bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulation of mts1 expression in mouse and human normal and tumor cells
Authors: Grigorian Mariam , Tulchinsky Eugene , Burrone Oscar , Tarabykina Svetlana , Georgiev Georgii , Lukanidin Eugene 
Published: May 2005

AbstractThe mts1 gene, encoding small Ca2+‐binding protein of the S100‐family, is considered as a gene whose activity correlates with the manifestation of a metastatic phenotype of tumor cells. It was shown before that the mts1 is expressed not only in metastatic tumor cells but also in some normal tissues, namely in so‐called “lymphoid” organs: spleen, thymus; bone marrow. In this work we analyzed in more detail the expression of mts1 in human and mouse hematopoietic cells and cell lines. A high level of mts1 RNA was observed in T‐lymphocytes, neutrophils, monocytes/macrophages and in corresponding cell lines. Controversially, the mts1 gene was silent in B‐lymphocytes as well as in myeloma and erythroleukemia cell lines. The possibility of modulating the mts1 gene expression by the action of different agents was demonstrated. Mitogens, such as lipopolysaccharides (LPS), interferon (IFNγ), and concanavalin A (Con A), modulate the level of the mts1 gene expression in hematopoietic cells differently. Calcium ionophore, A23187, can also be regarded as a modulator of the mts1 gene expression, since its addition to the cells results in a substantial decrease of the mts1 RNA level. It was shown that the mts1 RNA's half‐life is relatively long, more than 24 h. We therefore believe that calcium ionophore can activate some ribonucleases which degrade the mts1 RNA. Cycloheximide prevents the effect of A23187 and stabilizes the mts1 RNA, probably by blocking the synthesis of these nucleases. Thus, the obtained data indicate that the agents which are capable of changing the physiological status of the cells also modulate the mts1 gene expression.
Several small GTP‐binding proteins are strongly down‐regulated in simian virus 40 (SV40) transformed human keratinocytes and may be required for the maintenance of the normal phenotype
Authors: Gromov Pavel S. , Celis Julio E. 
Published: May 2005

AbstractHigh resolution two‐dimensional (2‐D) gel electrophoresis in combination with the blot overlay nucleotide binding assay was used to reveal low molecular weight GTP‐binding proteins expressed by primary cultured, normal human keratinocytes. Forty one small GTP‐binding proteins (30 isoelectric focusing, IEF; and 11 nonequilibrium pH gradient electrophoresis, NEPHGE) ranging in molecular weights from 18000 to 30000 and isoelectric points from 4.4 to 8.0 were detected and mapped in the master human keratinocyte database. Four GTP‐binding proteins were identified by 2‐D gel immunoblotting and these correspond to rap 1 and 2 and two forms of rab6. ras Proteins are most likely present in the [α32P]GTP 2‐D gel blots but their levels may be too low to be detected by immunoblotting. Quantitative changes in the relative expression levels of [α32P]GTP‐binding proteins in normal proliferating and simian virus 40 (SV40) transformed human keratinocytes (K 14) were determined by scintillation counting of the radioactive spots excised from the nitrocellulose blots. The results showed that thirteen of these proteins were not expressed in transformed K14 keratinocytes, implying that they may play a role in the maintenance of the normal cell phenotype.
Apoptotic cell death analyzed at the molecular level by two‐dimensional gel electrophoresis
Authors: Robaye Bernard , Døskeland Anne Paulus , Suarez‐Huerta Nathalie , Døskeland Stein Ove , Dumont Jacques E. 
Published: May 2005

AbstractThe pattern of protein expression and phosphorylation after an apoptotic stimulus has been studied in two systems. Bovine aortic endothelial cells were induced to undergo apoptotic cell death by a combination of a cytokine (tumor necrosis factor, TNF) and inhibitors of protein synthesis, like cycloheximide. Two‐dimensional (2‐DE) electrophoresis of proteins from such cells revealed specific proteolysis of distinct proteins, some at an early stage of apoptosis and some at a later stage. These proteins may have antiapoptotic properties. In rat IPC‐81 promyelocytic leukemia cells, cAMP induced apoptosis. 2‐DE of such cells pulse‐labeled with [35S]methionine revealed two “novel” protein spots (of 30 kDa and 46 kDa, respectively), induced very rapidly by a posttranscriptional mechanism. It is proposed that “dysphosphorylation” may accompany apoptosis in general, since both endothelial cells treated with TNF/cycloheximide and IPC‐81 cells treated with cAMP analog or the apoptosis‐inducing phosphatase inhibitors okadaic acid or calyculin A all showed altered protein phosphorylation patterns, as revealed by 2‐DE electrophoresis of proteins from cells prelabeled with 32Pi.
Gel electrophoretic analysis of nuclear matrix fractions isolated from different human cell lines
Authors: Kallajoki Markku , Osborn Mary 
Published: May 2005

AbstractThe nuclear matrix is operationally defined as the structure that remains after nuclei are extracted with nonionic detergent and with high salt and are digested with nucleases. Thus the nuclear matrix protein composition is critically dependent on the isolation conditions. We have compared nuclear matrices isolated from human cell lines by two different methods. First, isolated nuclei were extracted as above to obtain a matrix fraction. This method showed a substantial contamination by cytoplasmic intermediate filaments but immunization of mice resulted in antibodies recognizing nuclei and the mitotic spindle apparatus. Second, a nuclear matrix fraction was made by extracting whole cells as above and dissolving the residue in urea and dialysing against an assembly buffer to precipitate intermediate filament proteins (Fey, E. G. and Penman, S., Proc. Natl. Acad. Sci. USA 1988, 85, 121–125). Such fractions showed complex protein patterns in silver‐stained two‐dimensional gels for four cell lines: HeLa, MCF‐7, SW13 and the U333CG/343MG glioma line. While some proteins in the nuclear matrix fraction were common to all cell lines, others appeared cell‐line specific. Two‐dimensional gels and the immunoresponse in mice again showed contamination of these preparations with cytoplasmic proteins. These results clearly show the difficulties associated with protein chemical analysis of nuclear matrices: the preparations have substantial cytoplasmic contamination, the polypeptide composition is extremely complex and the yield of individual polypeptides is low. Thus, without further experiments one cannot say which proteins are true nuclear matrix components. In addition, our results underline the value of dissecting the nuclear matrix by analyzing its individual components with monoclonal antibodie, and we show examples of the nuclear staining patterns of three different classes of monoclonal antibodies isolated by this approach.
Separation and analysis of pig pancreatic zymogen granules with free flow electrophoresis and lectins
Authors: Spaans M. Carina , Tobler Markus , Ammann Rudolf W. , Freiburghaus Andreas U. 
Published: May 2005

AbstractPurified pig pancreatic zymogen granules were subjected to free flow electrophoresis (FFE) in an acetate buffer system (acetic acid/NaOH, pH 5.5) to detect the presence or absence of more than one population or zymogen granules. Pig pancreatic zymogen granules were purified by differential and density gradient centrifugation and subjected to FFE. Fractions were analyzed for protein, α‐amylase (EC 3.2.1.1) and 5′‐nucleotidase (EC 3.1.3.5) as marker enzymes for zymogen granule content and membranes, respectively. Only one distinct peak, with coincident α‐amylase and 5′‐nucleotidase activity, and most protein was detected, which reflects the presence of a single population of intact zymogen granules. This was confirmed by electron microscopy. When the granules were incubated with different lectins before FFE, the one distinct peak representing intact zymogen granules was shifted towards the cathode in the case of concanavalin A (Con A) and Ricinus communis agglutinin 120 (RCA 120). No splitting of the peak occurred. Our results do not support the hypothesis of a coexistence of more than one distinct population of zymogen granules.
Specific, sensitive and accurate quantification of albumin, retinol binding protein and transferrin in human urine and serum by zone immunoelectrophoresis assay (zia)
Authors: Vesterberg Olof 
Published: May 2005

AbstractFor zone immunoelectrophoresis assay (ZIA) glass. tubes, ID 2 mm and 90 mm high, are filled to 2/3 with buffer containing agarose and antibodies against the protein to be quantified, each sample being pipetted on top of separate agarose gel rods. On electrophoresis at 35–150 V for several hours, the sample proteins enter the gel with resultant immunoprecipitates, visualized by staining. The extension of each immunoprecipitation zone from the upper gel surface (measured with a ruler) is directly proportional to the amount of protein in each sample and can easily be quantitated by comparison with a linear calibration curve. ZIA can be used for quantification of several proteins in blood serum and plasma as well as in urine, as is illustrated for albumin, retinol‐binding protein (RBP) and transferrin. The recovery of the pure proteins added to urine is often close to 100%. ZIA has many advantages: (i) simple apparatus and procedure (no gel punching nor cooling), (ii) minimal antiserum consumption (1 mL may allow > 1000 assays), (iii) electrophoresis can be performed within a few hours or overnight, (iv) low coefficient of variation (often < 4%), (v) linear calibration curves, (vi) low detection limit (< 20 ng/mL), (vii) wide concentration ranges, (viii) no kits nor unique antisera preparation are required, and (ix) good agreement with the results from other methods.
Capillary zone electrophoresis of polymerase chain reaction‐amplified DNA fragments in polymer networks: The case of GATT microsatellites in cystic fibrosis
Authors: Gelfi Cecilia , Orsi Antonia , Righetti Pier Giorgio , Brancolini Valeria , Cremonesi Laura , Ferrari Maurizio 
Published: May 2005

AbstractIn cystic fibrosis (CF), the most common mutation, delta F508 (a three‐base‐pair deletion) accounts for ca. 70% of mutations in the worldwide population. The majority of other mutations (more than 350 reported so far to the Genetic Analysis Consortium) have been detected in single cases, thus rendering quite cumbersome a molecular diagnostic approach for the identification of CF chromosomes. As an alternative, linkage analysis based on intragenic polymorphism can be useful for prenatal diagnosis and CF‐carrier detection, provided that the heterozygosity of the allelic forms is very high. For this purpose, DNA microsatellites, consisting of two to epta nucleotide repeat clusters, displaying a high degree of polymorphism, are being increasingly used as markers in linkage studies. Two main allelic forms, one hexameric (111 bp) and one heptameric (115 bp), of a tetranucleotide (GATT) repeat polymorphism, at the junction of intron IVS6a and exon 6b, have been amplified by PCR technology. These two alleles can be separated in a 10–20%T polyacrylamide gradient gel and detected by ethidium bromide staining. As an alternate procedure, these two fragments are efficiently separated by capillary zone electrophoresis in a viscous solution of 6%T linear polyacrylamide and detected by their intrinsic absorbance at 254 nm.
Capillary electrophoresis of polymerase chain reaction‐amplified products in polymer networks: The case of Kennedy's disease
Authors: Nesi Marina , Righetti Pier Giorgio , Patrosso Maria Cristina , Ferlini Alessandra , Chiari Marcella 
Published: May 2005

AbstractIn Kennedy disease (an X‐linked motoneuronal disorder associated with an increase in the number of (CAG)n triplet repeats in the first exon of the Androgen receptor gene; about twice as many as in normal conditions), polymerase chain reaction‐amplified genic products exhibit two DNA fragments in the heterozygous female carriers, one with a range between 468 and 495 bp in the normal polymorphic population and a fragment corresponding to the pathological state that reaches 573 bp. These fragments are easily separated by gel‐slab electrophoresis and detected by intercalating dye staining (ethidium bromide). As an alternative procedure, capillary zone electrophoresis in polymer networks, consisting of 8% polyacryloylaminoethoxyethanol at 0% cross‐linker, offers a simple procedure for separation and on‐line detection via UV absorbance at 254 nm, thus avoiding additional staining steps. The capillary column can be repeatedly used for up to 80–100 times and the electropherogram is stored on a magnetic support. Easy comparison among different runs is obtained by aligning all tracings to an internal standard of a 650 bp fragment added as a marker.
Studies of serum protein complexes with nickel using crossed immunoelectrophoresis
Authors: Nielsen Jeanet Løgsted , Poulsen Otto Melchior , Abildtrup Anne 
Published: May 2005

AbstractCrossed immunoelectrophoresis of human serum spiked with nickel in the range 0.85–24 mmol Ni/L was used to study nickel‐protein complexes. These high concentrations, which are far higher than the physiological level (approximately 7.8 nmol/L), were used to saturate both high and low affinity binding sites. Addition of increasing amounts of nickel resulted in dose‐dependent changes of the electrophoretic patterns of prealbumin, α‐1‐lipoprotein, α‐1‐antitrypsin and α‐2‐macroglobulin. Radioactive 63Ni was used for crossed immunoelectrophoresis autoradiography experiments for further identification of nickel‐protein complexes. When a 63Ni pulse of 740 kBq/application was used, many human serum proteins were labeled. When using a 63Ni pulse of 185 kBq/application only albumin and α‐1‐antitrypsin were visualized clearly. The binding of large amounts of nickel to albumin, visualized by autoradiography, may reflect the high abundance of albumin in human serum as compared to other serum proteins. Addition of nickel to serum proteins resulted in liquid phase precipitation of serum proteins, and rocket immuno‐electrophoresis was used to demonstrate that IgG in particular is precipitated. This precipitation of serum proteins may disturb the elution profile when chromatographic techniques are used to analyze nickel‐protein complexes. Consequently, immunoelectrophoretic methods may also be attractive alternatives to column chromatographic techniques. The present study demonstrated that, besides the nickel‐binding of albumin and α‐2‐macroglobulin, several other serum proteins have nickel‐binding affinity.
N,N,N′,N′‐Tetramethyl‐1,3‐butanediamine as effective running electrolyte additive for efficient electrophoretic separation of basic proteins in bare fused‐silica capillaries
Authors: Corradini Danilo , Cannarsa Gianfranco 
Published: May 2005

AbstractThe effect of N,N,N′,N′‐tetramethyl‐1,3‐butanediamine (TMBD) in the running electrolyte on the electroosmotic flow and the migration behavior of four standard basic proteins in bare fused‐silica capillaries was examined at pH 4.0, 5.5, and 6.5. Depending on the electrolyte pH and additive concentration the electroosmotic flow was either cathodic or anodic. A similar Langmuirian‐type dependence of the electroosmotic flow on the concentration of TMBD in the running electrolyte was found at the three experimented pH values, which may be indicative of the specific adsorption of the additive in the immobilized region of the electric double layer at the interface between the capillary wall and the electrolyte solution. Electrophoretic separations of the four standard basic proteins performed at the three above pH values, showed well‐resolved, efficient and symmetric peaks, demonstrating the utility of this additive for protein electrophoresis in bare fused‐silica capillaries. The variations in separation efficiency, peak capacity, resolution and reproducibility of migration times as a function of the additive concentration at pH 6.5 were also examined.
Capillary electrophoretic separations of biotechnology‐derived proteins in E. coli fermentation broth
Authors: Strege Mark A. , Lagu Avinash L. 
Published: May 2005

AbstractA capillary electrophoresis (CE) method was developed for the analysis of a recombinant DNA protein in a fermentation broth matrix. A polyacrylamidecoated capillary was employed to eliminate electroosmotic flow, facilitating relatively rapid run times. Selectivity and efficiency were enhanced significantly through the incorporation of magnesium sulfate and acetonitrile, an organic modifier, into the separation buffer. Both of these additives appeared to influence the separation by diminishing the interaction of the protein analyte with the charged micelles present in the buffer. The quantitation capabilities of the CE method were validated and found to be comparable to the standards set by chromatographic techniques previously developed for similar applications.
Quantitative analysis of major whey proteins by capillary electrophoresis using uncoated capillaries
Authors: Recio Isidra , Molina Elena , Ramos Mercedes , de Frutos Mercedes 
Published: May 2005

AbstractA method that allows separation and quantitation of the main whey proteins by capillary electrophoresis using uncoated capillaries is proposed. Separations are performed using 100 mM borate buffer, pH 8.2, containing 30 mM sodium sulfate. The use of high pH and high ionic strength buffer reduces adsorption of proteins on the capillary wall, making their separation possible. Reproducibility of migration times and areas of peaks are improved by optimizing the capillary equilibration protocol and by using an internal standard. Relative standard deviations ranging between 0.74 and 1.03% for migration times and 2.14 to 5.23% for areas of major peaks are obtained. Detection limits equal to or lower than 0.5 mg/100 mL are achieved. Linear relationships of peak area versus concentration have been used to quantitate bovine serum albumin (BSA), α‐LA (α‐lactalbumin), β‐LG A (β‐lactoglobulin A) and β‐LG B (β‐lactoglobulin B) in cow's milk subjected to different thermal treatments. Electrophoretic profiles of these milk samples show peaks from other peptides besides those from main proteins. Characteristic patterns for whey from different species are obtained.
Temperature‐gradient gel electrophoresis as a screening tool for polymorphisms in multigene families
Authors: Schlötterer Christian 
Published: May 2005

AbstractInformation about sequence variability between different copies of a multigene family is indispensable for understanding the evolutionary mechanisms acting on multigene families. However, their high copy number has been a major obstacle to systematic analysis. Exemplified by the internal transcribed spacer 1 (ITS1) of the rDNA in Drosophila melanogaster, it is shown how temperature gradient gel electrophoresis (TGGE) can be used to study sequence polymorphisms in a multigene family. Experimental conditions influencing the melting behavior of the ITS1 fragment are discussed as well as discrepancies between observed and calculated melting patterns.
Temperature gradient gel electrophoresis for analysis of a polymerase chain reaction‐based diagnostic clonality assay in the early stages of cutaneous T‐cell lymphomas
Authors: Menke Martin A. O. H. , Tiemann Markus , Vogelsang Dörthe , Boie Christina , Parwaresch Reza 
Published: May 2005

AbstractBy means of a multiplex polymerase chain reaction (PCR) we amplified rearranged T‐cell receptor γ chain genes to detect monoclonality in 370 formalin‐fixed skin biopsy specimens, showing histological features of parapsoriasis or mycosis fungoides. PCr products were analyzed by temperature gradient gel electrophoresis (TGGE). We selected 20 positive cases for use in a comparison of this technique with conventional agarose and polyacrylamide gel electrophoresis (PAGE). With TGGE the T‐cells had shown monoclonality in 272 of the 370 cases; with agarose electrophoresis they did so in only 5 of the 20 selected cases and with PAGE in 16. Where multiple biopsy specimens from the same patient were analyzed, PCR products showed identical rearrangement patterns in TGGE. TGGE is an efficient technique that works on routine material and can help to verify a histological diagnosis of cutaneous T‐cell lymphoma.
Effect of pH and sodium dodecyl sulfate concentration on the analytical window in the direct‐injection analysis of plasma samples by capillary electrophoresis
Authors: Wätzig Hermann , Lloyd David K. 
Published: May 2005

AbstractPlasma samples can be analyzed with direct injection onto a capillary electrophoresis system if interactions between the plasma proteins and capillary walls are minimized. This can be achieved using micellar electrokinetic capillary chromatography with a surfactant such as sodium dodecyl sulfate. The surfactant complexes with the proteins, giving them a net negative charge, and thus causes them to be repelled from the negatively‐charged fused‐silica capillary walls. Migrating as anions, the complexed proteins appear late in the electropherogram. In such analyses, it is important to manipulate the mobility of the analyte(s) of interest such that they migrate in the useful analytical time window before the plasma proteins. In this article, a study of the effect of buffer pH and surfactant concentration on the width of the migration time window is reported. It is shown that at pH 6 the migration time window is wide, but reduced electroosmosis can result in inordinately long run times; pH 7 gives an acceptable analytical window with acceptable run times over a wide range of sodium dodecyl sulfate (SDS) concentrations; pH 8 and higher are most useful when higher concentrations of surfactant (> 50 mM SDS) are employed.
Molecular characterization of Borrelia burgdorferi sensu lato strains by pulsed‐field gel electrophoresis
Authors: Busch Ulrich , Teufel Cecilia Hizo , Boehmer Reinhard , Wilske Bettina , Preac‐Mursic Vera 
Published: May 2005

AbstractThe large restriction fragment patterns (LRFP) and linear plasmid profiles of eight tick isolates of Borrelia burgdorferi sensu lato were investigated with pulsed‐field gel electrophoresis (PFGE). The whole cell lysate was examined with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The MluI LRFP differentiates two species of Borrelia burgdorferi sensu lato (Borrelia burgdorferi sensu stricto and Borrelia garinii). LRFP is a suitable method to demonstrate genetic hetero‐ or homogeneity of isolates within one species without subsequent hybridization utilizing diverse probes. Different strains with similar or identical LRFP can be further discriminated by plasmid profile analysis. Our results show that each strain analyzed had a different plasmid profile. Therefore the linear plasmid profile has a potential application as a strain typing procedure. SDS‐PAGE of whole‐cell lysate supports the findings of homology within the B. burgdorferi sensu stricto species and the heterology within the B. garinii species.