finally by BAC, which is sparingly H‐bonded at 2 °C, and fully uncomplexed at 30°C. It is suggested that polymerization at 0–4°C should be abandoned, since it leads to unhomogeneous and unreproducible pore size matrices. Since H‐bonds in Bis molecules are fully disrupted only at 60°C (a temperature unsuitable for gel polymerization, since it produces short chains and unelastic matrices), it is also suggested that the use of Bis should be discontinued in favour of better cross‐links, such as DHEBA. In all cases, best polymerization conditions are obtained at 25–30°C. For all three cross‐linkers, homogeneous gels are obtained in presence of 8 M urea or in pure formamide as solvent, since both agents fully disrupt H‐bonds.
Incorporation of methylamine in an ultrasensitive silver stain for detecting protein in thick polyacrylamide gels
Authors: Marshall Thomas , Latner Albert L.
Published: May 2005

An ultrasensitive silver stain is described for the detection of proteins in 4–20 % w/v polyacrylamide gradient gels of 3mm thickness, Maximal sensitivity was achieved by substituting methylamine for ammonia in the diamine preparation and adopting a strategy of overstain followed by destain. The prediamine steps were performed at 60°C to conserve time, and the intensity of final background stain was reduced by altering the method of diamine preparation. From our experience, the technique described is more sensitive and reliable than previously published methods when applied to gels of this thickness, In any case, the sensitivity in our hands was at least equal to that of the newly described photochemical technique [7].
Two‐dimensional polyacrylamide gel electrophoresis of water‐soluble erythrocyte membrane proteins
Authors: White Mark D. , Ralston Gregory B.
Published: May 2005

A method is reported for analyzing the oligomeric state of proteins in a mixture by multidimensional electrophoresis. Water‐soluble proteins prepared from human erythrocyte membranes were separated by means of gel filtration followed by electrophoresis in detergent‐free disc gels. The major bands in detergent‐free disc gels were identified by means of electrophoresis into a second‐dimensional slab gel containing sodium dodecyl sulphate. Detergent‐free disc gels were also subjected to two‐dimensional electrophoresis into detergent‐free slab gels consisting of a continuous gradient of polyacrylamide gel. It was shown that many of the water‐soluble proteins existed as distinct oligomers. Spectrin aggregates (tetramer and dimmer) accounted for the slowest moving bands in the detergent‐free disc gels. A water‐soluble protein of the component 3 region appeared to be present as a hexamer, while component 4.1 was present as a tetramer. Components 4.3, 4.9 and 5 appeared to be present in a large number of aggregated states. Components 7 and 8 formed a heteropolymeric protein complex.
Properdin factor B (Bf) distribution in North and Central American populations
Authors: Dykes Dale D. , Polesky Herbert F. , Crawford Michael H.
Published: May 2005

A study of properdin factor B (Bf) in 22 North and Central American populations demonstrated gene clusters delineating Old World Caucasian, New World and Black Ancestoral groups. Result of gene frequencies were comparable to data in the literature of European Caucasian and African Blacks. New World indigeneous population were clearly separated from other racial groups with hybrid population clustering in between the major genetic contributions.
High resolution ultrathin‐layer isoelectric focusing of PGM1‐subgroups in forensic blood typing
Authors: Pflug Werner , De La Vigne Ulf , Bruder Walter
Published: May 2005

Ultrathin‐layer isoelectric focusing in 100 um polyacrylamide gels is described for the separation of the isozymes of the PGM1‐locus. The PGM1‐subgroups are best resolved in pH 5–7 gradients obtained by mixing Ampholine and Servalyt carrier ampholytes in a 2:1 ratio. By using a high field strength (180 V/cm) in the final stage of isoelectric focusing, the isozyme bands are sharper than in conventional thin‐layer gels. This facilitates identification of the subgroups. Distortions of the pH gradient are avoided if low initial field strengths (15–20 V/cm) are used. The method is highly reproducible and equally applicable for the analysis of blood samples as well as blood stains.
Agarose gel electrophoresis of bacteriophages and related particles. I. Avoidance of binding to the gel and recognizing of particles with packaged DNA
Authors: Serwer Philip , Hayes Shirley J.
Published: May 2005

Two potentialobstacles to using agarose gel electrophoresis for the fractionation of viruses and related particles are: (a) adherence of the sample to the gel during electrophoresis, and (b) the prolonged procedures necessary for determining whether or not particles stained with nucleic acid‐specific stains have their nucleic acid in a packaged state. It is demonstrated that adherence of positively charged tail fibers of bacteriophage T7 to agarose gels is the probable cause of a previously described (Serwer, P. and Pichler, M. E., J. Virol. 1978, 28, 917–928) sample concentration‐dependent spreading of bands formed by bacteriophage T7 during agarose gel electrophoresis (concentration effect). Procedures for screening preparations of agarose for nonadherence of particles are described and use of these procedures has resulted in the finding that the concentration effect is decreased or eliminated for T7 in preparations of agarose designed for low electro‐osmosis. In addition, it has been shown that after electrophoresis, the intensity with which bacteriophage T7 (and other DNA bacteriophages) stains with the nucleic acid‐specific stain, ethidium bromide, is increased by ejecting DNA from the interior of T7; this increase in staining intensity is used to recognized bands formed by particles with packaged DNA.
Electrophoretic cell separation using polyacrolein microspheres
Authors: Kempner David H. , Smolka Adam J. K. , Rembaum Alan
Published: May 2005

The electrophoretic mobility of a number of microspheres of homo‐ or copolymerized acrolein has been studied. It was found that increasing amounts of methacrylic acid increased the negative mobility of the microspheres, whereas reaction of the microspheres with 1,6‐diaminohexane decreased the negative mobility. When coated with antibody and specifically bound to cells, these microspheres were found to alter the target cells' electrophoretic mobility. This process allowed the separation of synthetic mixtures of microsphere‐labeled and unlabeled cell populations. Over 90 % of the recovered cells were found in fractions that were essentially either pure labeled or unlabeled cells.
Applicability of the log MM ‐ √D relationship to linear polyacrylamide gradient gel electrophoresis under a wide range of experimental conditions
Authors: Rothe G. M.
Published: May 2005

Recently we reported about a linear correlation between the logarithm of the size of native proteins (log mol mass or log Stokes' radius) and the square root of their migration distance (‐ √D) in linear polyacrylamide (PAA)‐gradient gels (G. M. Rothe and H. Purkhanbaba, Electrophoresis 1982, 3, 33–42). The linearity between log MM and √D is not subject to time using homogeneous buffers in electrophoresis, no matter how the constants of the corresponding regression lines, slope and intercept change as a function of time. The realiability of this correlction has been re‐examined with 0.7 mm thin gel plates and extending the time of electrophoresis under non‐denaturating conditions from 2 to 95 h. It is demonstrated that the linear log size ‐ √D‐relationship is also applicable to sodium dodecyl sulfate (SDS) linear PAA gradient gel electrophoresis.
Rapid purification of antibodies to single band purity using recycling isoelectric focusing
Authors: Binion Steven B. , Rodkey L. Scott , Egen Ned B. , Bier Milan
Published: May 2005

Recycling isoelectric focusing (RIEF) was used to isolate monoclonal antibodies from an electrophoretically heterogeneous rabbit antibody population. Milligram quantities of antibodies were processed to single band purity in 4 h. The ease of operation, extremely short run times, and high resolution of the RIEF apparatus give this system great flexibility. The demonstrated capacity of the RIEF system to process large quantities of protein suggests that the system is applicable to the largescale purification of antibodies.
Steady‐state rheoelectrolysis ‐ a method for isoelectric focusing without carrier ampholytes III. The influence of internal liquid flow
Authors: Rilbe Harry
Published: May 2005

Rheoelectrolysis of a salt of a weak acid with a weak base with controlled liquid exchange between anolyte and catholyte has been found to give pH courses in grave disagreement with theory. In addition, they were constantly drifting towards lower pH values. These results illustrate experimental difficulties in effectuating conditions compatible with the postulates of the theory: the drifting pH course was due to successive anodic destruction of the base, which gradually disappeared from the system, contrary to one postulate of the theory. The disagreement between the unfavourable experimental and the favourable theoretical pH course was traced to an unintentional internal liquid flow from anode to cathode, in contrast to another postulate of the theory. For that reason, the differential equation for steady‐state rheoelectrolysis with internal liquid flow is deduced and solved. The concentration courses of the ion constituents are found to be exponential instead of linear, which explains the development of an almost constant pH within a great portion of the apparatus and a steep pH change within the remaining portion. Theoretical pH courses for rheoelectrolysis with internal liquid flow are constructed and found to resemble the experimental curves very much. Quantitative information on the maximum tolerable internal flow is given.
Two‐dimensional electrophoresis of proteins in human retinal pigment epithelial cells: Identification of cytoskeletal proteins
Authors: Haley James E. , Flood Mary T. , Kjeldbye Hild M. , Maiello Eda M. , Bilek Mary K. , Gouras Peter
Published: May 2005

Two‐dimensional gel (2‐D) electrophoresis coupled to fluorography was used to obtain the [35S]‐methionine labeled protein profiles of cultured human retinal pigment epithelial cells. These patterns revealed about 250 polypeptides in the largely acidic isoelectric focusing 2‐D profile and approximately 200 in the mainly basic non‐equilibrium pH gradient electrophoresis 2‐D profile. The isolation of a detergent‐resistant cytoskeletal preparation revealed an enriched content of intermediate‐sized (7–10 nm) filaments as shown by electron microscopy. Two‐dimensional analysis (acidic isoelectric focusing, 2‐D) of the cytoskeletal preparation showed about 30 major proteins including non‐muscle actin, which had their counterparts in the total retinal pigment epithelium protein profile. These studies have begun to establish the subcellular local (filaments) and identity of some of the currently visualized 450 different retinal pigment epithelium proteins.
Evaluation of stained and unstained electropherograms by means of photoacoustic spectroscopy
Authors: Möller Ulrich , Köst Hans‐Peter , Schneider Siegfried , Coufal Hans
Published: May 2005

It is demonstrated that photoacoustic spectroscopy is a superior tool in the evaluation of electropherograms. The locla distribution of proteins in ultrathin layers of polyacrylamide gels used for isoelectric focusing is mapped with a resolution equal to or better than a laster densitomer. Even unstained samples of colored proteins can be mapped with excellent sensitivity and resolution due to the fact that the photoacoustic effect is caused only by the absorbed fraction of the incident light; scapttered light therefore doesse not cause a signal. in situ photoacosutic spectra of previously localzed zones allow the identification of the prosthetic groups of these proteins.
Two‐dimensional (crossed) immunoelectrophoresis template procedure: Use of preformed media
Authors: Saravis Calvin A. , Cook Richard B. , Sampson Christian E. , Polvino William J.
Published: May 2005

Two‐dimensional (crossed) immunoelectrophoresis using a two‐chamber csting cassette and a high gel strenght, low gelling temperature agarose mixture, can now be done with preformed thin antibody‐containing second dimension slab gels in a procedure that is less skill‐dependent and less injurious to heat‐labile macromolecules.
Mouse mitochondrial protein IEF 24: Identification and immunohistochemical localization of mitochondria in various tissues
Authors: Larsen Peter Mose , Fey Stephen J. , Bravo Rodrigo , Celis Julio E.
Published: May 2005

The abundance of mitochondrial protein IEF 24 in various Balb C mouse tissues was determined using two‐dimensional gel electrophoresis and silver staining. We found that this protein is an abundant component of all mouse tissues so far studied and these included adult brain, heart, intestine, kidney, liver, lung, muscle, skin and tongue. Comparatively little vimenting was observed in most tissues. Indirect immunofluorescent staining of methanol: acetone fixed cryostat sections from various mouse tissues using IEF 24 antibodies confirmed the mitochondrial localization of this protein but did not reveal a preferential distribution of this organelle. In contrast to these results, primary cultures of well‐spread fibroblasts from various tissues as well as of kidney epithelial cells showed in all cases the appearance of rows of well‐aligned mitochondrial fragments. These results, together with the apparently increased number of vimentin filaments observed in cultured mouse kidney fibroblasts, supported the notion that intermediate‐sized filaments of the vimentin type may serve as an anchorage site for the mitochondria in cultured cells. The lack of preferential distribution of the mitochondria observed in kidney fibrolasts in situ, which also exhibited vimentin filaments, may be due to their characteristic morphology. Cultured mouse kidney epithelial cells on the other hand showed only background fluorescence when reacted with the vimentin antibodies but showed abundant intermediate‐sized filaments of the keratin type. Based on the above results and considering the fact that many cell types in situ do not synthesize vimentin, it is proposed that both vimentin and keratin type intermediate filaments may serve as an anchorage site for the mitochondria. This proposal does does not necessarily contradict reports which indicate that mitochondria are also bound to microtubules, a possibility also suggested by our studies.
A rapid laboratory method for developing contact prints of stained polyacrylamide gels
Authors: Dunbar Bohn D. , Bundman Donnie S. , Dunbar Bonnie S.
Published: May 2005

A rapid laboratory method for processing quality color prints of high resolution two‐dimensional polyacrylamide gels has been developed to obtain photographs for data storage or for the presentation of results in poster formats. This method involves the contact printing of a gel directly onto the film so that the final print will be an exact duplication of the size and color of the gel. A variety of papers or films can be used to produce color prints or transparencies. This method has been adapted for use with a color or with a black and white photographic enlarger.
Analysis of two‐dimensional electrophoretic protein patterns using a video camera and a computer. I. The resolution power of the video camera
Authors: Schneider Werner , Klose Joachim
Published: May 2005

The resolution power of a video camera (Hamamatsu) was investigated under the aspect of employing the camera for the densitometric evaluation of two‐dimensional electrophoretic patterns of proteins. The features of the camera were tested on the illuminated glass pane of a light box and on specimens such as a blank film (X‐ray film), a blank gel (polyacrylamide gel plate) and a bar raster. The digitized values were analysed by a computer. While the hardware permits a half‐tone resolution from 0 (dark) to 255 (bright) grey levels, the values obtained experimentally showed that only 90 (glass pane), 70 (gel) or 60 (film) reproducible grey levels can be resolved, depending on the features of the supporting material of the two‐dimensional electrophoresis pattern. The addressable spatial resolution of the camera comprises 1024 × 1024 pixels. Under experimental conditions, however, only 570 × 700 pixels were resolved. Further investigation in necessary to prove whether the resolution power of a video camera is sufficient for a precise analysis of two‐dimensional protein patterns.