The Rheb switch 2 segment is critical for signaling to target of rapamycin complex 1

The small GTPase Rheb is a positive upstream regulator of the target of rapamycin (TOR) complex 1 in mammalian cells and can bind directly to TOR complex 1. To identify the regions of the Rheb surface most critical for signaling to TOR complex 1, we created a set of 26 mutants wherein clusters of 1-5 putative solvent-exposed residues were changed to alanine, ultimately changing 65 residues distributed over the entire Rheb surface. The signaling function of these mutants was assessed by their ability, in comparison to wild type Rheb, to restore the phosphorylation of S6K1(Thr389) when expressed transiently in amino acid-deprived 293T cells. The major finding is that two mutants situated in the Rheb switch 2 segment, Y67A/I69A and I76A/D77A, exhibit a near total loss of function, whereas extensive replacement of the switch 1 segment and other surface residues with alanines causes relatively little disturbance of Rheb rescue of S6K1 from amino acid withdrawal. This is surprising in view of the minimal impact of guanyl nucleotide on Rheb switch 2 configuration. The loss of function Rheb switch 2 mutants are well expressed and exhibit partial agonist function in amino acid-replete cells. They are unimpaired in their ability to bind GTP or mammalian (m)TOR in vivo or in vitro, and the mTOR polypeptides retrieved with these inactive Rheb mutants exhibit kinase activity in vitro comparable with mTOR bound to wild type Rheb. We conclude that Rheb signaling to mTOR in vivo requires a Rheb switch 2-dependent interaction with an element other than the three known polypeptide components of TOR complex 1.     

Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants

Background

Obesity is a major health problem. Although heritability is substantial, genetic mechanisms predisposing to obesity are not very well understood. We have performed a genome wide association study (GWA) for early onset (extreme) obesity.

Methodology/Principal Findings

a) GWA (Genome-Wide Human SNP Array 5.0 comprising 440,794 single nucleotide polymorphisms) for early onset extreme obesity based on 487 extremely obese young German individuals and 442 healthy lean German controls; b) confirmatory analyses on 644 independent families with at least one obese offspring and both parents. We aimed to identify and subsequently confirm the 15 SNPs (minor allele frequency ≥10%) with the lowest p-values of the GWA by four genetic models: additive, recessive, dominant and allelic. Six single nucleotide polymorphisms (SNPs) in FTO (fat mass and obesity associated gene) within one linkage disequilibrium (LD) block including the GWA SNP rendering the lowest p-value (rs1121980; log-additive model: nominal p = 1.13×10⁻⁷, corrected p = 0.0494; odds ratio (OR)_CT 1.67, 95% confidence interval (CI) 1.22–2.27; OR_TT 2.76, 95% CI 1.88–4.03) belonged to the 15 SNPs showing the strongest evidence for association with obesity. For confirmation we genotyped 11 of these in the 644 independent families (of the six FTO SNPs we chose only two representing the LD bock). For both FTO SNPs the initial association was confirmed (both Bonferroni corrected p<0.01). However, none of the nine non-FTO SNPs revealed significant transmission disequilibrium.

Conclusions/Significance

Our GWA for extreme early onset obesity substantiates that variation in FTO strongly contributes to early onset obesity. This is a further proof of concept for GWA to detect genes relevant for highly complex phenotypes. We concurrently show that nine additional SNPs with initially low p-values in the GWA were not confirmed in our family study, thus suggesting that of the best 15 SNPs in the GWA only the FTO SNPs represent true positive findings.     

Das essenzielle Spurenelement Zink. Ein Metall in Biologie und Medizin

The essential trace element zinc The human body contains 2–3 grams of the essential trace element zinc, and uptake as well as excretion underlies a tight control. If this balance is disturbed, a number of biological processes are affected: among other tasks, zinc is important for the development and function of the central nervous system, the immune defense, and the production and function of insulin. This is based on over 300 different zinc‐containing enzymes, several other zinc proteins, and a role of zinc in intracellular signal transduction.     

Dynamic studies of phloem and xylein flow in fully differentiated plants by fast nuclear-magnetic-resonance microimaging

A fast nuclear-magnetic-resonance imaging method was developed in order to measure simultaneously and quantitatively the water flow velocities in the xylem and the phloem of intact and transpiring plants. Due to technical improvements a temporal resolution of 7 min could be reached and flow measurements could be performed over a time course of 12-30 h. The novel method was applied to the hypocotyl of 35- to 40-day-old, leafy plants of Ricinus communis which were subjected to different light-dark regimes. The results showed that the xylem flow velocities and the xylem volume flow responded immediately to light on-off changes. Upon illumination the flow velocity and the volume flow increased as expected in respect to literature. In contrast, the phloem flow velocity did not change in response to the light-dark regimes. Interestingly, though, the volume flow in the phloem increased during darkness. These findings can be explained by assuming that the conducting area of the phloem becomes enlarged during the dark period due to opening of sieve pores.     

Alterations in apoptosis and epithelial-mesenchymal transformation in an in vitro cleft palate model

The processes of apoptosis and epithelial-mesenchymal transformation have been identified as two major mechanisms by which secondary palatal shelves achieve fusion. The aim of this study was to investigate alterations in these mechanisms by changing the physical distance between paired palatal shelves in an in vitro model of palatogenesis. Wild-type palatal pairs were dissected from E13.5 CD1 mouse embryos and allowed to grow in tissue culture for 48 hours at various intershelf distances. During the fusion process, medial edge epithelial cell fate was assessed using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, to evaluate apoptosis, and carboxyfluorescence (carboxy-2,7'-dichlorofluorescein diacetate succinimidyl ester) labeling, to measure transformation to mesenchymal cells. Palatal pairs separated in culture greater than or equal to 0.4 mm failed to fuse. TUNEL staining showed that the number of apoptotic cells in the palatal shelves increased as the intershelf distance increased, becoming marked in shelves that did not achieve fusion. The amount of epithelial-mesenchymal transformation, however, decreased with increasing intershelf distance. These results suggest that the contribution of epithelial-mesenchymal transformation and apoptosis to palatal shelf development and fusion can be altered by physical proximity. Therefore, one mechanism behind clefting in utero may result from an imbalance in epithelial-mesenchymal transformation and apoptosis as observed in vitro where palatal shelves are challenged to fuse by physical separation. This effect could be significant in the understanding and treatment of developmental palatal abnormalities. Perhaps in utero manipulation of intershelf spacing or epithelial-mesenchymal transformation and/or apoptosis could reverse the clefting paradigm.     

Activated protein C inhibits the release of macrophage inflammatory protein-1-alpha from THP-1 cells and from human monocytes

Several lines of evidence have implicated activated protein C (APC) to be an endogenous inhibitor of the inflammatory septic cascade. APC may exhibit direct anti-inflammatory properties, independent of its antithrombotic effects. Chemokines influence the interaction of monocytes at the endothelium during infection and sepsis and are involved in the molecular events leading to an adverse and lethal outcome of sepsis. Defining regulatory mechanisms on the monocytic release profile of the proinflammatory C-C chemokines macrophage inflammatory protein-1-alpha (MIP-1-alpha) and monocyte chemoattractant protein-1 (MCP-1) might have therapeutic implications for the treatment of sepsis. We established a monocytic cell model of inflammation by the addition of lipopolysaccharide (LPS) and examined the effect of human APC on LPS-stimulated chemokine release from the monocytic cell line THP-1. We found that human APC in supra-physiological concentrations of 2.5-10 microg/ml inhibited the LPS-induced release of the chemokines MIP-1-alpha and MCP-1, as measured by enzyme-linked immunosorbent assays (ELISA) at 6 up to 24 h. In addition to experiments on THP-1 cells, recombinant human APC in concentrations of 50 ng/ml was found to have an inhibiting effect on the release of MIP-1-alpha from freshly isolated mononuclear cells of septic patients. The ability of APC to decrease the release of the C-C chemokine MIP-1-alpha from the monocytic cell line THP-1 and from human monocytes may identify a novel immunomodulatory pathway by which APC exerts its anti-inflammatory action and may contribute to control the inflammatory response in sepsis.     

What's in the genome of a filamentous fungus? Analysis of the Neurospora genome sequence

The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively. Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process. The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. The analysis of the 3218 identified open reading frames provides a first overview of the protein equipment of a filamentous fungus. Significantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism. While several of these enzymes are specific for filamentous fungi many are shared exclusively with prokaryotes.     

alpha3beta1 integrin-CD151, a component of the cadherin-catenin complex, regulates PTPmu expression and cell-cell adhesion

The beta1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for alpha3beta1 integrin in association with the tetraspanin CD151 as a component of a cell-cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin-tetraspanin complex affects epithelial cell-cell adhesion at the level of gene expression both by regulating expression of PTPmu and by organizing a multimolecular complex containing PKCbetaII, RACK1, PTPmu, beta-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.     

The receptor protein tyrosine phosphatase mu, PTPmu, regulates histogenesis of the chick retina

The formation of laminae within the retina requires the coordinate regulation of cell differentiation and migration. The cell adhesion molecule and member of the immunoglobulin superfamily, receptor protein tyrosine phosphatase Mu, PTPmu, is expressed in precursor and early, differentiated cells of the prelaminated retina, and later becomes restricted to the inner plexiform, ganglion cell, and optic fiber layers. Since the timing of PTPmu expression correlates with the peak period of retinal lamination, we examined whether this RPTP could be regulating cell adhesion and migration within the retina, and thus influencing retinal development. Chick retinal organ cultures were infected with herpes simplex viruses encoding either an antisense sequence to PTPmu, wild-type PTPmu, or a catalytically inactive mutant form of PTPmu, and homophilic adhesion was blocked by using a function-blocking antibody. All conditions that perturbed PTPmu dramatically disrupted retinal histogenesis. Our findings demonstrate that catalytic activity and adhesion mediated by PTPmu regulate lamination of the retina, emphasizing the importance of adhesion and signaling via receptor protein tyrosine phosphatases in the developing nervous system. To our knowledge, this is the first demonstration that an Ig superfamily RPTP regulates the lamination of any neural tissue.