Structural and functional protein network analyses predict novel signaling functions for rhodopsin

Proteomic analyses, literature mining, and structural data were combined to generate an extensive signaling network linked to the visual G protein-coupled receptor rhodopsin. Network analysis suggests novel signaling routes to cytoskeleton dynamics and vesicular trafficking

Neuritogenic and Neuroprotective Properties of Peptide Agonists of the Fibroblast Growth Factor Receptor

Fibroblast growth factor receptors (FGFRs) interact with their cognate ligands, FGFs, and with a number of cell adhesion molecules (CAMs), such as the neural cell adhesion molecule (NCAM), mediating a wide range of events during the development and maintenance of the nervous system. Determination of protein structure, in silico modeling and biological studies have recently resulted in the identification of FGFR binding peptides derived from various FGFs and NCAM mimicking the effects of these molecules with regard to their neuritogenic and neuroprotective properties. This review focuses on recently developed functional peptide agonists of FGFR with possible therapeutic potential

Paracrine-mediated neuroprotection and neuritogenesis of axotomised retinal ganglion cells by human dental pulp stem cells:Comparison with human bone marrow and adipose-derived mesenchymal stem cells

We have investigated and compared the neurotrophic activity of human dental pulp stem cells (hDPSC), human bone marrow-derived mesenchymal stem cells (hBMSC) and human adipose-derived stem cells (hAMSC) on axotomised adult rat retinal ganglion cells (RGC) in vitro in order to evaluate their therapeutic potential for neurodegenerative conditions of RGC. Using the transwell system, RGC survival and length/number of neurites were quantified in coculture with stem cells in the presence or absence of specific Fc-receptor inhibitors to determine the role of NGF, BDNF, NT-3, VEGF, GDNF, PDGF-AA and PDGF-AB/BB in stem cell-mediated RGC neuroprotection and neuritogenesis. Conditioned media, collected from cultured hDPSC/hBMSC/hAMSC, were assayed for the secreted growth factors detailed above using ELISA. PCR array determined the hDPSC, hBMSC and hAMSC expression of genes encoding 84 growth factors and receptors. The results demonstrated that hDPSC promoted significantly more neuroprotection and neuritogenesis of axotomised RGC than either hBMSC or hAMSC, an effect that was neutralized after the addition of specific Fc-receptor inhibitors. hDPSC secreted greater levels of various growth factors including NGF, BDNF and VEGF compared with hBMSC/hAMSC. The PCR array confirmed these findings and identified VGF as a novel potentially therapeutic hDPSC-derived neurotrophic factor (NTF) with significant RGC neuroprotective properties after coculture with axotomised RGC. In conclusion, hDPSC promoted significant multi-factorial paracrine-mediated RGC survival and neurite outgrowth and may be considered a potent and advantageous cell therapy for retinal nerve repair

Purification of Saccharomyces cerevisiae mitochondria by zone electrophoresis in a free flow device.

This chapter describes the isolation of yeast mitochondria by differential centrifugation followed by mitochondrial purification through zone electrophoresis (ZE) using a free flow device (FFE). Starting from a yeast colony, cultures are grown under respiratory conditions to logarithmic phase. Cells are collected, their cell walls enzymatically disintegrated and the resulting spheroplasts are homogenized. Mitochondria are pre-fractionated from this homogenate by differential centrifugation. With the focus on further purification, pre-fractionated mitochondria are subjected to ZE-FFE. In ZE-FFE, mitochondria are transported with the buffer flow through the separation chamber and purified from contaminants by specific deflection through a perpendicularly oriented electric field. The purified mitochondria can be collected by centrifugation and used for further experiments and analysis such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, 2-DE or electron microscopy

16-BAC/SDS-PAGE analysis of membrane proteins of yeast mitochondria purified by free flow electrophoresis.

Mitochondria are essential organelles in cellular metabolism. These organelles are bounded by two membranes, the outer and inner membrane. Especially the inner membrane comprises a high content of proteins, for example, the protein complexes of the respiratory chain. High-resolution separation and analysis of such membrane proteins, for example, by two-dimensional gel electrophoresis (2-DE), is hampered by their hydrophobicity and tendency for aggregation. Here, we describe the separation of mitochondrial membrane proteins of Saccharomyces cerevisiae by 16-benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis (16-BAC/SDS-PAGE). This method enables the separation of membrane proteins owing to the solubilizing power of the ionic detergents 16-BAC and SDS, respectively. Mitochondria were isolated from yeast cultures by differential centrifugation and were further purified by free flow electrophoresis (FFE) in zone-electrophoretic mode (ZE). Subsequently, membrane proteins from ZE-FFE-purified mitochondria were enriched by carbonate extraction and subjected to 16-BAC/SDS-PAGE. The resulting protein spot patterns were visualized by a highly sensitive fluorescence stain with ruthenium-II-bathophenantroline disulfonate chelate (RuBP), and by colloidal Coomassie staining. Proteins were identified by Maldi-Tof mass spectrometry and peptide mass fingerprinting

From quantitative protein complex analysis to disease mechanism

AbstractInterest in the field of cilia biology and cilia-associated diseases – ciliopathies – has strongly increased over the last few years. Proteomic technologies, especially protein complex analysis by affinity purification-based methods, have been used to decipher various basic but also disease-associated mechanisms. This review focusses on some selected recent studies using affinity purification-based protein complex analysis, thereby exemplifying the great possibilities this technology offers

Cholinesterases precede "ON-OFF" channel dichotomy in the embryonic chick retina before onset of synaptogenesis.

Retinal physiology is balanced by the interplay between an ON and an OFF channel. The formation of this wiring dichotomy is not understood. Two neuropil sublayers of the inner plexiform layer (IPL) represent levels of cholinergic synaptic circuitry. These two sublayers are better segregated in avians than in most vertebrates; in the chick they are called a and d and participate in the OFF and ON channel, respectively. Both levels can be detected easily in the mature retina by choline acetyltransferase (ChAT) or by acetylcholinesterase (AChE); however, the usefulness of these enzymes as developmental markers is restricted, since ChAT is detected too late, while AChE labels not only cholinergic neuropil. To establish that individual AChE+ cells indeed project into cholinergic subbands a or d (and thus are involved in either channel), in the first part of this study we used the AChE-specific monoclonal antibody 1A2 plus confocal microscopy. We show that at embryonic stage E17 two AChE+ cell types of the inner nuclear layer project into the cholinergic subband a, and another one projects into d. Moreover, two others project into noncholinergic subbands, b and c, which develop only from E14 onwards. In contrast to immunocytochemistry, sensitive AChE histochemistry allows us to trace back the establishment of subbands a and d before E10, with the first AChE subband appearing along with IPL differentiation at E7. The establishment of AChE subbands is preceded by butyrylcholinesterase subbands; with differentiation following a central-peripheral gradient. Since synapses do not develop before E12 in the chick, we conclude that retinal ON-OFF dichotomy is laid down long before the formation of chemical synapses

Two-dimensional electrophoresis of membrane proteins.

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes