140 research outputs found

    Comparative two-dimensional gel analysis and microsequencing identifies gelsolin as one of the most prominent downregulated markers of transformed human fibroblast and epithelial cells

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    A systematic comparison of the protein synthesis patterns of cultured normal and transformed human fibroblasts and epithelial cells, using two-dimensional gel protein analysis combined with computerized imaging and data acquisition, identified a 90-kD protein (SSP 5714) as one of the most striking downregulated markers typical of the transformed state. Using the information stored in the comprehensive human cellular protein database, we found this protein strongly expressed in several fetal tissues and one of them, epidermis, served as a source for preparative two-dimensional gel electrophoresis. Partial amino acid sequences were generated from peptides obtained by in situ digestion of the electroblotted protein. These sequences identified the marker protein as gelsolin, a finding that was confirmed by two-dimensional immunoblotting of human MRC-5 fibroblast proteins using specific antibodies and by coelectrophoresis with purified human gelsolin. These results suggest that an important regulatory protein of the microfilament system may play a role in defining the phenotype of transformed human fibroblast and epithelial cells in culture

    hnRNPs H, H′ and F behave differently with respect to posttranslational cleavage and subcellular localization

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    AbstracthnRNPs H, H′ and F belong to a subfamily of the hnRNPs sharing a high degree of sequence identity. Eukaryotic expression and specific C-terminal antibodies were used to demonstrate great variation in the intracellular fate of the proteins. hnRNPs H and H′ become posttranslational cleaved into C-terminal 35 kDa proteins (HC, H′C) and possibly into N-terminal 22 kDa proteins. No detectable cleavage was observed for hnRNP F. hnRNP H/H′ is almost exclusively localized to the nucleus of many cell types while hnRNP F varies from a predominant nuclear localization in some cells to a predominant cytoplasmic localization in other cells. The different fates may reflect differences in functional roles that so far only have included nuclear functions. The presence of significant quantities of hnRNP F in the cytoplasm of many cells indicates that it also may have a functional role here

    Proteomics and nucleotide profiling as tools for biomarker and drug target discovery

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    Proteomics has gone through tremendous development during recent decades [...

    Cloning and expression of a novel human profilin variant, profilin II

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    AbstractWe have isolated a 1.7 kbp cDNA encoding a 140 amino acid protein (15.1 kDa, pI 5.91) with a high sequence similarity (62%) to human profilin (profilin I). We have termed this variant profilin II. Northern blot analysis showed that profilin II is highly expressed in brain, skeletal muscle and kidney and less strongly in heart, placenta, lung and liver. In addition, three different transcript lengths were detected. Only one transcript of profilin I was found. The expression level of this was low in brain and skeletal muscle, medium in heart and high in placenta, lung, liver and kidney

    Identification and characterization of endonuclein binding proteins: evidence of modulatory effects on signal transduction and chaperone activity

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    <p>Abstract</p> <p>Background</p> <p>We have previously identified endonuclein as a cell cycle regulated WD-repeat protein that is up-regulated in adenocarcinoma of the pancreas. Now, we aim to investigate its biomedical functions.</p> <p>Results</p> <p>Using the cDNA encoding human endonuclein, we have expressed and purified the recombinant protein from <it>Escherichia coli </it>using metal affinity chromatography. The recombinant protein was immobilized to a column and by affinity chromatography several interacting proteins were purified from several litres of placenta tissue extract. After chromatography the eluted proteins were further separated by two-dimensional gel electrophoresis and identified by tandem mass spectrometry. The interacting proteins were identified as; Tax interaction protein 1 (TIP-1), Aα fibrinogen transcription factor (P16/SSBP1), immunoglobulin heavy chain binding protein (BiP), human ER-associated DNAJ (HEDJ/DNAJB11), endonuclein interaction protein 8 (EIP-8), and pregnancy specific β-1 glycoproteins (PSGs). Surface plasmon resonance analysis and confocal fluorescence microscopy were used to further characterize the interactions.</p> <p>Conclusions</p> <p>Our results demonstrate that endonuclein interacts with several proteins indicating a broad function including signal transduction and chaperone activity.</p

    Ca(2+ )binding to complement-type repeat domains 5 and 6 from the low-density lipoprotein receptor-related protein

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    BACKGROUND: The binding of ligands to clusters of complement-type repeat (CR)-domains in proteins of the low-density lipoprotein receptor (LDLR) family is dependent on Ca(2+ )ions. One reason for this cation requirement was identified from the crystal structure data for a CR-domain from the prototypic LDLR, which showed the burial of a Ca(2+ )ion as a necessity for correct folding and stabilization of this protein module. Additional Ca(2+ )binding data to other CR-domains from both LDLR and the LDLR-related protein (LRP) have suggested the presence of a conserved Ca(2+ )cage within CR-domains from this family of receptors that function in endocytosis and signalling. RESULTS: We have previously described the binding of several ligands to a fragment comprising the fifth and the sixth CR-domain (CR56) from LRP, as well as qualitatively described the binding of Ca(2+ )ions to this CR-domain pair. In the present study we have applied the rate dialysis method to measure the affinity for Ca(2+), and show that CR56 binds 2 Ca(2+ )ions with an average affinity of K(D )= 10.6 microM, and there is no indication of additional Ca(2+ )binding sites within this receptor fragment. CONCLUSIONS: Both CR-domains of CR56 bind a single Ca(2+ )ion with an affinity of 10.6 microM within the range of affinities demonstrated for several other CR-domains

    Animal Models Used to Simulate Retinal Artery Occlusion:A Comprehensive Review

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    Identification of differentially regulated proteins in a patient with Leber's Congenital Amaurosis – a proteomic study

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    BACKGROUND: To identify the pattern of protein expression in the retina from a patient with Leber's Congenital Amaurosis (LCA) secondary to a mutation in the AIPL1 gene. The retina from one eye of a patient with LCA and 7 control eyes were studied. The tissue was subjected to high resolution two-dimensional gel electrophoresis, image analysis and mass spectrometry, in an effort to identify differentially regulated proteins. RESULTS: In the LCA retina seven protein spots were differentially expressed. Six proteins were significantly up-regulated of which three could be identified as: αA-crystallin, triosephophate isomerase, and an N-terminal fragment of the β-chain of ATP synthase. One protein spot that was down-regulated in the LCA retina was identified as a C-terminal fragment of β-tubulin. CONCLUSION: Retinal tissue in LCA is characterised by an up-regulation of αA-crystallin, triosephosphate isomerase, and ATP synthase (β-chain fragment) and down-regulation of a fragment of β-tubulin. These proteins/protein fragments may play a crucial role for the retinal degeneration processes in LCA and other retinal dystrophies

    Ocular Proteomics with Emphasis on Two-Dimensional Gel Electrophoresis and Mass Spectrometry

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    The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis, gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT, iTRAQ, ICAT and SILAC) are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions
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