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In order to improve understanding of the heat-induced changes in muscle growth, we determined the expression of genes related to protein and energy metabolism in the pectoralis major muscle of chickens. We also explored the protein kinase B (PKB also called Akt)/p70 S6 kinase (S6K1)/S6 pathway that mediates anabolic signals thereby regulating metabolism and hypertrophic/atrophic balance. Four-week-old chickens were exposed to 32 or 22 degrees C for 1 week. Chickens from both groups were then fasted for 16 h or left fed, and submitted to an oral administration of glucose-arginine to induce an anabolic response (30-min treatment) or left untreated. High ambient temperature and the associated decrease in feed intake modified the expression of certain energy-related genes (e.g. -40% for PGC-1alpha) and protein metabolism (e.g. about +80% for atrogin-1), but the expression of several muscle metabolism-related genes considered here was unchanged. The capacity for muscle protein synthesis, i.e. RNA/protein ratio, was reduced in warm conditions (approximately -20%). Slightly lower activation of S6 induced by glucose-arginine treatment was found at 32 degrees C compared to 22 degrees C, which might indicate somewhat lower efficiency of mRNA translation. Analysis of glucose/insulin balance suggested changes in glucose metabolism under heat exposure. However, this remains to be characterized., (Copyright 2010 Elsevier Inc. All rights reserved.)

The complexome is essential for a better understanding of protein functions. In order to study protein complexes, an approach allowing the extraction and the analysis in native conditions is needed. Two-dimensional blue native/SDS-PAGE (2D BN/SDS-PAGE) technology is thus an interesting and powerful approach for this purpose. This report deals with the analysis and the identification of the organellar protein complexes of Caco-2 human cells using 2D BN/SDS-PAGE and HPLC-chip-MS. We identified 58 protein complexes (26 heteromultimeric and 32 homomultimeric complexes) and 4 monomeric proteins. Among them, 32 protein complexes were pointed out, providing insights into the function of previously uncharacterized human proteins.

The yeast Yarrowia lipolytica uses hydrophobic substrates, such as alkanes, fatty acids and oils, for its growth. It has developed a strategy for the use of such substrates, involving the production of hydrophobic binding structures called protrusions on the cell surface. These protrusions are resemble channels connecting the cell wall to the inside of the cell, and are probably involved in transport mechanisms that we do not yet fully understand. The complete genome of the haploid Y. lipolytica strain E150 (CLIB99) was sequenced in 2004 by the Génolevures Consortium. The availability of a complete genome sequence for this species has made it possible to carry out proteomic and other investigations, leading to the characterization of lipid bodies (LB) in terms of (i) their lipid composition, (ii) the major LB proteins, as identified by mass spectrometry, and (iii) differences in protein or lipid composition as a function of the carbon source used. Functional analyses would provide insight into the biological processes associated with these bodies and 2D BN/SDS-PAGE is a highly suitable method for the analysis of protein complexes. This report provides a first description of the analysis and identification of hydrophobic binding protein complexes in Y. lipolytica. For this purpose, we used 2D BN/SDS-PAGE for the separation of protein complexes and HPLC-chip-MS for protein identification. We separated and identified 40 protein complexes (11 heteromultimeric and 29 homomultimeric), providing insight into their function. This study represents a major step forward, as most previous studies identified proteins either on the basis of sequence similarity to proteins from other organisms (44% of the proteins identified in this study) or by prediction (50% of proteins identified in this study) alone., ((c) 2009 Elsevier B.V. All rights reserved.)

Avian uncoupling protein (avUCP) is orthologous to UCP3, which is suggested to be involved in fatty acid metabolism and to limit the mitochondrial production of reactive oxygen species in mammals. In the chicken, the role and regulation of avUCP remain to be clarified. The aim of this study was to explore the control of avUCP expression by the beta-adrenergic system, known to be involved in avian thermoregulation and lipid utilization, and in UCP expression in mammals. Therefore, we measured the expression of avUCP mRNA and protein in the Pectoralis major muscle of chickens injected with the beta(2) agonist isoproterenol, and we investigated the potential pathways involved in the regulation of avUCP mRNA expression. Avian UCP mRNA expression was increased 7-fold 4h after isoproterenol injection, leading to a tendency to a 40% increase in avUCP protein 24h post-injection. This increase was preceded, 30 min after isoproterenol injection, by changes in the chicken thyroid status and in the muscular expression of PPARalpha, PPARbeta/delta, and PPARgamma coactivator-1alpha (PGC-1alpha). Moreover, the analysis of the avUCP promoter sequence suggested potential binding sites for PPARs and for thyroid hormone receptors. We also detected the activation of AMP-activated protein kinase, which has recently been reported to be involved in UCP3 regulation in mammals. This study presents for the first time evidence of beta-adrenergic control on avUCP messenger expression in chicken muscle and suggests the potential involvement of AMPK and several transcription factors in this regulation., (Copyright 2010 Elsevier Inc. All rights reserved.)

To explore the mechanisms leading to excessive adiposity in chicken, we investigated the regulation of fatty acid oxidation depending on genotype-related body fatness and diet composition. mRNA expression and/or activity of proteins involved in mitochondrial energy metabolism were measured in liver and gastrocnemius muscle of genetically lean or fat chickens reared on a low-fat/high-protein diet or an isoenergetic high-fat/low-protein diet (HF/LP). Muscle expressions of the muscle isoform of carnitine-palmitoyltransferase 1 (M-CPT1) and PPARbeta/delta were higher in fat than in lean chickens. This was also observed in liver, although only with the HF/LP diet for M-CPT1. This could stimulate mitochondrial fatty acid oxidation in fat chickens. Up-regulations of liver and muscle CPT-1 hepatic isoform, and muscle cytochrome-c-oxidase mRNA expressions, and of beta-hydroxyacyl-CoA-dehydrogenase activities suggest higher fatty acid utilization with the HF/LP diet. PPARbeta/delta and PGC-1alpha could control fatty acid oxidation in muscle and liver, respectively. Regulation of avian uncoupling protein (avUCP) mRNA was tissue-dependent. Predominantly expressed in muscle, it was stimulated in fat and in HF/LP-fed chickens, where it could be associated to the special need in muscle anti-oxidant pathways of fatter animals. In liver it was lower in fat than in lean chickens, and its potential function remains to be clarified.

The increased incidence of autoantibodies in malignancies has been described since the 1970s. Thus the ability to determine molecular fingerprinting of autoantibodies (antibody signatures) may provide useful clinical diagnostic and prognostic information. This review describes the use of several proteomics approaches for the identification of antigens recognized by these autoantibodies. Serological proteome analysis combines separation of tumor cell proteins on two-dimensional gel electrophoresis gels, Western blotting with sera of patients and healthy subjects, and identification of the detected antigens by MS. Alternatively multiple affinity protein profiling combines isolation of the antigens recognized by patient antibodies by two-dimensional immunoaffinity chromatography and identification by MS/MS. The use and limitations of reverse phase protein microarrays for testing patient serum containing autoantibodies are also considered. Lastly the most important difficulty of any proteomically identified autoantibody signature is validation in patient cohorts or clinical samples.

Huntington's disease (HD) causes widespread CNS changes and systemic abnormalities including endocrine and immune dysfunction. HD biomarkers are needed to power clinical trials of potential treatments. We used multiplatform proteomic profiling to reveal plasma changes with HD progression. Proteins of interest were evaluated using immunoblotting and ELISA in plasma from 2 populations, CSF and R6/2 mice. The identified proteins demonstrate neuroinflammation in HD and warrant further investigation as possible biomarkers.

This work investigates the use of an HPLC-chip microfluidic device interfaced to an IT mass spectrometer to search for biomarker signatures. To that end, the identification of autoantigens is chosen as a model. It not only constitutes a proof of concept model but also the growing interest in autoantibodies and autoantigens as new markers of diseases provides a practical application at the same time. The peptides are separated by the HPLC-chip system allowing suitable resolution and reproducibility. The determination of two parameters that characterize a peptide sequence during LC-MS/MS analyses, retention time (RT) and m/z ratio, improves the identification of a number of peptides derived from protein digests. These findings illustrate that accurate RT measurement obtained in a microfluidic device is useful to obtain mass/retention time (MRT) pairs for a given peptide, which can contribute to the definition of biomarker signatures.

Cancer remains one of the leading causes of death worldwide. Thus, to identify any useful biomarkers is still a need. We performed "cancer immunomics" to identify autoantibody signatures produced in response to the presence of either breast or colorectal cancer. SERological proteome analysis (SERPA) was performed by two-dimensional (2-D) electrophoresis separation, immunoblotting, image analysis, and mass spectrometry. Alternatively, to identify the antigens recognized by the autoantibodies of cancer patients, we developed an approach combining 2-D immunoaffinity chromatography, enzymatic digestion of the isolated antigens, nano flow separation of the resulting peptides, and identification: MAPPing (multiple affinity protein profiling). By these approaches we identified both proteins recognized by autoantibodies independently of a cancer status, and a limited number of proteins reacting preferentially with cancer sera.

Patients with cancer produce specific autoantibodies against protein antigens present in limited amount among a large background of immunoglobulins (Igs), nonrelevant as biomarkers, including natural antibodies. Multiple affinity protein profiling (MAPPing) that combines 2-D immunoaffinity chromatography, enzymatic digestion of the isolated proteins, and identification by MS/MS, may facilitate the identification of these so far unknown patient antibodies. The first immunoaffinity chromatography is crucial, as it is used for selectively removing proteins (autoantigens) recognized by natural antibodies. Application of this depletion step to colon cancer cell proteins is specifically described along with the identification of the natural autoantigens, as well as the coupling of this depletion step with the next steps. By enabling to separate antibody-binding proteins recognized by either natural autoantibodies or patient-specific antibodies this approach may contribute significantly towards the definition of autoantibody signatures.

Specific and efficient selection of serum immunoglobulins, but not other proteins, on T-gel remains difficult. T-gel capacity was determined for different activation conditions and serum loadings. Mass spectrometry analysis was used to identify the proteins found in the flow-through and in the eluted fractions. Alpha-2-macroglobulin and albumin were the major contaminants of the eluates. The influence of the competition between immunoglobulins and the other serum proteins on the adsorption was also studied. Using a serum depleted in immunoglobulins (flow-through of a first chromatography on T-gel), many serum proteins were retained on the T-gel, including albumin. We conclude that T-gel selectivity is less than absolute and may reflect for a large part the experimental conditions of the adsorption.

Background: The MCF7 breast cancer cell line is a cellular model for breast cancer studies and marker discovery. Therefore, a better knowledge of its proteome is a prerequisite for a more efficient use of this model., Materials and Methods: Proteins expressed during the exponential growth phase of MCF7 cells were analyzed and mapped using two-dimensional gel electrophoresis and mass spectrometry., Results: From the spots excised from preparative gels of whole-cell extracts, a subset of 368 different polypeptides, corresponding to 249 different proteins, was identified. These polypeptides were positioned on a silver-stained gel to construct a reference map., Conclusion: The data allowed the construction of the most extensive reference map for MCF7 published to date, with 189 novel proteins, which had not been previously listed on maps, and are now accessible on World 2D-PAGE database, providing a basis for further studies on MCF7., (Copyright© 2006 International Institute of Anticaner Research (Dr. John G. Delinassios), All rights reserved.)

The identification of specific protein markers for breast cancer would provide the basis for early diagnosis. Particularly, membrane and membrane-associated proteins are rich in targets for antibodies that may constitute suitable biomarkers of carcinogenesis. However, membrane proteins separation using 2-DE remains difficult. In this work, the breast cancer cell line MCF7 was used as source of proteins for the screening of potential cell membrane-associated antigens recognized by autoantibodies in patients with breast cancer and healthy volunteers. The protein extract obtained using trifluoroethanol (TFE) as cosolvent was compared to a total cell lysate protein extract prepared by a current technique. After 2-DE separation of the two extracts, their protein patterns clearly differed. About 63% of the proteins identified in the TFE-extract were predicted to possess at least one transmembrane domain. 2-D blots probed with sera from cancer patients or from healthy volunteers showed that, as expected, additional antigens were provided in the TFE-extract. Thus, the method described here appeared well suited for proteomic investigation of potential biomarkers undetected by current techniques.

Nanoflow liquid chromatography/mass spectrometry (nanoLC/MS) has become a current tool in proteomics applications increasingly used in the search for new biomarkers. A new integrated microfluidic device (HPLC-Chip), coupled to ion trap mass spectrometry (ITMS), appears as an innovative and robust tool for improving the identifications commonly performed by nanoLC/MS/MS. We tested this device for the identification of proteins obtained from two-dimensional gel electrophoresis or chromatography. The chip allows the measurement of reproducible retention times that, in association with m/z ratios, was found useful for identifying peptide sequences without ambiguity. A sensitivity increase of a factor of at least 5-fold is obtained compared to the results obtained previously in our laboratory by conventional nanoLC/MS/MS on the same ion trap. We conclude that this recently available microfluidic device can be a valuable tool during biomarker discovery programs, particularly identifying low-abundance proteins., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Basic science research in hematology has been determining the functions of gene products using classical approaches that typically involve studying one or a few genes at a time. Proteomics, defined as the study of protein properties on a large scale, provides tools to globally analyze malignant hematologic cells. A major challenge in cancer therapy is the identification of drugs that kill tumor cells while preserving normal cells. Differential display via proteomics enables analysis of direct as well as side-effects of drugs at a molecular level. Proteomics also allows a better understanding of cell signaling pathways involved during apoptosis in hematologic cells. Storing the information in a 2D electrophoresis database enhances the efficiency of proteome research on malignant cells. Finally, the work needed to be carried out on proteomic analysis prior to routine clinical adoption is discussed, and the necessity for multi-institutional collaborations is emphasized.

This review provides an overview on recent studies in the field of proteome analysis of lymphoma cells, and highlights the potentials of such studies for a better knowledge of drug effects at the molecular level. After giving general information on the field of proteome analysis of lymphoma cells, some characteristics of the strategies used during this analysis are pointed out, such as cell extraction strategies and affinity captures. Therefore, the issue of proteome analysis of lymphoma cells content will be covered with respect to those protein extracts that can be prepared in saline solutions, such as cytoplasm proteins, or that are associated with the cell membranes. The question of which kinds of information have been retrieved from lymphoma-cell proteomics is discussed on the basis of several examples-lymphoma cell-mapping studies and constitution of protein databases, and comparative proteome analysis studies of the modifications that result from a drug treatment., (Copyright (c) 2004 Wiley Periodicals, Inc.)

Background: Proteins associated with cancer cell membranes represent targets of choice for humoral immune response as well as potential tumour marker proteins in human malignancies. However, proteomic analysis of these proteins, and more generally of low-soluble proteins, remains difficult., Materials and Methods: The breast cancer cell line MCF7 was selected to evaluate a sequential extraction method that enables simple fractionation of human cell proteins according to their subcellular localization, yielding subproteomes enriched in cytosolic and membrane-associated proteins, respectively. A crude plasma membrane preparation was followed by the solubilisation of proteins using trifluoroethanol (TFE) as co-solvent., Results: Cross-matching and statistical analysis performed for each set of two-dimensional electrophoresis (whole-cell, membrane and soluble extracts) and between the different sets highlighted the reproducibility of the extraction process and its usefulness for proteomic analysis. Eighty-three % of the spots of the gels corresponding to the membrane fraction were not found in the gels of the soluble fraction., Conclusion: Due to its simplicity, the approach described here appears well suited for membrane proteomic investigation of human cancer cells and detection of potential biomarkers undetected by current techniques., (Copyright© 2005 International Institute of Anticaner Research (Dr. John G. Delinassios), All rights reserved.)

Bone marrow-derived mesenchymal stem cells (MSCs) are known to interact with hematopoietic stem cells (HSCs) and immune cells, and are of potential interest to be used as therapeutic agents for enhancing allogenic hematopoietic engraftment and preventing graft-versus-host disease (GVHD). Galectin 1 (Gal1) belongs to a family of structurally related molecules expressed in many vertebrate tissues that exert their functions both by binding to glycoconjugates, and by interaction with protein partners. In this work using a proteomic approach, we looked for the presence and the localization of Gal1 in short- and long-term culture of human (h) hMSC. We first determined, that Gal1 is one of the major proteins expressed in hMSC. We futher demonstrated that its expression is maintained when hMSC are expanded through a subculturing process up to five passages. Moreover, Gal1 is secreted and found at the cell surface of MSC, participating in extra cellular matrix (ECM)-cell interactions. Given the immunomodulatory properties of Gal1, its potential involvement in immunological functions of hMSC could be suggested.

This study presents an improved method for the complete transfer of proteins separated by two-dimensional gel electrophoresis to a membrane, specifically designed for the screening and identification of antigens recognized by autoantibodies in patients with breast cancer (BCP) and healthy volunteers. This paper reports the evaluation of this technique using proteins from MCF7 as a source of antigens following 2-DE separation. The appropriate quantity of protein to be loaded on gels (150 microg) has been determined, the aim being a complete and reproducible recovery of all separated proteins onto the polyvinylidene fluoride membrane (2D-blot) after a semi-dry electrotransfer. Several different transfer methods were tested in parallel, resulting in the selection and optimisation of one using a discontinuous buffer system, based on the isotachophoresis theory. To facilitate the comparative analysis of the different sets of 2D-blots probed with individual sera from BCP and healthy volunteers, the 2D-blots were stained with colloidal gold following the immunodetection step. The gels and 2D-blots were scanned and analysed by imaging software. The matching permitted exact localisation of particular relevant protein spots hybridised by antibodies on the 2D-blots. These spots were subsequently located on preparative gels for identification by mass spectrometry. A set of 40 2D-blots was probed with 20 sera from patients with breast cancer and 20 sera from healthy volunteers. In the protein profiles submitted to immunodetection, 15 proteins were repeatedly immunodetected by both BCP and sera from healthy people. Those proteins were identified by mass spectrometry. Conversely, some protein isoforms were preferentially immunodetected by BCP sera and may reflect the presence of this cancer. The improved isotachophoretic method described in this study is suitable for comparing the overall profile of autoimmunity between different populations and for subsequent identification of relevant antigens.

With the increasing importance of proteome analysis, studying the phosphoproteome is a priority for functional studies. Therefore, a rational approach to simplifying the proteome is needed. In this work, we examined the use of immobilized metal affinity chromatography (IMAC) using ferric ions-chelated column for enriching crude cell extracts in phosphoproteins. The adsorption of the proteins on Fe(3+) was obtained at an acidic pH 5.6, and their elution at a more basic pH in Tris buffer. To evaluate the separation, western blots were performed with either anti-phosphotyrosine or anti-phosphoserine/threonine. The analysis of the eluates demonstrated the selectivity of the separation, particularly for proteins phosphorylated on serine or threonine. In conclusion, the advantages and the limits of this approach are discussed.

The aim of the present work was to develop a highly productive and simplified process for active human galectin-1 (Gal1) production. Gal1 is a beta-galactoside binding lectin that differentially affects biological and cellular functions such as immune surveillance and apoptosis. These effects have attracted the attention of researchers in cell biology, biochemistry and immunology. However, the production of sufficient amounts of recombinant human Gal1 (rhGal1) is needed to study of the effects of Gal1 during cell treatments. To this end, an high-yield expression of rhGal1 was achieved by high-cell density fed-batch cultivation using an exponential glycerol feeding strategy and rhGal1 was purified by a one-step purification scheme using affinity chromatography.

Renovascular hypertension is characterized by stenosis of the renal artery and high plasma renin levels. The renal phenotype is characterized by high levels of renin in the hypoperfused kidney due to the recruitment of renin-producing cells along the afferent arterioles. This increase in myoepithelioïd cells is due mainly to the differentiation of existing smooth muscle cells with acquisition of a secretory phenotype. To understand the molecular mechanisms involved in this recruitment, we used the established rat model of renovascular hypertension known as the two-kidney, one-clip model in the Lewis rat. Renal arterioles were isolated using magnetized iron suspension. Differential proteomic analysis was performed using 2-D polyacrylamide gel electrophoresis followed by mass spectrometry. Comparative analysis of soluble proteins extracted from afferent arterioles of clipped and contralateral kidneys showed 14 proteins significantly differentially expressed by at least a factor of 2. These proteins were identified by mass spectrometry. The most striking protein revealed by proteomics is troponin T, which is down-regulated in the afferent arterioles of the clipped kidney. Confocal microscopy showed that troponin T is specific of the smooth muscle phenotype and absent in the myoepithelioïd phenotype. Our data suggest that troponin T is only present in renal smooth muscle cells.

The use of robots has major effects on maximizing the proteomic workflow required in an increasing number of high-throughput projects and on increasing the quality of the data. In peptide mass finger printing (PMF), automation of steps downstream of two-dimensional gel electrophoresis is essential. To achieve this goal, the workflow must be fluid. We have developed tools using macros written in Microsoft Excel and Word to complete the automation of our platform. Additionally, because sample preparation is crucial for identification of proteins by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, we optimized a sandwich method usable by any robot for spotting digests on a MALDI target. This procedure enables further efficient automated washing steps directly on the MALDI target. The success rate of PMF identification was evaluated for the automated sandwich method, and for the dried-droplet method implemented on the robot as recommended by the manufacturer. Of the two methods, the sandwich method achieved the highest identification success rate and sequence coverage of proteins., (2004 John Wiley & Sons, Ltd.)

Renovascular hypertension is characterised by stenosis of the renal artery and high plasma renin levels due to the recruitment of renin-producing cells along the afferent arterioles. This increase in myoepithelioid cells is mainly a result of the differentiation of existing smooth muscle cells with acquisition of a secretory phenotype. To understand the molecular mechanisms involved in this recruitment, we used the model of renovascular hypertension known as the two-kidney, one-clip model in the Lewis rat. Renal arterioles were isolated using magnetised iron suspension. Differential proteomic analysis was performed using two-dimensional electrophoresis gel followed by mass spectrometry for identification. The most striking protein revealed by proteomics is troponin T, which is down-regulated in the afferent arterioles of the clipped kidney. Confocal microscopy showed that troponin T is specific to the smooth muscle phenotype and absent in the myoepithelioid phenotype.

5'-azacytidine (AZC) is a potent DNA demethylating agent used clinically for treatment of patients with malignant hemopathies. We have previously shown that AZC induces a halt in cell growth and a decrease of cell activity, without affecting cell viability. We have also shown using proteomics, that 35 polypeptides were differentially expressed in a cytoplasmic fraction. The aim of this study was to provide a more complete picture of modifications in AZC-treated cells using cell membrane preparations. Therefore the protein pattern changes following AZC treatment of the cell line DG75 were studied on a detergent-solubilized fraction obtained from these membranes. Results showed that 49 proteins were differentially expressed in the membrane fraction. Seven polypeptides were down-regulated, while 42 were up-regulated. The identity of most of these differentially expressed proteins was determined by mass spectrometry (liquid chromatography-tandem mass spectrometry or matrix-assisted laser desorption/ionization-time of flight), and the identified proteins were grouped based on cellular function and participation in biochemical and signaling pathways.

Proteomics based approaches, which examine the expressed proteins of a tissue or cell type, complement the genome initiatives and are increasingly used to address biomedical questions. Proteins are the main functional output, and post-translational modifications such as phosphorylation are very important in determining protein function. To address this question, we developed a method for specific immunoprecipitation using anti-phosphotyrosine antibodies. This method is directly compatible with two-dimensional gel electrophoresis (2-DE). In this report data are presented on B-lymphoblasts from a patient suffering of Scott syndrome. Scott syndrome is an orphan inherited hemorrhagic disorder due to a lack of exposure of procoagulant phosphatidylserine at the exoplasmic leaflet of plasma membrane of blood cells. We hypothesized that a consequence of the mutation is to alter phosphorylation of proteins involved in signal transduction leading to breakdown in cellular signaling pathways mediating phosphatidylserine exposure. An immunoprecipitation method combined with 2-DE was applied to search for modifications in the expression of phosphorylated polypeptides related to Scott syndrome phenotype. We report here the construction of a B-lymphoblast subproteomic map comprising of polypeptides observed after immunoprecipitation using antibodies to phosphotyrosine. The polypeptides were identified either by mass fingerprinting, by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and/or by matching with various lymphoid cell 2-DE maps included in the Laboratoire de Biochimie des Protéines et Protéomique 2-DE database. A differential analysis was further performed to explore several hundred proteins in Scott B-lymphoblasts in comparison with control B-lymphoblasts. Then, image analysis allowed detection of variations between control and Scott syndrome phenotype lymphoblasts. Five spots were specifically found on 2-DE from Scott syndrome phenotype lymphoblasts, and four only appeared on 2-DE from control cells. Protein identification was achieved using a combination of mass fingerprinting and peptide identification using LC-MS/MS.

Advances in genomics have led to the accumulation of an unprecedented amount of data, giving rise to a new field in biochemistry, proteomics. We used a combination of two dimensional gel electrophoresis, analysis and annotation using third-generation software, and mass spectrometry to establish the proteome maps of lymphoblastoid B-cells, a prerequisite for analysis of drug effects and lymphocyte cell diseases. About 1200 protein spots were detected and characterised in terms of their isoelectric point, molecular mass and expression. The present status of proteomic technologies, as well as a description of the usefulness of human hematopoietic cells proteomic database are discussed.

BACKGROUND: 5-AzaCytidine (AzaC) is a DNA demethylating drugs that has been shown to inhibit cell growth and to induce apoptosis in certain cancer cells. Induced expression of the galectin1 (Gal1) protein, a galactoside-binding protein distributed widely in immune cells, has been described in cultured hepatoma-derived cells treated with AzaC and this event may have a role in the effect of the drug. According to this hypothesis, we investigated the effect of AzaC and Gal1 on human lymphoid B cells phenotype. METHODS: The effect of AzaC and Gal1 on cell growth and phenotype was determined on the Burkitt lymphoma cell line BL36. An immunocytochemical analysis for detection of Gal1 protein expression was performed in AzaC-treated cells. To investigate the direct effects of Gal1, recombinant Gal1 was added to cells. RESULTS: Treatment of lymphoid B cells with AzaC results in: i) a decrease in cell growth with an arrest of the cell cycle at G0/G1 phase, ii) phenotypic changes consistent with a differentiated phenotype, and iii) the expression of p16, a tumor-suppressor gene whose expression was dependent of its promoter demethylation, and of Gal1. A targeting of Gal 1 to the plasma membrane follows its cytosolic expression. To determine which of the effects of AzaC might be secondary to the induction of Gal1, recombinant Gal1 was added to BL36 cells. Treated cells displayed growth inhibition and phenotypic changes consistent with a commitment toward differentiation. CONCLUSIONS: Altered cell growth and expression of the cell surface plasma cell antigen, CD138 are detectable in BL36 cells treated by AzaC as well as by Gal1. It seems that AzaC-induced Gal1 expression and consequent binding of Gal1 on its cell membrane receptor may be, in part, involved in AzaC-induced plasmacytic differentiation.

As two-dimensional (2-D) electrophoresis allows the separation of several hundred proteins in a single gel, this technique has become an important tool for proteome studies and for investigating the cellular physiology. In order to take advantage of information provided by the comparison of proteome pictures, the mass spectrometry technique is the way chosen for a rapid and an accurate identification of proteins of interest. Unfortunately, in the case of industrial yeasts, due to the high level of complexity of their genome, the whole DNA sequence is not yet available and all encoded protein sequences are still unknown. Nevertheless, this study presents here 30 lager brewing yeast proteins newly identified with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), tandem mass spectrometry (MS/MS) and database searching against the protein sequences of Saccharomyces cerevisiae. The identified proteins of the industrial strain correspond to proteins which do not comigrate with known proteins of S. cerevisiae separated on 2-D gels. This study presents an application of the MS technique for the identification of industrial yeast proteins which are only homologous to the corresponding S. cerevisiae proteins.

Hypermethylation is an important mechanism for repression of tumor gene suppressor in cancer. The drug 5'-azacytidine (AZC) has been used as demethylating agent to induce the expression of previously silencing genes. In the present work, we attempted to determine, using proteomics, the changes in protein expression profiles following a treatment of an Epstein Barr virus (EBV)-negative Burkitt lymphoma (BL) cell line DG 75. The effects of the treatment in terms of cell viability and growth were first examined. The following observations were made: AZC treatment led to (i) a decrease in cell growth with an arrest of the cell at G0/G1 phase of the cell cycle, (ii) the expression of p16, a tumor-suppressor gene whose expression was dependent on its promoter demethylation. Proteomic study evidenced that AZC treatment affected protein expression in two different ways. Twenty-one polypeptides were down-expressed, while 14 showed an increased expression. Some of the upregulated proteins appeared related to the energy metabolism, to organization of cytoskeletal structures, and to cell viability and protein synthesis. We also established a reference map for proteins in DG 75 cell line, comprising 74 different polypeptides corresponding to 67 proteins. This map will be accessible via Internet as a resource for proteome analyses of B-cells. Taken together, the results presented here highlight new insights into lymphoma cell gene regulations following a treatment of lymphoma cells with AZC and illustrate a use of proteomics to evidence the direct and indirect effects of a drug and the pathways it possibly regulates.

The BPP (protein biochemistry and proteomics) two-dimensional electrophoresis (2-DE) database (http://www-smbh.univ-paris13.fr/lbtp/Biochemistry/Biochimie/bque.htm) was established in 1998. The current release contains 11 reference maps from human hematopoietic and lymphoid cell line samples. These reference maps have now 255 identified spots, corresponding to 84 protein entries. The World Wide Web (WWW) presentation is designed to allow public access to the available 2-DE data together with logical connections to databases providing complementary information.

Lymphoblastoid cell lines correspond to in vitro EBV-immortalized lymphocyte B-cells. These cells display a suitable model for experiments dealing with changes in protein expression occurring upon B-cell differentiation, after drug treatment, or after inhibition of some transcription factors. For all these reasons we have undertaken an effort aimed at developing a hematopoietic cell line protein two-dimensional electrophoresis (2-DE) database, containing B-lymphoblastoid 2-DE maps. In this work, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting analysis was adopted for protein identification. The peptide mass fingerprinting identification and the sequence coverage obtained on colloidal Coomassie blue (CBB) stained gel was close to that obtained using zinc-imidazole staining. Everything considered, CBB being more comfortable for subsequent spot manipulations, CBB staining was chosen for identification of a larger number of polypeptides. The results suggest that reticulation of the gel can interfere preventing the uptake of the enzyme during the in-gel digestion step. Consequently, low molecular mass proteins appear more difficult to identify by mass fingerprinting. Finally, the information provided in this study allows the construction of a new annoted reference map of human lymphoblastoid cell proteins. Among the identified proteins 60% were not yet positioned on 2-DE maps in three of the most important well-documented databases. The annoted map will be accessible via Internet on the LBPP server at URL:http:// www-smbh.univ-paris13.fr/lbtp/index.htm.

Modern lager brewing yeasts used in beer production are hybrid strains consisting of at least two different genomes. To obtain information on the identity of the parental strains that gave rise to industrial lager yeasts, we used two-dimensional (2-D) gel electrophoresis and analysed the proteomes of different Saccharomyces species isolated from breweries. We found that the proteome of lager brewing yeasts and of the type strains of S. carlsbergensis, S. monacensis and S. pastorianus can be interpreted as the superimposition of two elementary patterns. One originates from proteins encoded by a S. cerevisiae-like genome. The other corresponds to a divergent Saccharomyces species whose best representative is a particular S. pastorianus strain, NRRL Y-1551. A map of industrial lager brewing yeasts has been established, with the individual origin of proteins and with identification of protein spots by comparison to known S. cerevisiae proteins. This 2-D map can be accessed on the Lager Brewing Yeast Protein Map server through the World Wide Web. This study provides the first example of the use of proteome analysis for investigating taxonomic relationships between divergent yeast species., (Copyright 2000 John Wiley & Sons, Ltd.)

It has been well established that Galectin-1 (GAL1), a beta-galactoside-binding protein, regulates the viability of lymphoid cells. However, the signaling pathway governed by the binding of GAL1 to the cell membrane is not understood. As a first step towards the elucidation of GAL1-initiated signaling events leading to a reduced viability of Burkitt lymphoma B cells, we tried to characterize the initial events induced by the binding of GAL1 to its receptor. This characterization was performed in BL36 cells, a Burkitt lymphoma cell line sensitive to GAL1. The results were as follows: (1) when solubilized cell membrane lysates were affinity bound to immobilized GAL1 and eluted by competition, the tyrosine phosphatase glyco-protein CD45 was found in the eluate, highlighting the role of CD45 as a receptor of GAL1; (2) the phosphatase activity of cell membranes diminished after incubation with GAL1; (3) immunoprecipitation experiments demonstrated that the phosphotyrosine kinase Lyn was dysregulated in cells that have been cultured in medium containing 700 nM GAL1, and (4) that the ratio between two isoforms of Lyn was modified during the treatment with GAL1. The regulation of Lyn therefore seems to be a key event in the action of GAL1.

The Epstein-Barr virus (EBV)-encoded latent membrane protein-1 induces NF-kappaB activity by targeting IkappaBalpha. To understand the role of NF-kappaB activation in EBV-related oncogenesis, we have subcloned mutated IkappaBalpha(32/36A) cDNA into a pHEBo vector containing doxycycline regulatory sequences and stably transfected this construct into a lymphoblastoid cell line. Two tightly regulated clones were obtained in which IkappaBalpha(32/36A) was inducible in a doxycycline dose-dependent manner. Levels of inducible IkappaBalpha(32/36A) peaked at day 2. Inhibition of NF-kappaB activity was closely correlated with levels of inducible IkappaBalpha(32/36A). Levels of 3 well-known NF-kappaB-dependent genes, CD54, p105, and endogenous IkappaBalpha, were decreased when IkappaBalpha(32/36A) was induced, and the growth of IkappaBalpha(32/36A)-induced EBV-infected cells was slightly reduced. Loss of NF-kappaB activity was associated with decreased Bcl-2 protein levels. Finally, the induction of apoptosis was strongly increased in IkappaBalpha(32/36A)-overexpressing cells. Together these results show that it is possible to control IkappaBalpha(32/36A) levels, ie, NF-kappaB activity, in EBV-infected B-lymphocytes using a doxycycline-inducible vector. Moreover, our results indicate that NF-kappaB can protect EBV-infected cells from apoptosis by Bcl-2. Finally, our results suggest that a cellular model with doxycycline-inducible IkappaBalpha(32/36A) may be useful in the identification of genuine NF-kappaB target genes in EBV-infected B cells. (Blood. 2000;95:2068-2075)

Galectin-1 (GAL1) is a beta-galactoside-binding protein that has been implicated in the regulation of viability of lymphoid cells. However, the signaling pathway governed by the binding of GAL1 to the cell membrane is not understood yet. As a first step toward the elucidation of GAL1-initiated signaling events, electrophoresis techniques such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional electrophoresis (2-DE) were used together with precipitation techniques. This allowed us to identify the membrane receptor of GAL1, and to characterize the signal resulting from the binding of GAL1 to this receptor. Our results demonstrate that the tyrosine phosphatase CD45 is the receptor for GAL1, and that the src-type tyrosine kinase Lyn is a target for the effects of GAL1/CD45 interactions in B-cells. Furthermore, these results show the usefulness of combined precipitation and electrophoresis techniques to analyze phosphotyrosine-dependent mechanisms during the study of cell functions.

Strategies are needed for conclusive interpretation of two-dimensional gel electrophoresis (2-D PAGE) maps in order to identify pertinent differences in protein expression during regulation of the transcription of discrete sets of genes. The model used in this study was a human lymphoblastoid cell line in which a functional repression of the transcription factors NFkappaB was obtained by induction of overexpression of IkappaBalpha, a physiological inhibitor of NFkappaB. The analytical methodology used relies on the comparison of two sets of 2-D PAGE maps for detecting differences in protein expression between samples overexpressing or not overexpressing IkappaBalpha. The analysis was based on a combination of an automatic computerized analysis, constituting an actual aid for deciding, and of an interactive visual validation, corresponding to the interpretation of computer propositions. This strategy is proposed as a rapid way to detect potential variations in protein expression applicable to any biological model. In this study, correspondence analysis data made it possible to discrimate between the samples overexpressing or not overexpressing IkappaBalpha, and pointed out some of the potential meaningful spots characterizing the samples in which NFkappaB was active. Then, after visual validation of the computer data, 53 polypeptides were considered to be different in the two classes of gels. Five polypeptides were specifically found in both samples overexpressing IkappaBalpha. The overexpression of IkappaB also induced a lower expression of 11 polypeptides. Finally, 15 polypeptides were only expressed in samples in which IkappaBalpha was not overexpressed and, consequently, in which NFkappaB factors were active. Thus, these polypeptides are candidates for further analysis as putative target gene products of NFkappaB.

Hair cell loss and a non-functional epithelial reorganization appeared in the organ of Corti after acoustic or toxic damage. Moreover, in the drug damaged organ of Corti, transient atypical cells were recently described with characteristics of both immature hair cells and/or non-sensory epithelial cells. The phenotype of these atypical cells has been now investigated by using the galectine 1 (GAL-1) antibody. In the normal organ of Corti, this antibody recognizes all the epithelial cells except the sensory hair cells and their supporting cells. At PD 21, transient atypical cells were not stained by GAL-1 antibody, suggesting that they were originated from hair cells or their supporting cells. Later, the organ of Corti was substituted by an epithelial scare, GAL-1 stained. This study also emphasizes the particular resistance of the cochlear apex to degeneration after antibiotic intoxication., (Copyright 1999 Elsevier Science B.V.)

The potential of bioaffinity chromatography as a tool for study of biological recognition mechanisms is gaining increasing recognition. Biochromatographic methods allow the separation of proteins according to both the structure of their polypeptidic chain and their post-translational modifications. Among the various post-translational modifications which proteins undergo, glycosylation has conducted to the development of original methods (glycotechnologies). This review discusses the applications of glycotechnologies in bioaffinity chromatography, and particularly the use of biochromatography to elucidate mechanisms involved in glycobiology.

Antisera raised against galectin-1 exhibit crossreactivities with other galectins or related molecules. In order to overcome this problem, a monoclonal antibody to human brain galectin-1 was obtained by selecting clones without reactivity toward galectin-3. This mAb specifically bound galectin-1 of various animal origins but neither galectin-2 nor galectin-3. Western-blotting analysis of soluble human brain extracts after 2D gel electrophoresis revealed only the two most acidic isoforms of galectin-1. The ability of this mAb to bind galectin-1/asialofetuin complexes indicates that its epitope is not localized in the carbohydrate recognition domain of galectin-1. This particularity induces with efficiency its monospecificity.

Galectin-1, a polypeptidic factor that can have major effects on cell growth and apoptosis, was overexpressed in E. coli. This protein was purified to homogeneity by affinity chromatography on lactose coupled to divinylsulfone-activated agarose. The recombinant galectin-1 (rGAL1) was compared with the homologous protein purified from human brain tissue using two-dimensional electrophoresis on immobilized pH gradient (IPG-DALT). rGAL1 had a major isoelectric point of 5.4 (major pI of tissular galectin-1, 5.1) and its subunit molecular mass was 14500. Addition of rGAL1 to Jurkat T-lymphoblastoid cells induced cell death in a concentration-dependent manner.

Galectin 1 (GAL1) is a beta-galactoside-binding lectin involved in cell cycle progression. GAL1 overexpression is associated with neoplastic transformation and loss of differentiation. The gene encoding for human GAL1 resides on chromosome 22(q12; q13), and its expression is developmentally regulated. Although devoid of signal peptide GAL1 can be externalized from cells by a mechanism independent of the normal secretory process. We report here on a study of the effects of erythroid differentiation of the human leukemia cell line K562 on GAL1 protein expression. In undifferentiated K562 cells, GAL1 was expressed into the cytosol. However, the amount of GAL1 was surprisingly weaker in K562 cells than in other leukemia cell lines such as TF-1 or KG1a. Treatment of K562 cells with erythropoietin (EPO) or with aphidicolin (APH), an inhibitor for DNA polymerase alpha, induced an erythroid phenotype and led to the externalization of cytosolic GAL1 which was then bound to ligands on cell surface in a galactoside-inhibitable fashion. Our results demonstrate that acquisition of an erythroid phenotype is associated with an externalization of GAL1. The autocrine binding of GAL1 to cell surface ligands of non adherent cells such as K562 suggest that GAL1 is implicated rather in signal transduction than in cell-cell or cell-matrix interaction. Moreover, the reciprocal translocation involving chromosomes 9 and 22 t(9;22) present in K562 cells might explain the weak expression of GAL1 in K562 leukemia cells.

The presence of autoantibodies to human brain galectin-1 was investigated in serum from patients with multiple sclerosis, patients with or without evidence of other neurological disorders, and healthy controls, using an ELISA on purified brain galectin-1. Levels of autoantibodies to galectin-1 were significantly higher in patients than in healthy controls. Comparison of levels of anti-galectin-1 and anti-idiotypic antibodies mimicking human brain galectin-1 (L-IgG) showed that the highest levels of autoantibodies were present in patients with low levels of L-IgG. This finding can be explained by hypothesizing that the concentration of autoantibodies to galectin-1 is possibly associated with impairment of the regulation of the immune system.

Our previous studies have characterized an endogenous lectin from human brain identified as galectin-1. A soluble ligand of galectin-1 was purified from human brain by affinity chromatography and preparative electrophoresis. The purified ligand (termed HBGp82, for human brain galectin-1-binding polypeptide of 82,000 daltons) has an apparent molecular mass of 82 kDa and is glycosylated by N-linked biantennary complex structures. HBGp82 was partially characterized by microsequencing of peptide fragments. Similar peptides were found in a heat shock of protein of 90,000 daltons, hsp90. However, comparison of apparent molecular weights and matrix-assisted laser desorption mass spectrometry clearly showed that HBGp82 differs to some degree from hsp90.

With the systematic sequencing of the yeast genome, yeast biology has entered a new era where novel challenges have to be faced. One challenge is the identification of the function of the several hundred novel genes discovered by genome sequencing. Another is to understand how all yeast genes act in concert to ensure and maintain cell organization. Two-dimensional (2-D) gel electrophoresis is the technique of choice to take up these challenges because it provides the opportunity of obtaining an overall view of genome expression. In prospect of these studies we have undertaken the construction of a yeast 2-D gel protein database that contains information on polypeptides of the yeast protein map. In this paper we report the information presently contained in this database. The reported information includes the identification of 250 protein spots and the characterization of polypeptides corresponding to N-terminal acetylated proteins, mitochondrial proteins, glucose-repressed proteins, heat shock induced proteins and proteins encoded by intron-containing genes. In all, 600 spots are annotated. These data can be accessed on the Yeast Protein Map server through the World Wide Web network.

Vertebrate soluble beta-galactoside-binding lectins form a growing protein family that recently have been named galectins. Seven different galectins have been sequenced and characterized in mammals, and there is compelling evidence for the existence of other members of this lectin family. Three among six galectins are homodimers with (i) an identical subunit of a relative molecular mass of about 14500, and (ii) amino acid sequence homologies giving rise to possible immunochemical cross-reactivities. They are indistinguishable from each other by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), even when followed by immunoblotting. However, their different isoelectric points allow their identification using isoelectric focusing and two-dimensional (2-D) polyacrylamide gel electrophoresis. A strategy was developed to identify these galectins in crude extracts from cells and tissues, based on the two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt) analysis of the specific spots of purified galectins and of the spots of crude extracts, after silver staining. In addition, 2-D immunoblotting using anti-galectin 1 (Gal-1) and anti carbohydrate-binding protein 15 (CPB15) antibodies were performed on brain and leukemia cells (HL60) allowing an identification of related polypeptides. Our results indicate that the use of IPG-Dalt provides a suitable reproducibility and allows the detection of galectins or other galactoside-binding proteins even at basic pIs.

Histological brain sections were probed with human oligoclonal lectin-like IgGs (L-IgG) purified from normal serum. In intact brain, antigenic determinants for these IgG were restricted to some blood vessel endothelial cells. By contrast, during the inflammatory reaction following a surgical injury, these determinants were detected at the cell surface of different cell types, within and near the lesion site. The cells reacting with L-IgG consisted of endothelial cell, mature astrocytes, activated microglial and ependymal cells.

We have recently identified oligoclonal IgG antibodies that are related to a human brain lectin (HBL14) from serum and cerebrospinal fluid of patients with neurological disorders. They were termed lectin-like IgG (L-IgG) (Joubert-Caron et al., 1994a,b). In this paper, the occurrence of antibodies reactive both towards HBL14 and L-IgG was investigated. Binding of antibodies to HBL14 was demonstrated by solid-phase ELISA and chromatography on immobilized HBL14. Fab fragments of these antibodies were also shown to bind to HBL14. The specificity of the antibodies towards HBL14 was studied using a panel of different antigens. Our data show that individual sera from healthy people as well as a pool of immunoglobulins from 80 blood donors contain an IgG autoreactivity to HBL14, while no IgM autoreactivity was detected. Anti-HBL14 antibodies from sera were purified using affinity chromatography on immobilized HBL14. Affinity chromatography further allowed us to demonstrate that the binding of anti-HB14 antibodies was mediated through their Fab fragments. A higher amount of anti-HBL14 antibodies was purified using a L-IgG-depleted fraction of sera. The binding of anti-HBL14 antibodies to L-IgG was confirmed by ELISA. Finally, anti-HBL14 antibodies were found to be polyreactive. These results indicate the occurrence of a novel class of natural antibodies reactive towards a human brain lectin and suggest that these antibodies may participate in immunoregulatory mechanisms probably though idiotypic/anti-idiotypic interaction.

A method for the purification and biotinylation of Fab fragments, using thiophilic adsorption (T-gel), is described. The T-gel was used to purify an IgG fraction directly in the buffer suitable for biotinylation, and to adsorb intact IgGs and papain after enzymatic digestion. For the final step, Fc fragments were removed with a protein A column.