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Proteomic Study (proteomic + study)
Selected AbstractsProteomic study of DBA/2J mice retina: Down-regulation of Integrin ,7 correlated with retinal ganglion cell deathPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 21 2009Takashi Kanamoto Abstract To identify and determine the function of the proteins associated with the death of retinal ganglion cells (RGCs) in DBA/2J mice, an animal model of glaucoma, retinas of DBA/2J mice, were analyzed by proteomics at 5-, 7-, and 11-months-of-age. The proteins showing significant alterations were selected for identification by MS and 18 proteins were differentially expressed and the identified proteins included cell membrane receptors and proteins associated with intracellular signaling pathways. Among of identified proteins, the expression of Integrin ,7 at 7-months-of-age was decreased by about 89% of that at 5-months-of-age. Integrin ,7 was expressed in the RGCs. The effect of glutamate toxicity on the expression pattern of Integrin ,7 in a RGC line was also investigated and the glutamate-induced death of RGC was inhibited by the RNA knockdown of Integrin ,7. Our data showed also that the expression of 18 proteins in the DBA/2J was significantly altered in DBA2 mice and down-regulation of Integrin ,7 may have a protective effect on glutamate-induced death of RGCs. [source] Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dyePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2005Kazuyasu Fujii Abstract To identify the proteomic alterations associated with carcinogenesis of hepatocellular carcinoma (HCC), we compared the protein expression profiles of nine HCC cell lines with those of primary cultured hepatocytes established from five individuals. A differential proteomic study was performed by two-dimensional difference gel electrophoresis, in which protein samples are labeled with different fluorescent dyes and separated according to their isoelectric point and molecular weight. To label the protein samples, we used a newly developed and highly sensitive fluorescent dye, which reacts with all reduced cysteine residues of proteins. Principal component analysis based on the intensity of 1238,protein spots indicated that the HCC cells and the normal hepatocytes had distinct proteomic profiles. The Wilcoxon test was used to determine the protein spots whose intensity was differentially regulated in the HCC cells compared with the normal hepatocytes, and mass spectrometric analysis was used to identify the proteins corresponding to the spots. The proteins identified are involved in cell cycle regulation, binding to a tumor-suppressor gene product, fatty acid binding, and regulation of translation. Western blotting with specific antibodies revealed the overexpression of PCNA, EB1 and E-FABP in HCC tissues compared with noncancerous tissues. Aberrant regulation of EB1 and E-FABP has not previously been implicated in the development of HCC. [source] An efficient protein preparation for proteomic analysis of developing cotton fibers by 2-DEELECTROPHORESIS, Issue 22 2006Yuan Yao Abstract Preparation of high-quality proteins from cotton fiber tissues is difficult due to high endogenous levels of polysaccharides, polyphenols, and other interfering compounds. To establish a routine procedure for the application of proteomic analysis to cotton fiber tissues, a new protocol for protein extraction was developed by optimizing a phenol extraction method combined with methanol/ammonium acetate precipitation. The protein extraction for 2-DE was remarkably improved by the combination of chemically and physically modified processes including polyvinylpolypyrrolidone (PVPP) addition, acetone cleaning, and SDS replacement. The protocol gave a higher protein yield and vastly greater resolution and spot intensity. The efficiency of this protocol and its feasibility in fiber proteomic study were demonstrated by comparison of the cotton fiber proteomes at two growth stages. Furthermore, ten protein spots changed significantly were identified by MS/tandem MS and their potential relationships to fiber development were discussed. To the best of our knowledge, this is the first time that a protocol for protein extraction from cotton fiber tissues appears to give satisfactory and reproductive 2-D protein profiles. The protocol is expected to accelerate the process of the proteomic study of cotton fibers and also to be applicable to other recalcitrant plant tissues. [source] Improved 2-DE of microorganisms after acidic extractionELECTROPHORESIS, Issue 8 2006Ben R. Herbert Professor Abstract 2-DE separations of protein extracts sometimes have problems with poor resolution and streaking. This problem is particularly apparent with microorganisms, most notably those with a large cell wall. Here we describe a novel, rapid protocol for the extraction of microorganisms in acidic conditions, leading to increased resolution and 2-D gel quality. The efficiency of the protocol is demonstrated with extracts of bacteria, Escherichia coli and Bacillus subtilis; fungus, Trichoderma harzianum and yeast, Saccharomyces cerevisiae. We also demonstrate using a membrane centrifugal filtration, that large acidic molecules in excess of 100,kDa, probably including cell wall material, are responsible for the separation difficulties. A range of acidic extraction conditions were investigated, and it was found that optimal extraction is achieved using an extraction solution acidified to pH,3 by 80,mM citric acid. These findings have significant implications for the proteomic study of many medically, agriculturally and environmentally significant microorganisms, as the cell walls of these organisms are often considerably more complex than many commonly studied laboratory strains. [source] A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth modeFEMS MICROBIOLOGY LETTERS, Issue 1 2002Frédéric Trémoulet Abstract Escherichia coli 0157:H7 biofilms were studied by a new method of cultivation in order to identify some of the proteins involved in the biofilm phenotype. A proteomic analysis of sessile or planktonic bacteria of the same age was carried out by two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. Comparison of two-dimensional gels showed clear differences between protein patterns of sessile and planktonic cells. Fourteen proteins increased in biofilms, whereas three decreased. From these 17 proteins, 10 were identified by MALDI-TOF-MS and could be classified into four categories according to their function: (1) general metabolism proteins (malate dehydrogenase, thiamine-phosphate pyrophosphorylase), (2) sugar and amino acid transporters (d -ribose-binding periplasmic protein, d -galactose-binding protein, YBEJ), (3) regulator proteins (DNA starvation protein and H-NS) and (4) three proteins with unknown function. The results of this study showed that E. coli O157:H7 modified the expression of several proteins involved in biofilm growth mode. [source] Comparative proteomic analysis of primary mouse liver c-Kit,(CD45/TER119), stem/progenitor cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2007Yu-Fei He Abstract Liver stem/progenitor cells play a key role in liver development and maybe also in liver cancer development. In our previous study a population of c-Kit,(CD45/TER119), liver stem/progenitor cells in mouse fetal liver, was successfully sorted with large amount (106,107) by using immuno-magnetic microbeads. In this study, the sorted liver stem/progenitor cells were used for proteomic study. Proteins of the sorted liver stem/progenitor cells and unsorted fetal liver cells were investigated using two-dimensional electrophoresis. A two-dimensional proteome map of liver stem/progenitor cells was obtained for the first time. Proteins that exhibited significantly upregulation in liver stem/progenitor cells were identified by peptide mass fingerprinting and peptide sequencing. Nineteen protein spots corresponding to 12 different proteins were identified as showing significant upregulation in liver stem/progenitor cells and seem to play important roles in such cells in cell metabolism, cell cycle regulation, and stress. An interesting finding is that most of the upregulated proteins were overexpressed in various cancers (11 of 12, including 6 in human hepatocellular carcinoma (HCC)) and involved in cancer development as reported in previous studies. Some of the identified proteins were validated by real-time PCR, Western blotting, and immunostaining. Taken together, the data presented provide a significant new protein-level insight into the biology of liver stem/progenitor cells, a key population of cells that might be also involved in liver cancer development. J. Cell. Biochem. 102: 936,946, 2007. © 2007 Wiley-Liss, Inc. [source] Anti-cancer properties of anthraquinones from rhubarbMEDICINAL RESEARCH REVIEWS, Issue 5 2007Qing Huang Abstract Rhubarb has been used as a traditional Chinese medicine since ancient times and today it is still present in various herbal preparations. In this review the toxicological and anti-neoplastic potentials of the main anthraquinones from Rhubarb, Rheum palmatum, will be highlighted. It is interesting to note that although the chemical structures of various anthraquinones in this plant are similar, their bioactivities are rather different. The most abundant anthraquinone of rhubarb, emodin, was capable of inhibiting cellular proliferation, induction of apoptosis, and prevention of metastasis. These capabilities are reported to act through tyrosine kinases, phosphoinositol 3-kinase (PI3K), protein kinase C (PKC), NF-kappa B (NF-,B), and mitogen-activated protein kinase (MAPK) signaling cascades. Aloe-emodin is another major component in rhubarb found to have anti-tumor properties. Its anti-proliferative property has been demonstrated to be through the p53 and its downstream p21 pathway. Our recent proteomic study also suggests that the molecular targets of these two anthraquinones are different. However, both components were found to be able to potentiate the anti-proliferation of various chemotherapeutic agents. Rhein is the other major rhubarb anthraquinone, although less well studied. This compound could effectively inhibit the uptake of glucose in tumor cells, caused changes in membrane-associated functions and led to cell death. Interestingly, all three major rhubarb anthraquinones were reported to have in vitro phototoxic. This re-evaluation of an old remedy suggests that several bioactive anthraquinones of rhubarb possess promising anti-cancer properties and could have a broad therapeutic potential. © 2006 Wiley Periodicals, Inc. Med Res Rev, 27, No. 5, 609,630, 2007 [source] Differential expression of skeletal muscle proteins in high-fat diet-fed rats in response to capsaicin feedingPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2010Dong Hyun Kim Abstract In this study, the effects of capsaicin on expression of skeletal muscle proteins in Sprague,Dawley rats fed with a high-fat diet (HFD) were investigated. Rats were fed a HFD with or without capsaicin treatment for 8,wk. After HFD feeding, capsaicin-treated rats weighed an average of 8% less than those of the HFD control group. Gastrocnemius muscle tissue from lean and obese rats with or without capsaicin treatment was arrayed using 2-DE for detection of HFD-associated markers. Proteomic analysis using 2-DE demonstrated that 36 spots from a total of approximately 600 matched spots showed significantly different expression; 27 spots were identified as gastrocnemius muscle proteins that had been altered in response to capsaicin feeding, and 6 spots could not be identified by mass fingerprinting. Expression of various muscle proteins was determined by immunoblot analysis for the determination of molecular mechanisms, whereby capsaicin caused inhibition of adipogenesis. Immunoblot analysis revealed increased uncoupling protein 3 (UCP3) protein expression in HFD-fed rats, whereas contents were reduced with capsaicin treatment. Compared with the HFD control group, capsaicin treatment increased phosphorylation of AMP-activated protein kinase (AMPIC) CP3 and acetyl-CoA carboxylase (ACC). To support this result, we also analyzed in vitro differential protein expression in L6 skeletal muscle cells. These data suggest that the AMPK-ACC-malonyl-CoA metabolic signaling pathway is one of the targets of capsaicin action. To the best of our knowledge, this is the first proteomic study to report on analysis of diet-induced alterations of protein expression that are essential for energy expenditure in rat muscle. [source] Comparative proteomics of human embryonic stem cells and embryonal carcinoma cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2010Raghothama Chaerkady Abstract Pluripotent human embryonic stem cells (ESCs) can be differentiated in vitro into a variety of cells which hold promise for transplantation therapy. Human embryonal carcinoma cells (ECCs), stem cells of human teratocarcinomas, are considered a close but malignant counterpart to human ESCs. In this study, a comprehensive quantitative proteomic analysis of ESCs and ECCs was carried out using the iTRAQ method. Using two-dimensional LC and MS/MS analyses, we identified and quantitated ,1800 proteins. Among these are proteins associated with pluripotency and development as well as tight junction signaling and TGF, receptor pathway. Nearly ,200 proteins exhibit more than twofold difference in abundance between ESCs and ECCs. Examples of early developmental markers high in ESCs include ,-galactoside-binding lectin, undifferentiated embryonic cell transcription factor-1, DNA cytosine methyltransferase 3, isoform-B, melanoma antigen family-A4, and interferon-induced transmembrane protein-1. In contrast, CD99-antigen (CD99), growth differentiation factor-3, cellular retinoic acid binding protein-2, and developmental pluripotency associated-4 were among the highly expressed proteins in ECCs. Several proteins that were highly expressed in ECCs such as heat shock 27,kDa protein-1, mitogen-activated protein kinase kinase-1, nuclear factor of , light polypeptide gene enhancer in B-cells inhibitor like-2, and S100 calcium-binding protein-A4 have also been attributed to malignancy in other systems. Importantly, immunocytochemistry was used to validate the proteomic analyses for a subset of the proteins. In summary, this is the first large-scale quantitative proteomic study of human ESCs and ECCs, which provides critical information about the regulators of these two closely related, but developmentally distinct, stem cells. [source] A further proteomic study on the effect of iron in the human pathogen Trichomonas vaginalisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 12 2007Jose Batista De Jesus Dr. Abstract Iron is an essential element to support the growth and survival of Trichomonas vaginalis. It plays a critical role in the host,parasite interaction, and modulates the expression of virulence factors in this protozoan. In this work, parasites grown in iron-rich and iron-depleted media were analyzed by (i) light and scanning electron microscopy and (ii) 2-DE and MS. Withdrawal of iron from the culture medium resulted in dramatic changes in both the morphology and in the proteome pattern of T. vaginalis. Trophozoites underwent transformation from ellipsoid or amoeboid forms to rounded cells, whose flagella and axostyle were internalized. Forty-five proteins differentially expressed in parasites cultivated in the absence of iron were identified. In iron-depleted parasites, enzymes involved in energetic metabolism, proteolysis and hydrogenosomal iron-sulfur (Fe-S) proteins were down-regulated or even suppressed. Among up-regulated proteins, six isoforms of actin were detected. In addition, phosphoenolpyruvate carboxykinase, putative lactate dehydrogenase, and putative adenosine triphosphatase were also up-regulated or were exclusively observed in gels related to iron-depleted parasites. Our data demonstrate that iron has a pivotal role in the regulation of the morphological transformation of T. vaginalis and modulates the expression of both Fe-S and non-Fe-S proteins in the parasite. [source] Hypophosphorylation of the architectural chromatin protein DEK in death-receptor-induced apoptosis revealed by the isotope coded protein label proteomic platformPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 21 2006Anja Tabbert Abstract During apoptosis nuclear morphology changes dramatically due to alterations of chromatin architecture and cleavage of structural nuclear proteins. To characterize early events in apoptotic nuclear dismantling we have performed a proteomic study of apoptotic nuclei. To this end we have combined a cell-free apoptosis system with a proteomic platform based on the differential isotopic labeling of primary amines with N -nicotinoyloxy-succinimide. We exploited the ability of this system to produce nuclei arrested at different stages of apoptosis to analyze proteome alterations which occur prior to or at a low level of caspase activation. We show that the majority of proteins affected at the onset of apoptosis are involved in chromatin architecture and RNA metabolism. Among them is DEK, an architectural chromatin protein which is linked to autoimmune disorders. The proteomic analysis points to the occurrence of multiple PTMs in early apoptotic nuclei. This is confirmed by showing that the level of phosphorylation of DEK is decreased following apoptosis induction. These results suggest the unexpected existence of an early crosstalk between cytoplasm and nucleus during apoptosis. They further establish a previously unrecognized link between DEK and cell death, which will prove useful in the elucidation of the physiological function of this protein. [source] Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2005Arnaud Bruneel Dr. Abstract We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com. [source] Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2005Hong-Lin Chan Abstract Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N -hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response. [source] Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dyePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2005Kazuyasu Fujii Abstract To identify the proteomic alterations associated with carcinogenesis of hepatocellular carcinoma (HCC), we compared the protein expression profiles of nine HCC cell lines with those of primary cultured hepatocytes established from five individuals. A differential proteomic study was performed by two-dimensional difference gel electrophoresis, in which protein samples are labeled with different fluorescent dyes and separated according to their isoelectric point and molecular weight. To label the protein samples, we used a newly developed and highly sensitive fluorescent dye, which reacts with all reduced cysteine residues of proteins. Principal component analysis based on the intensity of 1238,protein spots indicated that the HCC cells and the normal hepatocytes had distinct proteomic profiles. The Wilcoxon test was used to determine the protein spots whose intensity was differentially regulated in the HCC cells compared with the normal hepatocytes, and mass spectrometric analysis was used to identify the proteins corresponding to the spots. The proteins identified are involved in cell cycle regulation, binding to a tumor-suppressor gene product, fatty acid binding, and regulation of translation. Western blotting with specific antibodies revealed the overexpression of PCNA, EB1 and E-FABP in HCC tissues compared with noncancerous tissues. Aberrant regulation of EB1 and E-FABP has not previously been implicated in the development of HCC. [source] Carbon starvation survival of Listeria monocytogenes in planktonic state and in biofilm: A proteomic studyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2003Emmanuelle Helloin Abstract The proteomes of Listeria monocytogenes expressed in suspension and biofilm state, in the presence and absence of a caron source, were analysed by two-dimensional electrophoresis with the help of computer software. The up-regulated proteins in each case were identified by peptide sequencing using electrospray ionisation tandem mass spectrometry and a database search against the Listeria genome was performed. Relevant functions could be attributed to a number of the induced proteins which contibute to the understanding of the mechanisms of starvation survival of L. monocytogenes in planktonic state and in biofilm. [source] Serum biomarkers of hepatitis B virus infected liver inflammation: A proteomic studyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2003Qing-Yu He Abstract Hepatitis B virus (HBV), a serious infectious and widespread human pathogen, represents a major health problem worldwide. Chronic HBV infection has a very high risk of evolving into hepatocellular carcinoma. Although considerable progress was made during the recent past, the pathogenesis of HBV infection is still elusive and a definite diagnosis of HBV infected liver information still relies on biopsy histological test. In this report, we used proteomics technology to globally examine HBV infected serum samples aiming at searching for disease-associated proteins that can be used as serological biomarkers for diagnosis and/or target proteins for pathogenetic study. By comparing with normal and HBV negative serum samples, we found that at least seven proteins were significantly changed in HBV infected sera. These greatly altered proteins were identified to be haptoglobin , and ,2 chain, apolipoprotein A-I and A-IV, ,1-antitrypsin, transthyretin and DNA topoisomerase II,. The alteration of these proteins is displayed not only in quantity but also in patterns (or specificity), which can be correlated with necroinflammatory scores. In particular, apolipoprotein A-I presents heterogeneous change in expression level with different isoforms and ,1-antitrypsin produces evidently different fragments implying diverse cleavage pathways. These unique phenomena appear specific to HBV infection. A combination simultaneously considering the quantities and isoforms of these proteins could be a useful serum biomarker (or index) for HBV diagnosis and therapy. [source] Modulation of gene expression by extracellular pH variations in human fibroblasts: A transcriptomic and proteomic studyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2003Maja A. Bumke Abstract Homeostasis of the intracellular ionic concentration, in particular that of hydrogen ions, is pivotal to the maintenance of cell function and viability. Nonetheless, pH fluctuations in both the intracellular and the extracellular compartments can occurr during development, in physiological processes and in disease. The influence of pH variations on gene expression has been studied in different model systems, but only for a limited number of genes. We have performed a broad range analysis of the patterns of gene expression in normal human dermal fibroblasts at two different pH values (in the presence and in the absence of serum), with the aim of getting a deeper insight into the regulation of the transcriptional program as a response to a pH change. Using the Affymetrix gene chip system, we found that the expression of 2068 genes (out of 12,565) was modulated by more than two-fold at 24, 48 or 72 h after the shift of the culture medium pH to a more acidic value, stanniocalcin 1 being a remarkable example of a strongly up-regulated gene. Genes displaying a modulated pattern of expression included, among others, cell cycle regulators (consistent with the observation that acidic pH abolishes the growth of fibroblasts in culture) and relevant extracellular matrix (ECM) components. Extracellular matrix protein 2, a protein with a restricted pattern of expression in adult human tissues, was found to be remarkably overexpressed as a consequence of serum starvation. Since ECM components, whose expression is controlled by pH, have been used as targets for biomolecular intervention, we have complemented the Affymetrix analysis with a two-dimensional polyacrylamide gel electrophoresis analysis of proteins which are differentially secreted by fibroblasts at acidic or basic pH. Mass spectrometric analysis of more than 650 protein spots allowed the identification of 170 protein isoforms or fragments, belonging to 40 different proteins. Some proteins were only expressed at basic pH (including, for instance, tetranectin), while others (e.g., agrin) were only detectable at acidic pH. Some of the identified proteins may represent promising candidate targets for biomedical applications, e.g., for antibody-mediated vascular targeting strategies. [source] Consistency of a two clinical site sample collection: A proteomics studyPROTEOMICS - CLINICAL APPLICATIONS, Issue 8-9 2010Cedric Wiesner Abstract Purpose: We investigated the ability to perform a clinical proteomic study using samples collected at different times from two independent clinical sites. Experimental Design: Label-free 2-D-LC-MS proteomic analysis was used to differentially quantify tens of thousands of peptides from human plasma. We have asked whether samples collected from two sites, when analyzed by this type of peptide profiling, reproducibly contain detectable peptide markers that are differentially expressed in the plasma of disease (advanced renal cancer) patients relative to healthy normals. Results: We have demonstrated that plasma proteins enriched in disease patients are indeed detected reproducibly in both clinical collections. Regression analysis, unsupervised hierarchical clustering and PCA detected no systematic bias in the data related to site of sample collection and processing. Using a genetic algorithm, support vector machine classification method, we were able to correctly classify disease samples at 88% sensitivity and 94% specificity using the second site as an independent validation set. Conclusions and clinical relevance: We conclude that multiple site collection, when analyzed by label-free 2-D-LC-MS, generates data that are sufficiently reproducible to guide reliable biomarker discovery. [source] The Ca2+ -dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation in ArabidopsisTHE PLANT JOURNAL, Issue 3 2010Norbert Mehlmer Summary Plants use different signalling pathways to respond to external stimuli. Intracellular signalling via calcium-dependent protein kinases (CDPKs) or mitogen-activated protein kinases (MAPKs) present two major pathways that are widely used to react to a changing environment. Both CDPK and MAPK pathways are known to be involved in the signalling of abiotic and biotic stresses in animal, yeast and plant cells. Here, we show the essential function of the CDPK CPK3 (At4g23650) for salt stress acclimation in Arabidopsis thaliana, and test crosstalk between CPK3 and the major salt-stress activated MAPKs MPK4 and MPK6 in the salt stress response. CPK3 kinase activity was induced by salt and other stresses after transient overexpression in Arabidopsis protoplasts, but endogenous CPK3 appeared to be constitutively active in roots and leaves in a strictly Ca2+ -dependent manner. cpk3 mutants show a salt-sensitive phenotype comparable with mutants in MAPK pathways. In contrast to animal cells, where crosstalk between Ca2+ and MAPK signalling is well established, CPK3 seems to act independently of those pathways. Salt-induced transcriptional induction of known salt stress-regulated and MAPK-dependent marker genes was not altered, whereas post-translational protein phosphorylation patterns from roots of wild type and cpk3 plants revealed clear differences. A significant portion of CPK3 was found to be associated with the plasma membrane and the vacuole, both depending on its N -terminal myristoylation. An initial proteomic study led to the identification of 28 potential CPK3 targets, predominantly membrane-associated proteins. [source] Gene expression in Large White or Duroc-sired female and castrated male pigs and relationships with pork qualityANIMAL GENETICS, Issue 6 2009A. Kwasiborski Summary This study assessed expression of 12 genes in 24 pig longissimus samples earlier subjected to a proteomic study by our group. Genes were selected on the basis of the earlier proteomic results. Pigs differed in rearing environment (indoors or outdoors), sire breed (Duroc or Large White) and gender (female or castrated male). At slaughter they experienced different stress conditions. The proportion of gene expression changes influenced by treatment factors was consistent with the proportion of protein changes in an earlier proteomic analysis of the same pigs. Expression levels of genes were often correlated. Gene expression was generally not correlated with the levels of the corresponding protein. Finally, most meat quality traits were correlated with the expression of at least one of the studied genes. The most meaningful of these was the association of a slower pH decline with lower levels of HSP72 expression and higher levels of HSP72 protein. ANXA2 and cMDH expression were also associated with various meat quality traits. These relationships may be related to pre-slaughter stress levels and fibre type composition. [source] | |||||||||||||