Comprehensive analysis of CLE polypeptide signaling gene expression and overexpression activity in Arabidopsis

Intercellular signaling is essential for the coordination of growth and development in higher plants. Although hundreds of putative receptors have been identified in Arabidopsis (Arabidopsis thaliana), only a few families of extracellular signaling molecules have been discovered, and their biological roles are largely unknown. To expand our insight into the developmental processes potentially regulated by ligand-mediated signal transduction pathways, we undertook a systematic expression analysis of the members of the Arabidopsis CLAVATA3/ESR-RELATED (CLE) small signaling polypeptide family. Using reporter constructs, we show that the CLE genes have distinct and specific patterns of promoter activity. We find that each Arabidopsis tissue expresses at least one CLE gene, indicating that CLE-mediated signaling pathways are likely to play roles in many biological processes during the plant life cycle. Some CLE genes that are closely related in sequence have dissimilar expression profiles, yet in many tissues multiple CLE genes have overlapping patterns of promoter-driven reporter activity. This observation, plus the general absence of detectable morphological phenotypes in cle null mutants, suggest that a high degree of functional redundancy exists among CLE gene family members. Our work establishes a community resource of CLE-related biological materials and provides a platform for understanding and ultimately manipulating many different plant signaling systems.     

A role for PKCε during C2C12 myogenic differentiation

In a previous report we have demonstrated that PLCγ1 is involved in the differentiation process of C2C12 myoblasts, induced by insulin administration. In order to identify the downstream targets of PLCγ1-dependent signalling, we have analyzed the expression of DAG-dependent PKC isoforms during muscle differentiation. We show that during myotube formation, there is a marked increase of PKCε and η expression, and that PKCε is able to form a complex with PLCγ1. The increase in PKCε amount during myogenic differentiation is associated to an increase in PKCε activity as well. Immunofluorescence analysis indicated that in growing C2C12 cells both PLCγ1 and PKCε localize in the cytoplasm with a distinct perinuclear accumulation. In insulin-treated cells, the expression of PLCγ1 and PKCε increases and the two proteins are still distributed mainly in the perinuclear region of the myotubes. We show that PLCγ1–PKCε complex co-localizes with protein 58 K, a specific Golgi marker. Moreover, our results indicate that the Golgi-associated PKCε form, i.e. PKCε phosphorylated at Ser 729, is increased in differentiated myoblasts. Since it has been previously demonstrated that in C2C12 cells after insulin administration cyclin D3 levels could be modulated by PLCγ1, we analyzed the effect on cyclin D3 expression of either PKCε overexpression or silencing, in order to investigate whether PKCε could also affect cyclin D3 expression. The results showed that either a modification of PKCε expression or a change in its catalytic activity determines a variation of cyclin D3 levels and muscle differentiation in terms of myogenin expression. These data support a role for PKCε in regulating insulin inositide-dependent PLCγ1 signalling in skeletal muscle differentiation.     

Crystallization and preliminary x-ray diffraction studies of a psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis

The Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) produces a cold-active iron superoxide dismutase (SOD). PhSOD is a homodimeric enzyme, that displays a high catalytic activity even at low temperature. Using hanging-drop vapour-diffusion technique, PhSOD has been successfully crystallized in two different crystal forms. Both crystal forms are monoclinic with space group P2(1) and diffract to 2.1 A resolution. Form I has unit-cell parameters a=45.49A b=103.63A c=50.37A beta=108.2 degrees and contains a homodimer in the asymmetric unit. Form II has unit-cell parameters a=50.48A b=103.78A c=90.25A beta=103.8 degrees and an asymmetric unit containing two PhSOD homodimers. Structure determination has been achieved using molecular replacement. The crystallographic study of this cold-adapted enzyme could contribute to the understanding of the molecular mechanisms of cold-adaptation and of the high catalytic efficiency at low temperature.     

A muscle cell line from dystrophic mice expressing an altered phenotype in vitro

The isolation and characterization of a myogenic cell line from C57BL/6J/dydy mice is described. This line (DyA4) maintains the morphological, biochemical and electrophysiological characteristics of the primary cultured cells, at least for 20 passages. The cells actively divide as long as they are subcultured in media supplemented with horse serum and embryo extract. If the cells are not subcultured for a few days, they fuse into multinucleated contracting myotubes, which readily synthesize specific muscle products such as acetylcholinesterase and acetylcholine receptor. This dystrophic cell line expresses in vitro the same altered phenotype that is characteristic of dystrophic muscle cells in primary cultures, namely reduced acetylcholine sensitivity and reduced acetylcholine receptor expression. Because they can be grown in large amounts, and represent a pure muscle cell population which express an altered phenotype in an in vitro aneural avascular environment, DyA4 cells provide a very useful model system for investigating the pathogenesis of murine muscular dystrophy.     

The physiology and pathology of inositide signaling in the nucleus

Nuclear inositide signaling is nowadays a well‐established issue and a growing field of investigation, even though the very first evidence came out at the end of the 1980's. The understanding of its biological role is supported by the recent acquisitions dealing with pathology and namely hematological malignancies. Here, we review this issue highlighting the main achievements in the last years. J. Cell. Physiol. 226: 14–20, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular and functional properties of the psychrophilic elongation factor G from the Antarctic Eubacterium <Emphasis Type="Italic">Pseudoalteromonas haloplanktis</Emphasis> TAC 125

The molecular and functional properties of the elongation factor (EF) G from the psychrophilic Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) were studied. PhEF-G catalyzed protein synthesis in vitro that was inhibited by fusidic acid, an antibiotic specifically acting on EF-G. The EF interacted with GDP only in the presence of P. haloplanktis ribosome and fusidic acid with an affinity similar to that displayed by Escherichia coli EF-G. The psychrophilic translocase elicited a ribosome-dependent GTPase that was competitively inhibited by GDP, the slowly hydrolyzable GTP analog GppNHp, and the protein synthesis inhibitor ppGDP. The temperature dependence of the activity of PhEF-G reached its maximum at least 26°C beyond the growth temperature of P. haloplanktis (4–20°C). The heat inactivation profile of the ribosome-dependent GTPase of PhEF-G gave a temperature for half inactivation (46°C), significantly lower than that for half denaturation measured by either UV- (57°C) or fluorescence-melting (62°C). This finding was attributed to a different effect of the temperature on the catalytic domain with respect to that elicited on the other domains constituting the EF, thus confirming the differential molecular flexibility present in psychrophilic enzymes. A molecular model, based on the 3D coordinates of a thermophilic EF-G, showed differences only in connecting loops. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.