bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of Concern: Up‐regulation of Wnt‐1 and β‐catenin production in patients with advanced metastatic prostate carcinoma
Authors: Chen Gaoping , Shukeir Nicholas , Potti Anil , Sircar Kanishka , Aprikian Armen , Goltzman David , Rabbani Shafaat A. 
Published: August 2004

AbstractBACKGROUNDWnt‐1 and β‐catenin expression levels play an important role in several malignancies. The authors determined the level of production of Wnt‐1 and β‐catenin in normal and malignant human prostate carcinoma cell lines. Surgical pathology specimens from primary prostatic adenocarcinoma, lymph node metastases, and skeletal metastases were used to establish a correlation between the level of Wnt‐1/β‐catenin expression, Gleason score, serum prostate‐specific antigen (PSA) status, and androgen receptor (AR) status.METHODSImmunohistochemical analysis was used to investigate the expression of Wnt‐1 and β‐catenin in human prostate carcinoma cell lines and in paraffin embedded sections of archival samples from 67 patients with prostate carcinoma. Comparison was made with the expression of tumoral AR and lymph node and skeletal metastases. These results were used to establish a correlation with the clinicopathologic status of patients with prostate carcinoma.RESULTSLevels of both Wnt‐1 and β‐catenin were low in normal prostate cells and were expressed highly in human prostate carcinoma cell lines. Wnt‐1 and cytoplasmic/nuclear β‐catenin expression was observed in 52% and 34%, respectively, of primary prostate carcinoma specimens. High levels of expression of Wnt‐1 and β‐catenin were seen in 77% of lymph node metastases and in 85% of skeletal metastases. These increased levels of expression were related directly to the Gleason score and to serum PSA levels in these patients. Maximum levels of Wnt‐1 and β‐catenin production were observed in skeletal metastases, whereas normal prostatic tissue failed to exhibit any detectable nuclear staining for β‐catenin.CONCLUSIONSHigh levels of Wnt‐1 and β‐catenin expression were associated with advanced, metastatic, hormone‐refractory prostate carcinoma, in which they can serve as markers of disease progression. Cancer 2004. © 2004 American Cancer Society.
Low Temperature Synthesis of Zeolite Films on Glassy Carbon: Towards Designing Molecularly Selective Electrochemical Devices
Authors: Walcarius Alain , Ganesan Vellaichamy , Larlus Olivier , Valtchev Valentin 
Published: August 2004

AbstractFaujasite‐type zeolite films have been grown on glassy carbon electrode surfaces embedded in an epoxy resin matrix by a quasi ambient temperature hydrothermal synthesis. The resulting zeolite modified electrodes are mechanically stable and provide an advantageous combination of electrochemical detection with charge and size selectivities at the molecular level, as demonstrated with the aid of some model electroactive probes or analytes (Ru(NH3)63+, Fe(CN)63−, Ru(bpy)32+, dopamine, ascorbic acid). To act efficiently as a physical molecular sieve barrier between the electrode surface and the external solution, these films must have a thickness of about 0.7 μm. These results open the door for further development of molecular sieving electrochemistry.
Mechanistic – Kinetic Scheme of Oxidation/Reduction of TEMPO Involving Hydrogen Bonded Dimer. RDE Probe for Availability of Protons in Reaction Environment
Authors: Janiszewska Anna Maria , Grzeszczuk Maria 
Published: August 2004

AbstractMechanistic – kinetic studies on the electrochemical oxidation/reduction process of radical TEMPO (2,2,6,6‐tetramethylpiperidine‐1‐oxyl) under ionic strength (0.1 M, 1.0 M) and pH (0, 7) of aqueous perchlorate electrolyte (NaClO4‐HClO4) have been undertaken. Analytical and/or digital simulation methods for voltammetry at stationary (CV) and rotating electrode (RDE) have allowed one to determine numerical values of twelve parameters characterizing two electrode reactions (oxidation and reduction of the radical) and three chemical reactions (protonation, disproportionation, dimerization involving the radical and/or electrogenerated species). A potential window of the measurements was 0.6 V and it corresponded to that where the oxidation wave of TEMPO in neutral aqueous solution is situated. To account for the observed pH effect, the hydrogen bonded dimer resulting from the radical reactant and the protonation product of its reduction has been postulated to form in solution near the electrode surface. The RDE voltammetric discernables of the TEMPO process (i.e., absolute RDE wave current, zero RDE current potential, oxidation and reduction limiting RDE currents) can be considered good candidates for a use to follow acidity of complex reactive media.
Detection of chondroitin sulfate‐binding proteins on the membrane
Authors: Saito Akio , Munakata Hiroshi 
Published: August 2004

AbstractChondroitin sulfate is a ubiquitous component of proteoglycans that is present both in the extracellular matrix and at the cell surface of various tissues. Until recently, chondroitin sulfate has attracted less attention than heparan sulfate and dermatan sulfate, owing to the limited number of known chondroitin sulfate‐binding proteins. To determine the biological function of chondroitin sulfate, biotinylated probes were prepared and used to search for binding proteins. Chondroitin sulfates A, C, D, and E were biotinylated through either the uronic acid or the residual core peptide. Lysates from mouse Lewis lung carcinoma (3LL) cells were blotted onto a nitrocellulose membrane after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and the membrane was treated with the biotinylated chondroitin sulfates. Among the chondroitin sulfate variants, the E type showed the most intense bands upon visualization of the membrane with avidin‐conjugated alkaline phosphatase and the appropriate substrates. The binding of chondroitin sulfate E to proteins in the cell lysate was not affected by the A, C or D variants but was reduced by treatment with dermatan sulfate. Lysates from 3LL cells were also treated with biotinylated chondroitin sulfate E and, after two‐dimensional (2‐D) electrophoresis and blotting, several chondroitin sulfate E‐binding proteins including lamins and heterogeneous nuclear ribonucleoproteins were identified by mass spectrometry.
Sensitive silver staining of protein in sodium dodecyl sulfate‐polyacrylamide gels using an azo dye, calconcarboxylic acid, as a silver‐ion sensitizer
Authors: Jin Li‐Tai , Hwang Sun‐Young , Yoo Gyurng‐Soo , Choi Jung‐Kap 
Published: August 2004

AbstractA highly sensitive silver staining method for detecting proteins in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was developed. It is based on the silver nitrate staining method but also employs an azo dye, calconcarboxylic acid (NN), as a silver‐ion sensitizer. It increases silver binding on protein bands or spots by the formation of a silver‐dye complex and also increases the reducing power of silver ions to metallic silver by NN itself with formaldehyde. After a 2 h gel fixing step, the protocol including sensitization, silver‐ion impregnation, and reduction steps can be completed in 1 h. The sensitivity is superior to that of silver stain with glutardialdehyde as a silver‐ion sensitizer. The detection limit of NN‐silver stain is 0.05–0.2 ng protein. Considering the high sensitivity without using glutardialdehyde, the NN‐silver stain would be useful for routine silver staining of proteins.
The utility of a two‐color fluorescence electrophoretic mobility shift assay procedure for the analysis of DNA replication complexes
Authors: Jing Debra , Beechem Joseph M. , Patton Wayne F. 
Published: August 2004

AbstractProtein‐DNA and protein‐protein interactions play essential roles in many biologic processes such as transcription, replication, recombination, and DNA repair. One of the most popular approaches to investigate specific protein‐nucleic acid interactions is the electrophoretic mobility shift assay (EMSA). We have developed a new nonradioactive method to detect both nucleotides and protein in EMSA. Nucleic acids are detected with SYBR® Green EMSA dye, while proteins are subsequently detected with SYPRO® Ruby EMSA dye. All fluorescent staining steps are performed after the entire gel‐shift experiment is completed, so there is no need to prelabel either the nucleic acids or the protein. The two‐color fluorescence dye staining procedure is fast, simple, and allows independent quantitative determination of either free or bound nucleic acids and protein. The interactions between lac repressor‐operator, and among the T4 primase‐helicase, primase‐DNA, helicase‐DNA, and within T4 [ssDNA‐primase‐helicase6] primosome, were used to demonstrate the advantages of this two‐color fluorescence detection EMSA method.
Ultrafiltration for proteomic sample preparation
Authors: Chernokalskaya Elena , Gutierrez Sara , Pitt Aldo M. , Leonard Jack T. 
Published: August 2004

AbstractProteome analysis represents significant challenges to the existing sample preparation techniques. Traditional methods, such as two‐dimensional electrophoresis, typically separate high‐molecular‐weight proteins while discarding low‐molecular‐weight species. This approach is well justified considering the complexity of any proteome. However, it is desirable to extract the maximum amount of information from each sample to investigate the entire range of biomolecules. We have demonstrated that ultrafiltration not only improves two‐dimensional electrophoresis (2‐DE) resolution of the protein fraction but also yields the low‐molecular‐weight fraction amenable for further analysis by high‐resolution mass spectrometry. This approach was successfully adapted to the variety of biological samples including cell and tissue lysates and serum. Therefore, ultrafiltration offers an alternative sample preparation technique that enables more thorough analysis of a proteome.
Selective proteome‐wide detection of hydrophobic integral membrane proteins using a novel fluorescence‐based staining technology
Authors: Hart Courtenay , Schulenberg Birte , Patton Wayne F. 
Published: August 2004

AbstractIntegral proteins containing two or more α‐helical membrane‐spanning domains are underrepresented in two‐dimensional gels. While sodium dodecyl sulfate (SDS)‐polyacrylamide gels separate these proteins, staining profiles are usually dominated by high‐abundance hydrophilic proteins in the specimen. A fluorescence‐based stain is presented that selectively highlights integral proteins containing two or more α‐helical transmembrane domains but does not detect lipoproteins or proteins with hydrophobic pockets, such as albumin. The stain detects as little as 5–10 ng of bacteriorhodopsin, a seven‐helix transmembrane protein. Stained proteins are detected using a laser scanner or charge‐coupled device (CCD) camera imaging system. Fluorescence intensity of stained bands is linear with protein quantity over at least two orders of magnitude. After visualizing transmembraneous proteins, the total protein profile may be revealed using a general protein stain. Analysis of the multisubunit protein F1F0 ATP synthase revealed selective staining of the a and c subunits, polypeptides known to possess 5 and 2 transmembrane domains, respectively.
Flow and evaporation cells for the detection of proteins on membranes with the peroxyoxalate chemiluminescent reaction in organic media
Authors: Castro‐Hartmann Pablo , Daban Joan‐Ramon 
Published: August 2004

AbstractThe high‐energy intermediates generated in the reaction of bis(2,4,6‐trichlorophenyl)oxalate (TCPO) with H2O2 can excite electronically different fluorophores with a high quantum yield in organic solvents. We have previously applied this peroxyoxalate chemiluminescent reaction to the detection of proteins labeled with the fluorescent dye 2‐methoxy‐2,4‐diphenyl‐3(2H)‐furanone (MDPF) on polyvinylidene difluoride (PVDF) membranes. In this work, we have investigated the possibility to enhance the sensitivity of this detection method using specially designed cells in which the reagents TCPO and H2O2 in acetone are continuously renewed. In the flow cell, two syringes are used to renew the reagents in the reaction chamber containing the PVDF membrane with blotted proteins labeled with MDPF. In the evaporation cell, a fresh solution of reagents continuously replaces the volume of acetone evaporated in the reaction chamber. Both cells show a low emission background but the observed elution of proteins from the membrane produced by the flow of reagents in acetone limits the maximum sensitivity attainable with these cells. The best result (detection of 1 ng of MDPF‐labeled protein) has been obtained with the evaporation cell.
Optimized conditions for diluting and reusing a fluorescent protein gel stain
Authors: Ahnert Nancy , Patton Wayne F. , Schulenberg Birte 
Published: August 2004

AbstractThis study elucidates the optimum conditions at the minimum cost for using SYPRO Ruby protein gel stain. It deals with the effects of gel fixation and staining times, as well as dilution and reuse of SYPRO Ruby protein gel stain in one‐dimensional (1‐D) gels. Signal strength and dynamic range were highest in gels that were fixed thoroughly before staining, followed by overnight staining. Using the optimized protocol, dilution or reuse of the stain reduces the dynamic range and signal intensity. Sensitivity remains high if the stain is reused up to two times, but signal intensity is reduced up to 2.5‐fold in twice used stain. Sensitivity also remains high if the stain is diluted 1:2 in water, but signal intensity is reduced up to 6‐fold. Of the two options, reuse or dilution, reuse better retains signal intensity and dynamic range.
Comparison of fluorescent stains: Relative photostability and differential staining of proteins in two‐dimensional gels
Authors: Smejkal Gary B. , Robinson Myra H. , Lazarev Alexander 
Published: August 2004

AbstractThe fluorescence of proteins stained with Deep Purple and SYPRO Ruby was measured over a time course of UV transillumination to determine the relative photostability of each stain. Mean spot fluorescence (n = 200 matched spots) in gels stained with Deep Purple decreased 27% following 2 min of UV transillumination, compared to SYPRO Ruby, which decreased 17%. After 19 min, an 83% decrease in Deep Purple fluorescence was observed, compared to 44% for SYPRO Ruby. By interpolation, the half‐life of Deep Purple fluorescence was estimated to be approximately 6 min. The half‐life of SYPRO Ruby fluorescence was not reached during the 19 min time course. Further, differential staining of proteins was observed in gels stained with Deep Purple and SYPRO Ruby as compared to colloidal Coomassie Brilliant Blue and silver staining.
Focused proteomics: Monoclonal antibody‐based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain
Authors: Murray James , Marusich Michael F. , Capaldi Roderick A. , Aggeler Robert 
Published: August 2004

AbstractWe have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post‐translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro‐Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post‐translational modifications in control and patient samples using only small amounts of tissue.
Characterization of dynamic and steady‐state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry
Authors: Schulenberg Birte , Goodman Terrie N. , Aggeler Robert , Capaldi Roderick A. , Patton Wayne F. 
Published: August 2004

AbstractProtein phosphorylation plays a vital role in the regulation of most aspects of cellular activity, being key to propagating messages within signal transduction pathways and to modulating protein function. Pro‐Q® Diamond phosphoprotein gel stain is suitable for the fluorescence detection of phosphoserine‐, phosphothreonine‐, and phosphotyrosine‐containing proteins directly in sodium dodecyl sulfate (SDS)‐polyacrylamide gels. The technology is especially appropriate for profiling steady‐state and dynamic phosphorylation on a proteome‐wide scale, as demonstrated through detection of the native phosphorylation of cardiac mitochondrial phosphoproteins and changes in this profile arising from the activity of a protein kinase. For example, Pro‐Q Diamond phosphoprotein gel stain was employed to demonstrate that among the 46 subunits of the mitochondrial respiratory chain complex, NADH:ubiquinone oxidoreductase (complex I), a 42 kDa subunit is phosphorylated in the steady‐state. However, exposure of mitochondria to cAMP‐dependent protein kinase increases phosphorylation of this 42 kDa subunit and results in de novo phosphorylation of an 18 kDa subunit as well. Since Pro‐Q Diamond dye binds to phosphorylated residues noncovalently, the staining technology is fully compatible with modern microchemical analysis procedures, such as peptide mass profiling by matrix assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry and post‐source decay analysis of peptide phosphorylation.
Combining microscale solution‐phase isoelectric focusing with Multiplexed Proteomics® dye staining to analyze protein post‐translational modifications
Authors: Schulenberg Birte , Patton Wayne F. 
Published: August 2004

AbstractPreviously, a strategy for rapidly identifying mitochondrial phosphoproteins was presented that involves prefractionating multisubunit complexes by sucrose gradient centrifugation, followed by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis and selective staining of phosphoproteins and total protein with fluorescent dyes [1]. Though suitable for evaluating the mitochondrial proteome, which consists of numerous multisubunit complexes, the strategy is not generally applicable to other complex proteomes. We determined that prefractionating samples by solution‐phase isoelectric focusing is an effective alternative to sucrose‐gradient fractionation that can be applied equally well to the analysis of mitochondrial and plasma proteins. Fluorescence‐based multiplexing dye technologies greatly extend the capacity of SDS‐polyacrylamide gel electrophoresis with respect to the investigation of proteome‐wide changes in protein expression and post‐translational modification, such as phosphorylation and glycosylation [2]. Overall, the prefractionation/Multiplexed Proteomics® staining technology permits rapid, higher throughput screening of specimens for the identification of potentially interesting fractions that can subsequently be evaluated more thoroughly by two‐dimensional gel electrophoresis.
A two‐dimensional electrophoresis map of Chinese hamster ovary cell proteins based on fluorescence staining
Authors: Hayduk Eric J. , Choe Leila H. , Lee Kelvin H. 
Published: August 2004

AbstractWe report on the development of a detailed two‐dimensional electrophoresis map of Chinese hamster ovary (CHO) cell proteins based on fluorescence staining and tandem time‐of‐flight (TOF/TOF)‐mass spectrometry. We observed a 71% success rate in the identification of proteins even though the CHO genome is not sequenced. The map consists of 224 protein identifications present in 274 two‐dimensional gel electrophoresis (2‐DE) gel spots. We have also initiated a study of the phosphoproteome using a commercially available phosphoprotein‐specific fluorescent stain. Using this stain, we observe 672 phosphorylated proteins, including many proteins known to be phosphorylated, which is 36% of the proteins we visualized with a total protein stain and consistent with expectations.
Microscale fractionation facilitates detection of differentially expressed proteins in Alzheimer's disease brain samples
Authors: Lopez Mary F. , Mikulskis Alvydas , Golenko Eva , Melov Simon , Bennett David , Cherkasskiy Alex , Kuzdzal Scott , Kramer Susan , Sissors Dan 
Published: August 2004

AbstractFractionation enhances the resolution of proteins with similar characteristics by reducing the number of proteins that comigrate in gels, thus facilitating the detection of lower‐abundance proteins and the accurate determination of quantitative and qualitative differences in disease and normal samples. An efficient, reproducible microscale fractionation protocol for complex protein mixtures using novel ion‐exchange membrane chromatographic substrates (PerkinElmer, Boston, MA, USA; Vivascience, Carlsbad, CA, USA) is described. The fractionation techniques were used in combination with two‐dimensional (2‐D) gels and orthogonal matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry to identify differentially expressed proteins in brain samples from persons with and without Alzheimer's disease.
Towards two‐dimensional electrophoresis mapping of the cerebrospinal fluid proteome from a single individual
Authors: Finehout Erin J. , Franck Zsofia , Lee Kelvin H. 
Published: August 2004

AbstractWe test the ability of state‐of‐the‐art two‐dimensional electrophoresis (2‐DE) technology to enable the proteome mapping of ante mortem cerebrospinal fluid (CSF) from a single individual. Using the sensitive technologies of a fluorescent protein stain and fluorescence laser scanning of 2‐DE gels, combined with matrix‐assisted laser desorption/ionization‐time of flight/time of flight‐mass spectrometry (MALDI‐TOF/TOF‐MS) for protein identification, a highly detailed 2‐DE map of the CSF proteome was created. The 2‐DE map contains 600 identified spots representing 82 different proteins. Of the 82 proteins identified, 25 have not appeared in any previously published 2‐DE map of CSF, and 11 have not been previously reported to exist in CSF. Most of the identifications originate from an ante mortem CSF sample collected from a single hydrocephalus patient. A supplemental map created from neurologically normal patients is also presented. A webpage with protein identification and scoring information from both maps is available at http://www.leelab.org/csfmap.