bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of impact of physico‐chemical drug properties on monitoring drug levels by micellar electrokinetic capillary chromatography with direct serum injection
Authors: Schmutz André , Thormann Wolfgang
Published: May 2005

The impact of physico‐chemical properties of 25 compounds, including antiepileptic, anti‐inflammatory and beta‐blocking drugs, on their determination by micellar electrokinetic capillary chromatography (MECC) with direct serum injection (DSI) is discussed. Having a pH 9.2 buffer containing 75 mM sodium dodecyl sulfate (SDS), elution is dependent on hydrophobicity, the order of emergence being basically according to increasing octanol/water partition coefficients (logP values). Peak shape is determined by the dissociation behavior (expressed by pKa) and plasma protein binding (PPB). Sharp peaks are produced by compounds having low PPB and, independently of PPB, by drugs with pKa values which are similar to the buffer pH. Broad or double peaks are established by drugs of low pKa values and significant (> about 40%) PPB. In order to evaluate the effective amount of a protein‐bound drug measured by MECC‐DSI, serum levels of drugs with different PPB, namely ethosuximide (no PPB), phenobarbital (PPB of about 50%) and naproxen (PPB > 99%) have been determined by both MECC‐DSI and MECC with extract injection (MECC‐EXI). In each case, with more than 40 sera, there is good agreement between the two sets of data. Thus, employing MECC‐DSI, total amounts of drugs are determined, i.e. a complete release of the drugs from the proteins is effected by the impact of dodecyl sulfate on the sampled proteins.
Determination of benzodiazepines by micellar electrokinetic chromatography
Authors: Bechet Isabelle , Fillet Marianne , Hubert Philippe , Crommen Jacques
Published: May 2005

A method for the separation and determination of benzodiazepines by micellar electrokinetic chromatography (MEKC) has been developed. Separation buffers consisted of aqueous solutions of glycine and triethanolamine (pH 9.0), containing sodium dodecyl sulfate (SDS) as surfactant and methanol as organic modifier. The effect of the concentration of SDS, methanol, glycine and triethanolamine on migration times and resolution was studied. Ten benzodiazepines were baseline separated at a 25 mM SDS concentration and 20% v/v methanol in a 75mM glycine‐250 mM triethanolamine buffer. Under these conditions, the within‐day reproducibilities were 0.3–0.5% for migration times and 1.7–1.9% for peak areas at a concentration of 10 μg/mL. The limits of detection and quantification for oxazepam were 0.2 and 0.7 μg/mL, respectively, using an injection time of 5s.
Analysis of lower aliphatic aldehydes in water by micellar electrokinetic chromatography with derivatization to 2,4‐dinitrophenylhydrazones
Authors: Takeda Sahori , Wakida Shin‐ichi , Yamane Masataka , Higashi Kunishige
Published: May 2005

The analysis of lower aliphatic aldehydes in water following derivatization to 2,4‐dinitrophenylhydrazones, was investigated by micellar electrokinetic chromatography (MEKC). The effects of the buffer pH and acetonitrile addition on the separation were investigated. Under optimized conditions the 2,4‐dinitrophenylhydrazine (DNPH) reagent was completely separated from four DNPH derivatized aldehydes with high separation efficiency. DNPH‐formaldehyde and DNPH‐acetaldehyde were also completely separated from one another. Their relative standard deviation of migration times and peak areas were within 1.0% and 3.3%, respectively. The reproducibility of peak areas in this MEKC analysis was as good as that of high‐performance liquid chromatography (HPLC) analysis with the same derivatization method. The recovery values from tap and river water were in the range of 97–102%. The detection limits of formaldehyde and acetaldehyde were 0.05 mg/L and 0.08 mg/L respectively.
Enantiomeric separation of drugs by affinity electrokinetic chromatography using dextran sulfate
Authors: Nishi Hiroyuki , Nakamura Kouji , Nakai Hideo , Sato Tadashi , Terabe Shigeru
Published: May 2005

Dextran sulfate (sodium salt), which is a mixture of a linear α‐(1,6)‐linked D‐glucose polymer having a sulfate group in the molecule, has been employed as a chiral selector in affinity electrokinetic chromatography (EKC) for the separation of enantiomers of drugs. Enantiomers of trimetoquinol hydrochloride and its positional isomer have been successfully separated by the method using a 3–5% dextran sulfate buffer solution of pH 5.5 within 20 min. The effects of buffer pH, concentration of dextran sulfate, and additives (such as an organic solvent, urea and a surfactant) on the enantioselectivity were investigated. The choice of pH and the concentration of dextran sulfate has been important for the improvement in selectivity.
Altered electrophoretic behavior of DNA due to short‐time UV‐B irradiation
Authors: Mäueler Winfried , Kyas Andreas , Bröcker Frank , Epplen Jörg T.
Published: May 2005

UV‐B irradiation is often inevitable for visualization of DNA fragments after ethidium bromide staining. Three different simple‐repeat‐containing, double‐stranded genomic DNA fragments were analyzed for UV‐B (312 nm) damage using different gel electrophoretic systems. The effects of UV‐B light were obvious after 5 min (31.5 kJ/m2) of irradiation in native polyacrylamide gel electrophoresis (PAGE). Standard single‐strand conformation analyses revealed no alterations while a modification did. Sodium dodecyl sulfate (SDS)‐PAGE was found to be highly sensitive with regard to the detection of damages and their time/dosage dependency. In addition, SDS‐PAGE analysis pointed to different events occurring during UV‐B irradiation. Alterations in DNA conformation were detected in every single strand analyzed after 1 min (6.3 kJ/m2) of UV‐B exposure. Gel retardation analyses revealed significant changes of protein binding to target DNAs after 2 min of irradiation – possibly stemming from structural modifications and/or originating from binding sites for proteins involved in DNA repair.
Capillary electrophoresis separation of oligosaccharides: I. Effect of operational variables
Authors: Guttman András , Cooke Nelson , Starr Christopher M.
Published: May 2005

The influence of operating variables on the separation of labeled oligosaccharide molecules in capillary electrophoresis with polymer networks is presented. In this study, an equation was derived that relates the effects of electrophoresis variables such as field strength, temperature, molecular weight and gel concentration to the migration velocity. As a model system, 8‐aminonaphthalene‐1,3,6‐trisulfonate ANTS‐labeled wheat starch digest was examined to show the validity of the equation. As an illustration of the high resolving power achievable with capillary electrophoresis, oligosaccharide molecules up to the degree of polymerization of 25 were separated with the very high efficiency of N>106 and efficiency generation rate of 3500 plates/m/s for maltose. Separation based on the size of the oligosaccharides was not obtained with either entangled or unentangled polymer solutions.
Isoelectrc focusing in immobilized pH gradient of melon (Cucumis melo L.) seed protein: Methodical and genetic aspects, and application in breeding
Authors: van den Berg Bartel M. , Gabillard Daniel
Published: May 2005

Genetic variability of melon seed proteins was studied by separation of seed protein by isoelectric focusing in immobilized pH gradient (IEF‐IPG) under denaturing conditions. A routine procedure was developed for IEF‐IPG of hundreds of individual melon seeds per day. A group of 74 accessions from 19 morphologically distinct groups and from different geographic origin were studied by IEF‐IPG using pH gradients of 4–10, 4–7 and 6–10. The electrophoretic analysis of the 74 accessions showed 270 reproducible seed protein bands of which 70 were variable. Genetic evaluation led to the conclusion that at least 20 loci govern the variation found. The phylogenetic trees constructed using the protein data on one hand and the morphological data on the other hand were compared and their use was evaluated. A number of commercial Cantaloup F1 hybrid descendants derived from the F1 hybrids by diplohaploidization or single plot descent were studied by IEF‐IPG using pH gradients of 4–7 and 6–10. Among the F1 hybrids and their descendants 265 reproducible protein bands could be identified of which 72 were variable as to presence versus absence. The genetic interpretation of the protein pattern as found by IEF‐IPG and the use of IEF‐IPG in plant breeding was discussed. It was concluded that IEP‐IPG of melon seed proteins is a valuable tool in breeding.
Response of bacteria and fungi to high‐pressure stress as investigated by two‐dimensional polyacrylamide gel electrophoresis
Authors: Groß Michael , Kosmowsky Ilona J. , Lorenz Rainer , Molitoris Hans Peter , Jaenicke Rainer
Published: May 2005

In an attempt to generalize previous observations (Jaenicke et al., Appl. Environ. Microbiol. 1988, 54, 2375–2380) and to find a convenient model system for studies of the pressure response, we tested the suitability of Escherichia coli and Thermotoga maritima (bacteria), and of five different eukaryotic species including the filamentous fungi Asteromyces cruciatus and Dendryphiella salina., and the marine yeasts Debaryomyces hansenii, Rhodosporidium sphaerocarpum, and Rhodotorula rubra. Using two‐dimensional polyacrylamide gel electrophoresis, detailed investigations on the pressure response were carried out with E. coli and Rhodosporidium sphaerocarpum. In the former organism, major pressure response proteins could not be detected, although there are significant differences in expression of some proteins as well as some minor components that are found in all of the high pressure cell extracts but not in extracts from cultures grown at atmospheric pressure. In Rhodosporidium sphaerocarpum, no change in protein expression patterns was observed between 0.1 and 20 MPa. However, approaching the limit of viability of 50 MPa, additional protein spots became detectable at 45 MPa. This finding correlates with the observation of abnormal growth forms of the organism at this pressure (Lorenz, R. et al. manuscript in preparation).
Protein studies of human non‐Hodgkin's B‐lymphoma: Appraisal by two‐dimensional gel electrophoresis
Authors: Mohammad Ramzi M. , Maki Auday , Vistisen Kerry , Al‐Katib Ayad
Published: May 2005

We have utilized two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) coupled with silver stain to identify cellular proteins in human non‐Hodgkin's B‐lymphoma (NHL). Five cell lines (SKDHL2B, WSU‐DLCL2, WSU‐NHL, WSU‐FSCCL and SKLN1), representing four different NHL maturational stages and a normal Epstein‐Barr virus (EBV)‐transformed line of B‐cell origin (SKLN1) were studied. The NHL lines were immunophenotyped using flow cytometry with lineage associated monoclonal antibodies. Whole cell lysates of the cell lines were subjected to 2‐D PAGE analyses. The gels were analyzed with an image scanning computer and the qualitative differences of protein patterns were studied. Results revealed great similarities in patterns of the NHL lines. A master map containing common NHL‐protein spots was constructed. When the map of each tumor line was compared to the master map, several protein spots were associated with each NHL‐grade. Search for these proteins in the normal EBV‐transformed B‐cell line showed that only one of the proteins (S3; Mr/pI 19/5.9) was present. Proteins that were detected in malignant NHL, but not in the normal EBV‐line, could provide important information regarding the human NHL B‐lymphocyte databases. Whether or not these proteins are definite malignant markers to distinguish between different NHL maturational stages needs further exploration through electroblotting and microsequencing.
Sponge‐like electrophoresis media: Mechanically strong materials compatible with organic solvents, polymer solutions and two‐dimensional electrophoresis
Authors: Harrington Michael G. , Lee Kelvin H. , Bailey James E. , Hood Leroy E.
Published: May 2005

A new range of sponge‐like media, called “electrophoresis sponges”, is presented. They differ from electrophoresis gels primarily in that they are mechanically stronger, providing a permanent structure of directly measurable pore size dimensions. The new media are similar to capillary electrophoresis in terms of pore size range, they are mechanically strong with directly definable walls, and are compatible with polymer solutions. The sponges differ from capillary electrophoresis in that they provide large numbers of channels, with a corresponding high load capacity for simultaneous runs in multiple channels and they are compatible directly with multi‐dimensional separations, such as high resolution two‐dimensional electrophoresis. Furthermore, they can be molded (or cut) to any shape and retain that shape, they can be handled more easily than gels, they can be reused if necessary, they can be distributed in the same format between labs easily, and they can be stored indefinitely. Chemically, they can be hydrophilic or hydrophobic, with capability ranging from inert to reactive surfaces. Pore sizes can range from the sub‐nanometer to 100 micron scale. Results with various hydrophobic sponges are reported for the carrier ampholyte‐based isoelectric focusing of proteins. Broad and narrow pH gradients are established in the sponges that are more linear than those achieved with polyacrylamide gels. One‐ and two‐dimensional electrophoresis of proteins has been achieved, for example with high resolution of the charge isomers of the haptoglobin beta chain, using sponge‐based isoelectric focusing. Isoelectric focusing is about threefold faster in the tested sponges than in equivalent polyacrylamide gels. This improved speed is probably related to the larger sponge pores. Moreover, both the quantity of sample entry of the hydrophobic protein zein and its resolution after isoelectric focusing in the electrophoresis sponges (in the presence of organic solvent) was superior to that achieved in polyacrylamide gels. Experimentation and the application of these electrophoresis sponges is still preliminary, but the sponges appear to have potential as alternatives to the existing media used for electrophoresis.
Capillary electrophoresis combined with 252Cf plasma desorption and electrospray mass spectrometry for the structural characterization of hydrophobic polypeptides using organic solvents
Authors: Weinmann Wolfgang , Parker Carol E. , Baumeister Kerstin , Maier Claudia , Tomer Kenneth B. , Przybylski Michael
Published: May 2005

Capillary electrophoresis (CE) conditions have been developed for the separation of hydrophobic polypeptides, such as fatty acid‐acylated peptides, and their subsequent structural identification by 252Cf plasma desorption (PDMS) and electrospray mass spectrometry (ESMS). Salt‐ and detergent‐free aqueous acetic acid buffers containing up to 20% 2‐propanol or 25% acetonitrile were employed for CE separations of hydrophobic peptides with (i) untreated, and (ii) 3‐aminopropyltrimethoxysilane‐derivatized fused silica capillaries. For both capillary types, electroosmotic flow rates suitable for sample isolation and transfer were determined, and CE separations of polypeptide mixtures were compared for aqueous buffers containing 2‐propanol or acetonitrile. For the mass spectrometric identification of CE‐separated peptides, a sheath flow sample isolation method was developed for subsequent transfer to PDMS. This procedure enabled the efficient isolation of peptide fractions for PDMS analysis, or alternative microanalytical techniques.

Presented in part at the International Electrophoresis Forum, Munich, October 26–28, 1992.
Native electrophoresis to monitor chemical modifications of human interleukin‐3
Authors: Smit Victor
Published: May 2005

Native electrophoresis on the Pharmacia PhastSystem using a new buffer system is used to monitor chemical modifications of human interleukin‐3. The method is faster, more gentle and more convenient than amino acid analysis. Micrograms of protein suffice in contrast with milligrams required in spectro‐photometric methods. As compared to generally used native electrophoresis, the combination of a changed conductivity, lower pH and the low density matrix results in a much better separation and detection of both the reaction products containing a high charge and the reaction products containing a low charge. This enables monitoring of extensive modifications as well as mild modifications. Our method also provides indications for denaturation of the protein. It has proven to be highly reproducible for at least 10 different modification reactions. Information can be obtained about the presence of the various distinct forms of the modified molecule, for instance leading to the average number of groups on the molecule. It also detects modifications not detectable with generally used native electrophoresis.
Glucose, fructose, mannose and/or glucose‐1‐phosphatereleasing activity stains for glycosidases and glycosyltransferases in gels after isoelectric focusing
Authors: Mukasa Hidehiko , Tsumori Hideaki , Uezono Yoshiko
Published: May 2005

β‐Fructofuranosidase, α‐glucosidase, β‐glucosidase, α‐mannosidase, β‐mannosidase, sucrose phosphorylase, glucosyltransferase and fructosyltransferase were separated by isoelectric focusing and sensitively detected to be slightly diffuse and insoluble spots in thin‐layer gels, supported by a glass plate, by release of monosugars or a sugar phosphate, followed by conversion to glucose‐6‐phosphate (G6P) and then by reduction of NADP+ to NADPH, terminated by the formation of reduced Nitroblue Tetrazolium (NBT). Approximately 1‐10 mU of enzyme was focused and the gel, after washing with a buffer, was partially dried and directly stained by uniformly spreading on the gel surface a staining medium containing sucrose or nitrophenyl glycosides as substrates, intermediary enzymes such as hexokinase, mutase and/or isomerase, NADP+, ATP, Mg+, phenazine methosulfate (PMS) and NBT. Specific staining procedures for each of these activities, on sucrose or on the glycosides as substrates, and staining procedures for multiple activities are described, with the conditions necessary for optimal development.
Analysis of cellular phosphoproteins by two‐dimensional gel electrophoresis: Applications for cell signaling in normal and cancer cells
Authors: Guy Graeme R. , Philip Robin , Tan Y. H.
Published: May 2005

Two‐dimensional (2‐D) gel electrophoresis has been used to map proteins from various cell types in an effort to eventually link such, maps to the sequencing of the entire human genome. While this analysis indicates the cellular disposition and expression of proteins, another application of 2‐D gels, the analysis of phosphoproteins, can provide much information as to the assembly and “wiring” of the signal transduction circuits within cells which appear to be enervated by phosphate exchange. The preparation and separation of 32P‐labeled proteins is described, as well as various analytical methods, including: the variety of gel systems available for specialist types of analyses, comparing 33P‐ and 32P‐labeling of proteins, imaging techniques, phosphoamino analysis, phosphopeptide separation, identifying the amino acid groups that are phosphorylated, and the identification of phosphoproteins on 2‐D gels by immunoprecipitation, corunning of purified proteins, comparative mapping and micro‐sequencing, and by Western blotting. Examples (in brackets) are given of applications in which 2‐D phosphogels can be applied, which offer advantages over other techniques. These include: (i) identifying in vivo substrates for kinases (protein kinase C activated by phorbol myristate acetate), (ii) investigating cytokine signaling pathways (tumor necrosis factor and interleukin −1), (iii) investigating the effects of drugs on signaling pathways (okadaic acid, menadione and cyclooxygenase inhibitors), (iv) characterization of specific phosphoproteins (heat‐shock protein Hsp27 and stathmin), (v) comparing normal and transformed cells (MRC‐5 human lung fibroblasts and their SV‐40‐transformed counterparts, MRC‐5 SV1 cells), (vi) purifying phosphoproteins, (vii) investigating the relationship of protein phosphorylation to stages in the cell cycle (stathmin), (viii) investigating protein/protein interactions, (ix) mapping in vitro kinase substrates (protein kinase C, protein kinase A, and mitogen activated protein kinase activated protein kinase 2), and (x) locating and identifying cellular phosphatases (Hsp27 phosphatase). It is possible that the mapping of phosphoproteins can be linked to other 2‐D gel databases and that information derived from these can be used in the future to better understand the signaling mechanisms of normal and cancerous cells.
Electrophoretic analysis of proteins associated with tumor cell invasion
Authors: Seftor Richard E. B.
Published: May 2005

Polyacrylamide gel electrophoresis is an extremely powerful tool for separating and analyzing proteins associated with different diseases and has been invaluable in the identification and analysis of proteins associated with characteristics unique to tumor cells. This study presents data demonstrating the application of conventional sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis and substrate‐incorporated SDS‐polyacrylamide gel electrophoresis (zymography) to obtain information about the proteins and catalytically active (or activatable) proteases associated with the process of tumor cell invasion using established human melanoma and breast carcinoma cell lines. Conventional SDS‐polyacrylamide gel electrophoresis was used to show that cells sequentially selected from a low invasive human melanoma cell line on the basis of their ability to invade in vitro have an increase and/or addition of six unique proteins on their cell surface. In a different application of SDS‐polyacrylamide gel electrophoresis, zymography was used to demonstrate that there is an increase in the levels of gelatinase A in the conditioned medium from three differently invasive human melanoma cell lines coincident with their ability to invade in vitro. Furthermore, the conditioned medium from the most invasive melanoma cell line demonstrated the greatest amount of gelatinase B activity. While the conditioned medium from three human breast carci noma cell lines contained low levels of both gelatinase A and B, one breast cell line also contained activity associated with stromelysin(s) not seen in the melanoma cell lines. The most invasive melanoma and breast cell lines contained appreciable levels of urokinase, while the less invasive melanoma cell lines contained low levels of tissue‐type plasminogen activator and the less invasive breast cell lines contained little‐to‐no plasminogen activator(s) activity. These observations can contribute to our understanding of the invasive process and the electrophoretic techniques used to monitor, test and analyze therapeutic approaches for metastatic diseases.
Two‐dimensional gel mapping of small GTPases reveals transformation‐specific changes during oncogenesis
Authors: Huber Lukas A. , Beug Hartmut , Simons Kai , Reichmann Ernst
Published: May 2005

Epithelial cells transformed by oncogenes in vitro change their gene expression program, thereby losing features of cell polarity and cell adhesion. Using ras‐transformed mammary epithelial cells, we have investigated the expression of other small GTP‐binding proteins by high‐resolution two‐dimensional gel electrophoresis and direct GTP ligand binding after renaturing transfer onto nitrocellulose, Ras‐transformed cells lost the expression of one epithelial‐specific GTP‐binding protein (21–22 kDa, pI 4.5–4.8) and instead expressed a fibroblast GTP‐binding protein (21–22 kDa, pI 4.8–5.0).
Genomic fingerprinting of Yersinia enterocolitica species by degenerate oligonucleotide‐primed polymerase chain reaction
Authors: Sayada Chalom , Picard Bertrand , Elion Jacques , Krishnamoorthy Rajagopal
Published: May 2005

A novel version of the polymerase chain reaction (PCR) termed degenerate oligonucleotide‐primed PCR (DOP‐PCR) was used to fingerprint the bacterial genome of Yersinia enterocolitica species. Eight well‐characterized reference strains of Y. enterocolitica (ribotype, serotype, biotype and zymotype) were examined. Optimal experimental conditions for reproducibility, sensitivity and specificity were obtained using a single DOP‐primer. All the strains gave distinct DOP‐PCR profiles composed of 12 to 19 fragments with sizes from 200 to 1.500 bp. Thus, the DOP‐PCR, which has so far been used to fingerprint human, mouse, fruitfly and plant DNA, is also well‐suited to bacterial DNA.
Double‐ and single‐strand conformation polymorphism analysis of point mutations and short tandem repeats
Authors: Barros Francisco , Muñoz‐Barús Ignacio , Lareu Maria V. , Rodriguez‐Calvo Maria S. , Carracedo Angel
Published: May 2005

Single point mutations in small DNA fragments and single unit differences in simple repetitive DNA can be detected as double‐strand conformation polymorphisms using polyacrylamide gel electrophoresis, with and without sodium dodecyl sulfate, even at temperatures as low as 3°C. Changes in a single base are distinguished by means of the analysis of the heteroduplexes, and changes in more than two bases can be distinguished in both homoduplexes and heteroduplexes. Polymerase chain reaction (PCR) conditions can be designed not only to amplify homoduplexes, heteroduplexes and singlestrand DNA at the same time, but also to focus the analysis on either singlestrand conformation polymorphism (SSCP) or double‐strand conformation polymorphism (DSCP). DSCP seems to be advantageous in typing DNA polymorphisms or mutations in loci with few variants, but, because it is necessary to have a simple pattern of all possible combinations of the alleles, it is not as advantageous in typing systems with many variants.
Electroosmosis of polymer solutions in fused silica capillaries
Authors: Bello Michael S. , De Besi Patrizia , Rezzonico Roberta , Righetti Pier Giorgio , Casiraghi Ernestina
Published: May 2005

The classical von Smoluchowski equation predicts that the electroosmotic mobility generated by the wall zeta potential could be suppressed if the viscosity of the solution adjacent to the wall were extremely high. When performing runs in capillaries filled with polymer solutions (2% methyl cellulose solutions with viscosities of 25 cP), however, one consistently finds that the quenching of electroosmotic mobility is substantially less than predicted by the von Smoluchowski relationship. The electroosmotic flow is progressively suppressed with subsequent electrophoretic runs, suggesting a “dynamic coating” of the polymers onto the capillary wall. This progressive reduction of electroosmotic mobility tends to a plateau value which is still substantially higher than the value derived on the basis of the von Smoluchowski relationship. The following explanation is proposed: due to the very high shear rate in the electric double layer, the polymer molecules change their orientation and/or conformation, which lowers the fluid viscosity in this region. A scaling equation for electroosmotic mobility taking into account the non‐Newtonian properties of polymer solutions is derived. It predicts electric field dependence of the electroosmotic mobility as the shear rate in the double layer is proportional to the electric field. Experimental measurements confirm the dependence of the electroosmotic mobility on the electric field.
Characterization of the acute phase serum protein response in pigs
Authors: Lampreave Fermín , González‐Ramón Nieves , Martínez‐Ayensa Silvio , Hernández Miguel‐Angel , Lorenzo Hans‐Kristian , García‐Gil Agustín , Piñeiro Andrés
Published: May 2005

Acute inflammation was induced in pigs using a single subcutaneous turpentine injection. The acute phase serum protein response was analyzed using crossed immunoelectrophoresis and immunodiffusion. The concentration of C reactive protein and haptoglobin increases 5–7 times 48 h after the injection, whereas the concentration of an α2‐globulin, named pig major acute phase protein (pig‐MAP), increases at least 15‐fold. A molecular mass of 115 kDa for pig‐MAP was determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. This protein did not crossreact with antisera to human hemopexin, ceruloplasmin, H‐kininogen and complement factor C3. Albumin and α‐lipoprotein were negative acute phase proteins because their concentration significantly decreased during inflammation. Finally, the concentration of α1‐acid glycoprotein, fetuin, α1‐protease inhibitor, transferrin and α2‐macro‐globulins, as well as total proteins, did not change significantly during inflammation.
An informativity index for multilocus DNA fingerprints
Authors: Krawczak Michael , Lubjuhn Thomas
Published: May 2005

An informativity index for multilocus DNA fingerprints, r, was developed. It is based upon the Shannon information that a paternal DNA fingerprint conveys about the offspring phenotype pattern. Both simulation and empirical data reveal that the index r is strongly correlated with the mean log likelihood ratio (paternity vs. nonpaternity) expected in trio cases of true paternity. Since r can be estimated from DNA fingerprints of unrelated individuals in advance, it will provide an easy means to assess the potential utility of a given probe/enzyme combination in kinship testing.
Detection of pectinesterase in polyacrylamide gels
Authors: Alonso Jesús , Rodríguez M. Teresa , Canet Wenceslao
Published: May 2005

A rapid and sensitive method of detecting pectinesterase activity following electrophoresis or isoelectric focusing in polyacrylamide gels is described. The method uses ruthenium red and requires no addition of substrate when making the gels, thus obviating direct enzyme‐substrate contact during electrophoresis. Because of its versatility, the method can be used in a wide variety of applications, such as plant and microbial taxonomy, enzyme purification and characterization, or as an analytical method in fresh and processed plant technology.
Analysis of recombinant human tumor necrosis factor beta by capillary electrophoresis
Authors: Yao Yi Ju , Loh Kean Chong , Chung Maxey C. M. , Li Sam F. Y.
Published: May 2005

Capillary electrophoresis was used for the analysis of recombinant human tumor necrosis factor beta (rhTNF‐β). Reproducible separation of the protein could be achieved in Polybrene‐coated capillaries. Optimum conditions for separation were achieved with a pH 4.5 sodium acetate buffer. Besides Polybrene‐coated capillaries, other commercially available column and column‐coating reagents were also examined for the separation of rhTNF‐β. Capillary electrophoresis was found to be a potentially useful method for product analysis and process monitoring in recombinant‐DNA technology.
Trapping gel electrophoresis of end‐labeled DNA: An analytical model for mobility and diffusion
Authors: Slater Gary W. , Desruisseaux Claude , Villeneuve Christine , Guo Hong L. , Drouin Guy
Published: May 2005

As shown by Ulanovsky, Drouin and Gilbert (Nature 1990, 343, 190–192), the gel electrophoretic migration of DNA is severely reduced by steric trapping when streptavidin is attached to one end of the polyelectrolyte. We present a model that allows us to calculate both the mobility and the diffusion coefficient, hence the resolution factor of the resulting separation. We compare our results to those of Défontaines and Viovy (Electrophoresis 1993, 14, 8–17) and we show that the averages over the molecular conformations must be done carefully. We also show that trapping increases diffusion substantially and that this makes constant‐field trapping electrophoresis incapable of increasing the number of bases read per sequencing run. Finally, we conclude that severe trapping may lead to highly anomalous transport behavior where one cannot define a velocity or a diffusion constant.
Detection of two hypervariable (ATTTT)n loci in the human genome
Authors: Dürr Christine , Hinney Anke , Luckenbach Christine , Ritter Horst
Published: May 2005

Object of this investigation was the isolation of a single‐locus probe from a multi‐locus fingerprint. Individual specific multi‐locus fingerprints in man were generated by using the oligonucleotide probe (ATTTT)5. An isolated (ATTTT)5‐positive DNA fragment was analyzed using polymerase chain reaction (PCR) cycle‐sequencing and nonradioactive direct‐blotting electrophoresis. A digoxigenated oligonucleotide synthesized according to this sequence was used as a single‐locus probe. Two hypervariable loci were detected on Southern blots. Formal genetic investigations for the two loci were performed in order to estimate the allele frequencies. Locus 1 shows an individual‐specific banding pattern with an autosomal‐codominant inheritance and can be used for forensic investigations. Locus 2 also represents a polymorphic pattern, but the inheritance is not according to the Mendelian rules. Probably we have detected a highly mutagenic locus in the human genome.
Temperature gradient gel electrophoresis for analysis of clonal evolution in non‐Hodgkin's lymphoma of the thyroid
Authors: Tiemann Markus , Menke Martin A. O. H. , Asbeck Robert , Wacker Hans‐Heinrich , Parwaresch Reza
Published: May 2005

To analyze the transition of an autoimmune disease into a mucosa‐associated lymphoid tissue‐non‐Hodgkin's lymphoma (MALT‐NHL), we investigated a total of 27 cases of clinically diagnosed autoimmune thyroiditis with lymphoid hyperplasia. Three cases of thyroid hyperplasia served as controls. Monoclonal B cells were detected by studying rearrangement patterns of the hypervariable CDR III regions within the immunoglobulin heavy chain gene locus and the T‐cell receptor γ chain gene (TCRG). We used a seminested polymerase chain reaction (PCR) to demonstrate immunoglobulin rearrangements and a multiplex PCR for TCRG rearrangements. The PCR products were analyzed by temperature gradient gel electrophoresis to expand mixtures of homo‐ and hetero‐duplices within heterogeneous populations of B cells. With this approach we found monoclonality in 14 of the 27 cases of Hashimoto's disease. In a reinvestigation we discovered additional histological and immunohistochemical features of MALT‐NHL in 17 cases. The 14 cases of thyroiditis with clonally expanded B cells clearly demonstrate the transition from autoimmune disease to non‐Hodgkin's lymphoma.
Quantification of oxidative phosphorylation enzymes after blue native electrophoresis and two‐dimensional resolution: Normal complex I protein amounts in Parkinson's disease conflict with reduced catalytic activities
Authors: Schägger Hermann
Published: May 2005

Blue native polyacrylamide gel electrophoresis (BN‐PAGE), a method for the isolation of native membrane proteins from biological membranes, was adapted to the isolation of oxidative phosphorylation (OXPHOS) enzymes from milligram amounts of human tissues. Combined with Tricine‐sodium dodecyl sulfate (SDS)‐PAGE in the second dimension, the protein subunits of OXPHOS complexes could be analyzed and quantified. The characteristics of the technique are described and protocols for processing different tissues are provided. The technique was applied for the analysis of defects of OXPHOS complexes in Parkinson's disease. A significant reduction of complex V was observed in one case. Absolutely normal complex I protein amounts were in contrast to reduced catalytic activities of complex I in Parkinson's disease. This discrepancy can be explained by binding of endogenous complex I inhibitors or by alterations of a protein subunit not affecting the assemblage of the complex but modifying the enzymatic properties.
Cyclodextrin modified micellar electrokinetic chromatography for the chiral direct resolution of (+), (−)‐trans‐1,2‐dihydrodiol metabolite of chrysene in vitro activated by rat liver microsome S9 fraction
Authors: Desiderio Claudia , Fanali Salvatore , Sinibaldi Massimo , Polcaro Chiara
Published: May 2005

A γ‐cyclodextrin (γ‐CD) modified electrokinetic micellar capillary chromatography (MEKC) method was used for the enantiomer separation of a racemic trans‐1,2‐dihydro‐1,2‐dihydroxy‐chrysene (chry‐trans‐1,2‐diOH) mixture. The chiral resolution was strongly influenced by several important parameters: surfactant concentration and addition of organic modifier to the background electrolyte (BGE). An optimized electrophoretic system was used, consisting of the following conditions: 25 mM phosphate buffer, pH 7.8, 50 mM sodium dodecyl sulfate, 20 mM γ‐CD, 7.4% V/V 2‐propanol as BGE; the applied voltage, 18 kV, corresponded to 37 μA at a constant temperature of 25°C. This electrophoretic method was applied for monitoring the chry‐trans‐1,2‐diOH enantiomer formation in a real sample, obtained from in vitro metabolic activation of chrysene by phenobarbital‐ß‐naphthoflavone‐induced rat microsomes. The (+) and (‐) enantiomers were identified by the racemate and the single enantiomer standard addition method and by spectra comparison with the synthetic compound. Under the experimental conditions used for chrysene activation, the (+) optical isomer was the prevailing form. The CD‐MEKC system showed high reproducibility and selectivity, allowing a fast and interference‐free analysis even of the in vitro metabolic sample extract, without any pretreatment.
Separation of jumper ant (Myrmecia pilosula) venom allergens: A novel group of highly basic proteins
Authors: Donovan Gregory R. , Street Michael D. , Baldo Brian A.
Published: May 2005

The sting of the jumper ant (Myrmecia pilosula) causes severe allergic reactions, including anaphylaxis in sensitized individuals. Two of the major allergens, Myr p I and Myr p II, have been cloned, immunocharacterized and nucleotide‐sequenced and they encode 112 and 75 residue polypeptides, respectively. Both allergens are highly basic proteins having isoelectric point values greater than 10. However, electrophoretic analysis has generated conflicting results as to the actual sizes of the allergens in the native venom. Electrophoretic, immunological and N‐terminal analyses suggested that these allergens undergo extensive post‐translational processing to final forms of 45 and 27 residues, respectively. The results highlight the difficulties in the study of small, basic proteins and polypeptides by electrophoretic techniques.
Expression of the human urokinase‐type plasminogen activator receptor in E. coli and Chinese hamster ovary cells: Purification of the recombinant proteins and generation of polyclonal antibodies in chicken
Authors: Magdolen Viktor , Rettenberger Peter , Lopens Antje , Oi Hidekazu , Lottspeich Friedrich , Kellermann Josef , Creutzburg Sabine , Goretzki Lothar , Weidle Ulrich H. , Wilhelm Olaf , Schmitt Manfred , Graeff Henner
Published: May 2005

The receptor for urokinase‐type plasminogen activator (uPAR) may contribute to the invasive and metastatic capacity of tumor cells by focusing the serine protease urokinase‐type plasminogen activator (uPA) to the cell surface. uPA activates plasminogen to plasmin which in turn degrades extracellular matrix proteins or activates other proteases. Mature uPAR is a heavily glycosylated protein of about 284 amino acids attached to the plasma membrane via a glycosyl‐phosphatidylinositol (GPI) anchor. A set of different polyclonal uPAR antibodies has been generated in order to investigate the role of uPAR in tumor spreading in more detail. For this purpose, uPAR (lacking the GPI anchor) was expressed in E. coli and Chinese hamster ovary (CHO) cells. Recombinant uPAR from E. coli (corresponding to amino acids 1–284 of human uPAR) was expressed with an N‐terminal histidine‐tag insertion and purified by nickel chelate affinity chromatography. Soluble uPAR, synthesized by CHO cells (corresponding to amino acids 1–277 of human uPAR), was isolated by ligand (uPA) affinity chromatography. Expression in E. coli led to a nonglycosylated form of uPAR, whereas uPAR produced by CHO cells seemed to be glycosylated to a similar extent as the naturally occurring human form of uPAR (as analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis). Prior to immunization the N‐termini of the recombinant uPAR variants were determined by amino acid sequence analysis. Polyclonal antibodies were generated in chickens and purified from egg yolk. The reaction patterns of these antibodies were analyzed by Western blot analyses and flow cytofluorometry.
Differentiation of scombroid fish species (tunas, bonitos and mackerels) by isoelectric focusing, titration curve analysis and native polyacrylamide gel electrophoresis of sarcoplasmic proteins
Authors: Rehbein Hartmut
Published: May 2005

Differentiation of scombroid fishes was possible by electrophoresis of sarcoplasmic proteins using either isoelectric focusing (IEF), titration curve analysis, or native polyacrylamide gel electrophoresis (PAGE). By IEF with Phast‐Gels 3–9 species specific patterns, characterized by a few bands in the cathodal part of the gels, were obtained. This type of gel was also used for titration curve analysis. Here, too, the closely related species Thunnus thynnus and T. albacares gave different protein patterns. Native PAGE, native cathodal electrophoreses in Clean Gel 10% with buffer pH 5.5, proved to be a fast and simple method for differentiation of scombroid fishes. As most of the prominent sarcoplasmic proteins of these species have pIs in the neutral or alkaline pH range, they are positively charged at pH 5.5 and move to the cathode.
Diffusion, Joule heating, and band broadening in capillary gel electrophoresis of DNA
Authors: Slater Gary W. , Mayer Pascal , Grossman Paul D.
Published: May 2005

We calculated the longitudinal and transverse diffusion coefficients for a DNA molecule undergoing gel electrophoresis in the limit where it is reptating through a dense polymer matrix. Our results indicate that both diffusion coefficients increase with the electric field intensity. The transverse and longitudinal diffusion coefficients are roughly equal for regular field intensities, but the former dominates for the high field intensities normally used for capillary gel electrophoresis (CE). This has important implications for the optimization of CE. Our results clearly show that the naive use of the zero‐field diffusion constant, the Einstein relation or the longitudinal diffusion constant when calculating the contribution of the parabolic temperature profile to band broadening may lead to large overestimates under typical CE conditions. Finally, we show that the field‐dependent diffusion coefficients may be responsible for the existence of an optimal field intensity for CE, even if Joule heating is neglected.
Phosphorylation of ribosomal protein L30 after herpes simplex virus type 1 infection
Authors: Simonin Denis , Diaz Jean‐Jacques , Kindbeiter Karine , Denoroy Luc , Madjar Jean‐Jacques
Published: May 2005

In addition to an irreversible stimulation of S6 ribosomal protein phosphorylation, there is a modification of a subset of ribosomal proteins by phosphorylation after herpes simplex virus type 1 (HSV‐1) infection. Moreover, in the course of this infection, three additional phosphorylated proteins can be extracted from ribosomes and separated by two‐dimensional electrophoresis (2‐DE) of total ribosomal proteins. One of them exhibits an identical molecular mass to L30, while being more acidic. This protein is phosphorylated on serine residues. The kinetics of appearance of this protein in the ribosomal fraction correlated with a decrease in L30 staining, as shown by 2‐DE. Determination of the N‐terminal amino acid sequence of this extra phosphoprotein and of L30‐derived peptides demonstrated the identity of these two proteins.
Association of protein polymorphism among pepper (Capsicum annuum L.) inbred lines with agronomic performance of their crosses
Authors: Posch Anton , van den Berg Bartel M. , Görg Angelika
Published: May 2005

The genetic variation of 10 morphologically similar pepper (Capsicum annuum L.) inbred lines has been analyzed by two‐dimensional electrophoresis with immobilized pH gradient (IPG‐DALT) of seed proteins. For all pairs of inbred lines genetic distance indices were calculated on the basis of the IPG‐DALT analysis and a genetic tree was constructed. The protein polymorphism data of the 10 inbred lines were integrated into a pepper breeding program to validate the assumption that there is a higher chance of achieving better hybrid performance when the genetic distance between the parents is as great as possible. Field trials were performed in Turkey and consisted of a total of 27 crossings based on 9 inbred lines. Fifteen hybrids exceeded the crop yield of the better parent and 8 additional hybrids exceeded the mean crop yield of both parents. The genetic distance indices of the parental inbred lines based on protein polymorphism data were not significantly correlated to heterosis performance of the experimental hybrids (r = 0.20), indicating that protein polymorphism data add little to the prediction of single cross hybrid performance. Nevertheless, protein polymorphism data support the breeder in being more effective in finding out the best inbred combinations, because most of the less promising crossings can be omitted from the expensive and time‐consuming performance tests. Those hybrids whose distance indices exceeded the mean of all 27 distance values (0.52) were superior to the mean value of all heterosis performances by 19%. Moreover, 11 out of 15 hybrids having distance indices higher than the mean of the 27 crossings exceeded the mean of all heterosis values (304 g), but only 5 hybrids out of 12 showing distance values below the mean exceeded the overall mean of heterosis performance. We conclude that IPG‐DALT can be used as an efficient tool in vegetable hybrid breeding.
From image processing to classification: I. Modeling disturbances of isoelectric focusing patterns
Authors: Jensen Kirsten , Søndergaard Ib , Skovgaard Ib Michael , Nielsen Hans Bruun
Published: May 2005

In order to optimize the conditions for evaluation of isoelectric focusing (IEF) patterns by digital image processing, the sources of error in determination of the pI values were analyzed together with the influence of a varying background. The effects of band distortions, in the spectra of the individual lanes, were examined. In order to minimize the effect of these distortions, optimal conditions for handling IEF patterns by digital image processing were elucidated. The systematic part of the global deformation on the gels was investigated and an algorithm was developed by which it was possible to correct for a part of the individual distortions. The effects of various corrections for lane distortions were illustrated by classification, using different types of discriminant analysis. Finally the background disturbances were examined, and described by a mathematical model.
Capillary and slab gel electrophoresis profiling of oligosaccharides
Authors: Guttman András , Starr Christopher
Published: May 2005

Capillary electrophoresis with laser‐induced fluorescent detection, a one‐dimensional version of the well‐established planar analytical method of polyacrylamide gel electrophoresis, has been proven to be a powerful new microanalytical method for profiling complex carbohydrates. In this paper a comparison is presented between the planar high concentration polyacrylamide gel electrophoresis method and capillary electrophoresis of different carbohydrates with respect to performance and efficiency. N‐Linked oligosaccharides were released from several glycoproteins, including fetuin, human immunodeficiency virus (HIV) envelope recombinant glycoprotein (GP‐120), α1‐acid glycoprotein and ribonuclease B, using recombinant peptide‐N‐glycosidase F (PNGase F). Both separation methods involve labeling of the released carbohydrates at the reducing end with the fluorescent dye, disodium 8‐amino‐1,3,6‐naphthalene trisulfonate (ANTS). Fluorophore labeling was followed by separation of the labeled oligosaccharides either by high concentration polyacrylamide gel electrophoresis or capillary electrophoresis.