Activation of Cdk2 stimulates proteasome-dependent truncation of tyrosine phosphatase SHP-1 in human proliferating intestinal epithelial cells

SHP-1 is expressed in the nuclei of intestinal epithelial cells (IECs). Increased SHP-1 expression and phosphatase activity coincide with cell cycle arrest and differentiation in these cells. Suspecting the tumor-suppressive properties of SHP-1, a yeast two-hybrid screen of an IEC cDNA library was conducted using the full-length SHP-1 as bait. Characterization of many positive clones revealed sequences identical to a segment of the Cdk2 cDNA sequence. Interaction between SHP-1 and Cdk2 was confirmed by co-immunoprecipitations whereby co-precipitated Cdk2 phosphorylated SHP-1 protein. Inhibition of Cdk2 (roscovitine) or proteasome (MG132) was associated with an enhanced nuclear punctuate distribution of SHP-1. Double labeling localization studies with signature proteins of subnuclear domains revealed a co-localization between the splicing factor SC35 and SHP-1 in bright nucleoplasmic foci. Using Western blot analyses with the anti-SHP-1 antibody recognizing the C terminus, a lower molecular mass species of 45 kDa was observed in addition to the full-length 64-65-kDa SHP-1 protein. Treatment with MG132 led to an increase in expression of the full-length SHP-1 protein while concomitantly leading to a decrease in the levels of the lower mass 45-kDa molecular species. Further Western blots revealed that the 45-kDa protein corresponds to the C-terminal portion of SHP-1 generated from proteasome activity. Mutational analysis of Tyr(208) and Ser(591) (a Cdk2 phosphorylation site) residues on SHP-1 abolished the expression of the amino-truncated 45-kDa SHP-1 protein. In conclusion, our results indicate that Cdk2-associated complexes, by targeting SHP-1 for proteolysis, counteract the ability of SHP-1 to block cell cycle progression of IECs.     

Interaction of the RAGE cytoplasmic domain with diaphanous-1 is required for ligand-stimulated cellular migration through activation of Rac1 and Cdc42

Cellular migration is a fundamental process linked to diverse pathological states such as diabetes and its complications, atherosclerosis, inflammation, and cancer. The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule which binds distinct ligands that accumulate in these settings. RAGE-ligand interaction evokes central changes in key biological properties of cells, including proliferation, generation of inflammatory mediators, and migration. Although RAGE-dependent signal transduction is critically dependent on its short cytoplasmic domain, to date the proximate mechanism by which this RAGE domain engages and stimulates cytoplasmic signaling pathways has yet to be identified. Here we show that the RAGE cytoplasmic domain interacts with Diaphanous-1 (Dia-1) both in vitro and in vivo. We employed the human RAGE cytoplasmic domain as "bait" in the yeast two-hybrid assay and identified the formin homology (FH1) domain of Dia-1 as a potential binding partner of this RAGE domain. Immunoprecipitation studies revealed that the RAGE cytoplasmic domain interacts with the FH1 domain of Dia-1. Down-regulation of Dia-1 expression by RNA interference blocks RAGE-mediated activation of Rac-1 and Cdc42 and, in parallel, RAGE ligand-stimulated cellular migration. Taken together, these findings indicate that the interaction of the RAGE cytoplasmic domain with Dia-1 is required to transduce extracellular environmental cues evoked by binding of RAGE ligands to their cell surface receptor, a chief consequence of which is Rac-1 and Cdc42 activation and cellular migration. Because RAGE and Dia-1 are implicated in the regulation of inflammatory, vascular, and transformed cell migration, these findings highlight this interaction as a novel target for therapeutic intervention in inflammation, atherosclerosis, diabetes, and cancer.     

Flavonoids redirect PIN-mediated polar auxin fluxes during root gravitropic responses

The rate, polarity, and symmetry of the flow of the plant hormone auxin are determined by the polar cellular localization of PIN-FORMED (PIN) auxin efflux carriers. Flavonoids, a class of secondary plant metabolites, have been suspected to modulate auxin transport and tropic responses. Nevertheless, the identity of specific flavonoid compounds involved and their molecular function and targets in vivo are essentially unknown. Here we show that the root elongation zone of agravitropic pin2/eir1/wav6/agr1 has an altered pattern and amount of flavonol glycosides. Application of nanomolar concentrations of flavonols to pin2 roots is sufficient to partially restore root gravitropism. By employing a quantitative cell biological approach, we demonstrate that flavonoids partially restore the formation of lateral auxin gradients in the absence of PIN2. Chemical complementation by flavonoids correlates with an asymmetric distribution of the PIN1 protein. pin2 complementation probably does not result from inhibition of auxin efflux, as supply of the auxin transport inhibitor N-1-naphthylphthalamic acid failed to restore pin2 gravitropism. We propose that flavonoids promote asymmetric PIN shifts during gravity stimulation, thus redirecting basipetal auxin streams necessary for root bending.     

Denitration of 2,4,6-trinitrotoluene by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ESA-5 in the presence of ferrihydrite

Denitration of 2,4,6-trinitrotoluene (TNT) was evaluated in oxygen-depleted enrichment cultures. These cultures were established starting with an uncontaminated or a TNT-contaminated soil inoculum and contained TNT as sole nitrogen source. Incubations were carried out in the presence or absence of ferrihydrite. A significant release of nitrite was observed in the liquid culture containing TNT, ferrihydrite, and inoculum from a TNT-contaminated soil. Under these conditions, Pseudomonas aeruginosa was the predominant bacterium in the enrichment, leading to the isolation of P. aeruginosa ESA-5 as a pure strain. The isolate had TNT denitration capabilities as confirmed by nitrite release in oxygen-depleted cultures containing TNT and ferrihydrite. In addition to reduced derivatives of TNT, several unidentified metabolites were detected. Concomitant to a decrease of TNT concentration, a release of nitrite was observed. The concentration of nitrite peaked and then it slowly decreased. In the absence of TNT, the drop in the concentration of nitrite in oxygen-depleted cultures was lower when ferrihydrite was provided, suggesting that ferrihydrite inhibited the utilization of nitrite by P. aeruginosa ESA-5.     

Long-term drought modifies the fundamental relationships between light exposure, leaf nitrogen content and photosynthetic capacity in leaves of the lychee tree (Litchi chinensis)

Drought has dramatic negative effects on plants' growth and crop productivity. Although some of the responses and underlying mechanisms are still poorly understood, there is increasing evidence that drought may have a negative effect on photosynthetic capacity. Biochemical models of leaf photosynthesis coupled with models of radiation transfer have been widely used in ecophysiological studies, and, more recently, in global change modeling. They are based on two fundamental relationships at the scale of the leaf: (i) nitrogen content-light exposure and (ii) photosynthetic capacity-nitrogen content. Although drought is expected to increase in many places across the world, such models are not adapted to drought conditions. More specifically, the effects of drought on the two fundamental relationships are not well documented. The objective of our study was to investigate the effects of a long-term drought imposed slowly on the nitrogen content and photosynthetic capacity of leaves similarly exposed to light, from 3-year-old lychee trees cv. Kwa? Mi. Leaf nitrogen and non-structural carbohydrate concentrations were measured along with gas exchanges and the light-saturated rate of photosynthetic electron transport (J(max)) after a 5.5-month-long period of drought. Leaf nitrogen content on a mass basis remained stable, while the leaf mass-to-area ratio (LMA) increased with increasing water stress. Consequently, the leaf nitrogen content on an area basis (N(a)) increased in a non-linear fashion. The starch content decreased, while the soluble sugar content increased. Stomata closed and net assimilation decreased to zero, while J(max) and the ratio J(max)/N(a) decreased with increasing water stress. The drought-associated decrease in photosynthetic capacity can be attributed to downregulation of photosynthetic electron transport and to reallocation of leaf nitrogen content. It is concluded that modeling photosynthesis in drought conditions will require, first, the modeling of the effect of drought on LMA and J(max).     

Pleiotropy in the melanocortin system, coloration and behavioural syndromes

In vertebrates, melanin-based coloration is often associated with variation in physiological and behavioural traits. We propose that this association stems from pleiotropic effects of the genes regulating the synthesis of brown to black eumelanin. The most important regulators are the melanocortin 1 receptor and its ligands, the melanocortin agonists and the agouti-signalling protein antagonist. On the basis of the physiological and behavioural functions of the melanocortins, we predict five categories of traits correlated with melanin-based coloration. A review of the literature indeed reveals that, as predicted, darker wild vertebrates are more aggressive, sexually active and resistant to stress than lighter individuals. Pleiotropic effects of the melanocortins might thus account for the widespread covariance between melanin-based coloration and other phenotypic traits in vertebrates.     

Relaxivities of paramagnetic liposomes: on the importance of the chain type and the length of the amphiphilic complex

Nuclear magnetic relaxation dispersion (NMRD) profiles of unilamellar DPPC liposomes incorporating Gd-DTPA-bisamides with alkyl chains of 12 to 18 C atoms in their external and internal layers were recorded in order to study the influence that the chain length and structure of Gd-bisamides incorporated in the liposomal membrane have on their proton relaxivity. The NMRD profiles recorded at 310 K show that the relaxivity reaches a minimum value when the carbon chain lengths of the phospholipid and of the Gd complex match and is at a maximum in the presence of a carbon-carbon double bond. For these DPPC paramagnetic liposomes, the longer the aliphatic chains of the complex, the larger will be its immobilization in the membrane. In addition, the presence of an unsaturated carbon-carbon bond in the alkyl chain of the Gd complex induces an increase of its mobility and of its water exchange rate with, as a result, a much greater efficiency as an MRI contrast agent.     

Drosophila retinal pigment cell death is regulated in a position-dependent manner by a cell memory gene

The stereotyped organization of the Drosophila compound eye depends on the elimination by apoptosis of about 25% of the inter-ommatidial pigment cell precursors (IOCs) during metamorphosis. This program of cell death is under antagonistic effects of the Notch and the EGFR pathways. In addition, uncharacterized positional cues may underlie death versus survival choices among IOCs. Our results provide new genetic evidences that cell death is regulated in a position- dependent manner in the eye. We show that mutations in Trithorax-like (Trl) and lola-like/batman specifically block IOC death during eye morphogenesis. These genes share characteristics of both Polycomb-Group and trithorax-Group genes, in that they are required for chromatin-mediated repression and activation of Hox genes. However, Trl function in triggering IOC death is independent from a function in repressing Hox gene expression during eye development. Analysis of mosaic ommatidiae containing Trl mutant cells revealed that Trl function for IOC death is required in cone cells. Strikingly, cell death suppression in Trl mutants depends on the position of IOCs. Our results further support a model whereby death of IOCs on the oblique sides of ommatidiae requires Trl-dependent reduction of a survival signal, or an increase of a death signal, emanating from cone cells. Trl does not have the same effect on horizontal IOCs whose survival seems to involve additional topological constraints.     

Plant Sar1 isoforms with near-identical protein sequences exhibit different localisations and effects on secretion

In plants, differentiation of subdomains of the endoplasmic reticulum (ER) dedicated to protein export, the ER export sites (ERES), is influenced by the type of export-competent membrane cargo to be delivered to the Golgi. This raises a fundamental biological question: is the formation of transport intermediates at the ER for trafficking to the Golgi always regulated in the same manner? To test this, we followed the distribution and activity of two plant Sar1 isoforms. Sar1 is the small GTPase that regulates assembly of COPII (coat protein complex II) on carriers that transport secretory cargo from ER to Golgi. We show that, in contrast to a tobacco Sar1 isoform, the two Arabidopsis Sar1 GTPases were localised at ERES, independently of co-expression of Golgi-destined membrane cargo in tobacco cells. Although both isoforms labelled ERES, one was found to partition with the membrane fraction to a greater extent. The different distribution of fluorescent fusions of the two isoforms was influenced by the nature of an amino acid residue at the C-terminus of the protein, suggesting that the requirements for membrane association of the two GTPases are not equal. Furthermore, functional analyses based on the secretion of the bulk flow marker α-amylase indicated that over-expression of GTP-restricted mutants of the two isoforms caused different levels of ER export inhibition. These novel results indicate a functional heterogeneity among plant Sar1 isoforms.     

Sulphur-oxidizing extracellular bacteria in the gills of Mytilidae associated with wood falls

Six morphotypes of small mussels (Bivalvia: Mytilidae) were found attached to naturally sunken wood collected in the Bohol Sea (Philippines). These specimens are related to the large Bathymodiolus mussels that are found worldwide at cold seeps and hydrothermal vents. In these habitats, the mytilids harbour sulphur- and methane-oxidizing endosymbionts in their gills and depend on the energy and carbon provided by the symbionts. In this study, bacteria associated with the gills of wood-associated mussels are characterized using molecular and microscopic techniques. The existence of bacteria in the lateral zone of gill filaments in all specimens is demonstrated. Comparative analyses of 16S rRNA gene and adenosine 5'-phosphosulphate (APS) reductase gene sequences indicate that the bacteria are closely related to sulphur-oxidizing endosymbionts of Bathymodiolus. FISHs using specific probes confirm that sulphur oxidizers are by far the most abundant, if not the only bacteria present. Electron micrographs displayed mostly extracellular bacteria located between microvilli at the apical surface of host gill epithelial cells all along the lateral zone of each gill filament. In some specimens, occasional occurrence of intracellular bacteria with similar morphology was noted. This study provides the first molecular evidence for the presence of possible thiotrophic symbiosis in sunken wood ecosystems. With their epibiotic bacteria, wood-associated mussels display a less integrated type of interaction than described in their seep, vent and whale fall relatives.