Secretome profiling of primary human skeletal muscle cells

The skeletal muscle is a metabolically active tissue that secretes various proteins. These so-called myokines have been proposed to affect muscle physiology and to exert systemic effects on other tissues and organs. Yet, changes in the secretory profile may participate in the pathophysiology of metabolic diseases. The present study aimed at characterizing the secretome of differentiated primary human skeletal muscle cells (hSkMC) derived from healthy, adult donors combining three different mass spectrometry based non-targeted approaches as well as one antibody based method. This led to the identification of 548 non-redundant proteins in conditioned media from hSkmc. For 501 proteins, significant mRNA expression could be demonstrated. Applying stringent consecutive filtering using SignalP, SecretomeP and ER_retention signal databases, 305 proteins were assigned as potential myokines of which 12 proteins containing a secretory signal peptide were not previously described. This comprehensive profiling study of the human skeletal muscle secretome expands our knowledge of the composition of the human myokinome and may contribute to our understanding of the role of myokines in multiple biological processes. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Stable isotope labeling of Arabidopsis thaliana cells and quantitative proteomics by mass spectrometry

Quantitative analysis of protein expression is an important tool for the examination of complex biological systems. Albeit its importance, quantitative proteomics is still a challenging task because of the high dynamic range of protein amounts in the cell and the variation in the physical properties of proteins. Stable isotope labeling by amino acids in cell culture (SILAC) has been successfully used in yeast and mammalian cells to measure relative protein abundance by mass spectrometry. Here we show for the first time that proteins from Arabidopsis thaliana cell cultures can be selectively isotope-labeled in vivo by growing cells in the presence of a single stable isotope-labeled amino acid. Among the tested amino acids ([2H3]-leucine, [13C6]arginine, and [2H4]lysine), [13C6]arginine proved to be the most suitable. Incorporation of [13C6]arginine into the proteome was homogeneous and reached efficiencies of about 80%. [13C6]Arginine-labeled A. thaliana suspension cells were used to study the regulation of glutathione S-transferase expression in response to abiotic stress caused by salicylic acid and to identify proteins that bind specifically to phosphorylated 14-3-3 binding motifs on synthesized bait peptides in affinity purification experiments. In conclusion, the combination of stable isotope labeling of plant cells and mass spectrometry is a powerful technology that can be applied to study complex biological processes that involve changes in protein expression such as cellular responses to various kinds of stress or activation of cell signaling.     

Taproot promoters cause tissue specific gene expression within the storage root of sugar beet

The storage root (taproot) of sugar beet (Beta vulgaris L.) originates from hypocotyl and primary root and contains many different tissues such as central xylem, primary and secondary cambium, secondary xylem and phloem, and parenchyma. It was the aim of this work to characterize the promoters of three taproot-expressed genes with respect to their tissue specificity. To investigate this, promoters for the genes Tlp, His1-r, and Mll were cloned from sugar beet, linked to reporter genes and transformed into sugar beet and tobacco. Reporter gene expression analysis in transgenic sugar beet plants revealed that all three promoters are active in the storage root. Expression in storage root tissues is either restricted to the vascular zone (Tlp, His1-r) or is observed in the whole organ (Mll). The Mll gene is highly organ specific throughout different developmental stages of the sugar beet. In tobacco, the Tlp and Mll promoters drive reporter gene expression preferentially in hypocotyl and roots. The properties of the Mll promoter may be advantageous for the modification of sucrose metabolism in storage roots.     

Apoptosis in a tissue-like culture model of human colorectal adenoma cells

Tissue-like cultures of LT97 human colonic adenoma cells were produced by plating on reconstructed connective tissue supports (CTR) containing colon-associated fibroblasts to form mucosal monolayers and polyp-like structures closely resembling the in vivo situation. Tight cell-cell contacts and a high density of desmosomes were observed in 93.8+/-3.5% of the section area for CTR cultures, but only in 17.8+/-10.0% of the area for cultures on collagen gels (CG) without fibroblasts. While LT97 cultures on plastic shed apoptotic bodies into the medium, they collected at the basal pole of the cell layer in the CTR cultures as is also observed in adenoma tissue. Only a small proportion was released into the medium as shown by the detection of apoptosis specific keratin fragments, which could be increased by 100microM of quercetin and resveratrol. In addition, tissue-like structures altered the relative effectivity of test compounds. As the tissue-like conditions closely resemble the situation in vivo tissue-like cultures are an important step towards the establishment of tissue reconstructs that can replace animal models in the analysis of basic mechanisms of growth control and the testing of tumor promoting and chemopreventive compounds and may even be superior in terms of predictivity as they use human cells.     

Pyrrolidine Dithiocarbamate Activates p38 MAPK and Protects Brain Endothelial Cells From Apoptosis: A Mechanism for the Protective Effect in Stroke?

The antioxidant and inhibitor of nuclear factor κB pyrrolidine dithiocarbamate (PDTC) potently reduces infarct size in various experimental stroke models. In addition, it has been shown to have a favourable safety profile in humans. In this study, we further investigated the mechanistic actions of PDTC on cerebral microvascular endothelial cells as main components of the blood–brain barrier. We propose activation of p38 MAPK by PDTC as an additional protective mechanism. C57/BL6 mice were subjected to transient MCAO for 2 h and treated with PDTC (100 mg/kg) or vehicle i.p. before reperfusion. Infarct size was determined after 24 h. Apoptosis was induced in a cerebral microvascular endothelial cell line and the effect of pretreatment with PDTC and its dependency on p38 MAPK activity was assayed. PDTC administered 2 h after MCAO reduced infarct size by 61% (P < 0.05) and reduces the apoptotic death rate in vivo. In vitro, PDTC reduces the apoptotic death rate of bEnd.3 cells. p38 MAPK was activated by PDTC and its inhibition abrogated the protective effect of PDTC. PDTC reduces infarct size after stroke with a reasonable time window and decreases apoptotic cell death in vivo and in vitro. The attenuation of apoptotic cell death in brain microvascular endothelial cells is dependent on p38 MAPK activity.     

Unraveling Sterol-dependent Membrane Phenotypes by Analysis of Protein Abundance-ratio Distributions in Different Membrane Fractions Under Biochemical and Endogenous Sterol Depletion

During the last decade, research on plasma membrane focused increasingly on the analysis of so-called microdomains. It has been shown that function of many membrane-associated proteins involved in signaling and transport depends on their conditional segregation within sterol-enriched membrane domains. High throughput proteomic analysis of sterol-protein interactions are often based on analyzing detergent resistant membrane fraction enriched in sterols and associated proteins, which also contain proteins from these microdomain structures. Most studies so far focused exclusively on the characterization of detergent resistant membrane protein composition and abundances. This approach has received some criticism because of its unspecificity and many co-purifying proteins. In this study, by a label-free quantitation approach, we extended the characterization of membrane microdomains by particularly studying distributions of each protein between detergent resistant membrane and detergent-soluble fractions (DSF). This approach allows a more stringent definition of dynamic processes between different membrane phases and provides a means of identification of co-purifying proteins. We developed a random sampling algorithm, called Unicorn, allowing for robust statistical testing of alterations in the protein distribution ratios of the two different fractions. Unicorn was validated on proteomic data from methyl-β-cyclodextrin treated plasma membranes and the sterol biosynthesis mutant smt1. Both, chemical treatment and sterol-biosynthesis mutation affected similar protein classes in their membrane phase distribution and particularly proteins with signaling and transport functions.The plasma membrane incorporates a broad spectrum of proteins covering mainly different structural, signaling or transport functionalities. Being the first semipermeable cell barrier to its surrounding environment the plasma membrane is important for metabolite transport as well as initiation point of several signaling processes (1–4). To maintain cell homeostasis, protein activity as well as complex formation through protein protein interactions (PPI) need to be tightly regulated. The major regulating mechanisms are postranslational modification of proteins and modulated abundances of proteins present in the plasma membrane. Another potential regulating mechanism became apparent with the discovery of sterol and sphingolipid enriched domains (microdomains) in the plasma membrane (5–8, 3). Microdomain like structures have been shown to form spontaneously in artificial plasma membranes (9). After a decade of research on these structures, microdomains turned out to be particularly involved in signaling and transport processes incorporating a specific set of proteins. Microdomains provide subcompartments in the plasma membrane with specific physicochemical properties that on specific sterol protein interactions might alter protein activity or PPIs. With the discovery of microdomains the fluid lipid mosaic model was extended by distinguishing two plasma membrane phases, an ordered phase of lower density (L_o phase) enriched in sterols, sphingolipids and long chain fatty acids and a disordered phase of higher density (L_d phase). From isolated plasma membranes a lower density and a higher density membrane fraction can be separated in a sucrose gradient after treatment with non-ionic detergents. The resulting detergent resistant membrane fraction (DRM)¹ is related to L_o phase and high density detergent soluble membrane fraction (DSF) relates to L_o phases. Although it is still under debate how well DRMs represent native plasma membrane microdomains (10–12), research on protein-sterol interactions is possible by usage of sterol depleting agents like methyl-β-cyclodextrin mβcd (13). Therefore mβcd is suitable for detecting false positive cholesterol protein interactions in DRM studies (14–19). Proteins depleted on mβcd treatment are finally considered to be sterol dependent (15–17). To compare the mβcd treatment for disturbing the sterol distribution in the L_o fraction, we studied the sterol biosynthesis deficient mutant smt1. (20) smt1 carries a point mutation in the smt1 locus, encoding the sterol methyltransferase 1 and it exhibits a dwarf-like phenotype on whole plant level (20). In total, three sterol methyl transferases are encoded in Arabidopsis where SMT1 catalyzes the first step in the sterol biosynthesis by adding a methyl group at C24 of the sterol precursor cycloartenol. SMT2 and SMT3 act at later steps and were shown to be functionally redundant as C-24 sterol methyltransferases at the branching in sterol synthesis that either leads to sitosterol or campesterol (21). The total sterol composition in smt1 mutants was shown to be different from wild type, with the major phytosterols like sitosterol, stigmasterol, and brassicasterol being strongly depleted. In contrast, other sterol species remained unaltered and some even increased (20, 21). So far, it remains unclear how the altered sterol-composition of the smt1 mutant affects sterol-protein interactions. In this study, using the newly developed algorithm Unicorn, we compared changes in protein distributions between DRM and DSF after biochemical mβcd treatment and on endogenous alterations in sterol composition in smt1 to improve understanding of sterol–protein interactions.     

Adhesive mechanisms in cephalopods: a review

Abstract

Several genera of cephalopods (Nautilus, Sepia, Euprymna and Idiosepius) produce adhesive secretions, which are used for attachment to the substratum, for mating and to capture prey. These adhesive structures are located in different parts of the body, viz. in the digital tentacles (Nautilus), in the ventral surface of the mantle and fourth arm pair (Sepia), in the dorsal epidermis (Euprymna), or in the dorsal mantle side and partly on the fins (Idiosepius). Adhesion in Sepia is induced by suction of dermal structures on the mantle, while for Nautilus, Euprymna and Idiosepius adhesion is probably achieved by chemical substances. Histochemical studies indicate that in Nautilus and Idiosepius secretory cells that appear to be involved in adhesion stain for carbohydrates and protein, whilst in Euprymna only carbohydrates are detectable. De-adhesion is either achieved by muscle contraction of the tentacles and mantle (Nautilus and Sepia) or by secretion of substances (Euprymna). The de-adhesive mechanism used by Idiosepius remains unknown.     

The Osedax trophosome: organization and ultrastructure

The polychaete family Siboglinidae, which is currently construed as comprising the Frenulata, Monilifera (composed of Sclerolinum), Vestimentifera, and Osedax, has become known for its specialized symbiont-housing organ called the trophosome. This organ replaced the digestive system of the worms and is located in the elongated trunk region in Frenulata, Sclerolinum, and Vestimentifera. Currently two types of trophosomes have been described: in the taxa Frenulata and Sclerolinum the bacteriocytes originate from endoderm, and in Vestimentifera they originate from mesoderm. In Osedax, a trophosome was described as lacking (Rouse et al., 2004), but bacteriocytes are located in Osedax's characteristic root tissue. Here, we argue for a consistent name for the symbiont-housing tissue, namely trophosome, as in other siboglinids. In this study we provide morphological evidence that in Osedax the bacteriocytes are derived from somatic mesoderm. We show that the trophosome in Osedax is an apolar tissue composed of bacteriocytes and nonsymbiotic cells. As in vestimentiferans, a specific cell cycle was identified; however, in this case it is directed from the posterior to the anterior end of the worms instead of from the center toward the periphery. Comparison of all siboglinid trophosomes and re-evaluation of their body regions allows us to discuss whether the trophosomes are homologous and to hypothesize about the organization of the last common ancestor of Siboglinidae.     

MASCP Gator: an aggregation portal for the visualization of Arabidopsis proteomics data

Proteomics has become a critical tool in the functional understanding of plant processes at the molecular level. Proteomics-based studies have also contributed to the ever-expanding array of data in modern biology, with many generating Web portals and online resources that contain incrementally expanding and updated information. Many of these resources reflect specialist research areas with significant and novel information that is not currently captured by centralized repositories. The Arabidopsis (Arabidopsis thaliana) community is well served by a number of online proteomics resources that hold an abundance of functional information. These sites can be difficult to locate among a multitude of online resources. Furthermore, they can be difficult to navigate in order to identify specific features of interest without significant technical knowledge. Recently, members of the Arabidopsis proteomics community involved in developing many of these resources decided to develop a summary aggregation portal that is capable of retrieving proteomics data from a series of online resources on the fly. The Web portal is known as the MASCP Gator and can be accessed at the following address: http://gator.masc-proteomics.org/. Significantly, proteomics data displayed at this site retrieve information from the data repositories upon each request. This means that information is always up to date and displays the latest data sets. The site also provides hyperlinks back to the source information hosted at each of the curated databases to facilitate more in-depth analysis of the primary data.