The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-beta-catenin pathway by targeting Dishevelled for degradation

Dishevelled is a conserved protein that interprets signals received by Frizzled receptors. Using a tandem-affinity purification strategy and mass spectrometry we have identified proteins associated with Dishevelled, including a Cullin-3 ubiquitin ligase complex containing the Broad Complex, Tramtrack and Bric à Brac (BTB) protein Kelch-like 12 (KLHL12). This E3 ubiquitin ligase complex is recruited to Dishevelled in a Wnt-dependent manner that promotes its poly-ubiquitination and degradation. Functional analyses demonstrate that regulation of Dishevelled by this ubiquitin ligase antagonizes the Wnt-beta-catenin pathway in cultured cells, as well as in Xenopus and zebrafish embryos. Considered with evidence that the distinct Cullin-1 based SCF(beta-TrCP)complex regulates beta-catenin stability, our data on the stability of Dishevelled demonstrates that two distinct ubiquitin ligase complexes regulate the Wnt-beta-catenin pathway.     

Design and synthesis of substituted imidazole and triazole N-phenylbenzo[d]oxazolamine inhibitors of retinoic acid metabolizing enzyme CYP26

The design of N-phenylbenzo[d]oxazolamines as CYP26A1 inhibitors involved ligand docking experiments using molecular modeling (FlexX) and analysis of ligand interactions at the binding domain. The synthesis of the benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines was achieved by cyclisation of the corresponding isothiocyanates with subsequent introduction of the haem-binding heterocycle. Triazole and tetrazole derivatives were also prepared for comparison with the lead imidazole derivative. The benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines with small substituents in the phenyl ring were moderately potent CYP26A1 inhibitors (IC(50) 8 and 12 microM) and comparable with liarozole (IC(50) 7 microM).     

Competitive strategies of soft corals (Coelenterata: Octocorallia). II. Variable defensive responses and susceptibility to scleractinian corals

Interactions involving competition for space between several species of alcyonacean and scleractinian corals were assessed experimentally on Britomart Reef, central region of the Great Barrier Reef, Australia. Colonies of three soft coral species, Sarcophyton ehrenbergi Marenzeller, Nephthea brassica Kukenthal, and Capnella lacertiliensis Macfayden Forskal (Coelenterata:Alcyonacea) were relocated within stands of two scleractinian corals, Parités andrewsi Vaughan (= P. cylindrica Dana) and Pavona cactus Förskal (Coelenterata:Scleractinia). Undisturbed scleractinian and relocated alcyonacean controls were also monitored.Alcyonacean corals induced necrosis of tissue in scleractinian corals. Necrosis was significantly more pronounced when colonies were in contact but was also observed in the absence of contact, implicating the presence of active allelopathic agents. Scleractinian coral species varied in their susceptibility to the ill effects of alcyonaceans, with Pontes andrewsi being more susceptible than Pavona cactus. Of the soft corals, Nephthea caused the highest degree of mortality in the two scleractinian corals examined and Sarcophyton the least. Some soft corals appear to retain their toxins while others release them, implying a combination of anti-predatory and anti-competitor roles for the secondary metabolites. Scleractinian corals were often overgrown by soft corals.Both species of scleractinian corals were found to cause approximately equal amounts of tissue necrosis in alcyonaceans. These effects were more pronounced when colonies were in direct contact. The necrotic effects among alcyonacean corals were species-specific. Alcyonaceans also overgrew scleractinian corals and secreted a protective polysaccharide layer in areas proximal to scleractinians. Secretion of this layer was stimulated differentially by the two scleractinian species and also varied in frequency of occurrence among the alcyonaceans.High levels of tissue necrosis were observed in both groups of organisms within 3 wk of initiation of the experiment. Necrosis increased with time in the scleractinian corals and decreased in the alcyonaceans. The development of a protective polysaccharide layer in the alcyonaceans increased with time.     

The vastus lateralis neuromuscular activity during all-out cycling exercise

ObjectiveThe objective of this work was to study modifications in motor control through surface electromyographic (sEMG) activity during a very short all-out cycling exercise.MethodsTwelve male cyclists (age 23 ± 4 years) participated in this study. After a warm-up period, each subject performed three all-out cycling exercises of 6 s separated by 2 min of complete rest. This protocol was repeated three times with a minimum of 2 days between each session. The braking torque imposed on cycling motion was 19 N m. The sEMG of the vastus lateralis was recorded during the first seven contractions of the sprint. Time–frequency analysis of sEMG was performed using continuous wavelet transform. The mean power frequency (MPF, qualitative modifications in the recruitment of motor units) and signal energy (a quantitative indicator of modifications in the motor units recruitment) were computed for the frequency range 10–500 Hz.ResultssEMG energy increased (P ? 0.05) between contraction number 1 and 2, decreased (P ? 0.05) between contraction number 2 and 3 then stabilized between contraction number 3 and 7 during the all-out test. MPF increased (P ? 0.05) during the all-out test. This increase was more marked during the first two contractions.ConclusionsThe decrease in energy and the increase in the sEMG MPF suggest a large spatial recruitment of motor units (MUs) at the beginning of the sprint followed by a preferential recruitment of faster MUs at the end of the sprint, respectively.     

Synthesis of New Pyrrolo[1,2-a]quinoxaline Derivatives as Potential Inhibitors of Akt Kinase

Akt kinases are attractive targets for small molecule drug discovery because of their key role in tumor cell survival/proliferation and their overexpression/activation in many human cancers. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel Akt kinase inhibitors, based on a quinoxaline or pyrazinone scaffold. A series of new substituted pyrrolo[1,2-a]quinoxaline derivatives, structural analogues of these active quinoxaline or pyrazinone pharmacophores, was synthesized from various substituted 2-nitroanilines or 1,2-phenylenediamine via multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937 and HL60, and the breast cancer cell line MCF7. Three of these human cell lines (K562, U937 and MCF7) exhibited an active phosphorylated Akt form. The most promising active pyrroloquinoxalines were found to be 1a that inhibited K562 cell line proliferation with an IC₅₀ of 4.5 μM, and 1h that inhibited U937 and MCF7 cell lines with IC₅₀ of 5 and 8 μM, respectively. These two candidates exhibited more potent activities than the reference inhibitor A6730.     

Mirror self-recognition and symbol-mindedness

The view that mirror self-recognition (MSR) is a definitive demonstration of self-awareness is far from universally accepted, and those who do support the view need a more robust argument than the mere assumption that self-recognition implies a self-concept (e.g. Gallup in Socioecology and Psychology of Primates, Mouton, Hague, 1975; Gallup and Suarez in Psychological Perspectives on the Self, vol 3, Erlbaum, Hillsdale, 1986). In this paper I offer a new argument in favour of the view that MSR shows self-awareness by examining the nature of the mirror image itself. I argue, using the results of ‘symbol-mindedness’ experiments by Deloache (Trends Cogn Sci 8(2):66–70, 2004), that where self-recognition exists, the mirror image must be functioning as a symbol from the perspective of the subject and the subject must therefore be ‘symbol-minded’ and hence concept possessing. Further to this, according to the Concept Possession Hypothesis of Self-Consciousness (Savanah in Conscious Cogn 2011), concept possession alone is sufficient to demonstrate the existence of self-awareness. Thus MSR as a demonstration of symbol-mindedness implies the existence of self-awareness. I begin by defending the ‘mark test’ protocol as a robust methodology for determining self-recognition. Then follows a critical examination of the extreme views both for and against the interpretation of MSR as an indication of self-awareness: although the non-mentalistic interpretation of MSR is unconvincing, the argument presented by Gallup is also inadequate. I then present the symbol-mindedness argument to fill in the gaps in the Gallup approach.     

The role of Mg2+ in the hydrolytic activity of the isolated chloroplast ATPase: Study by high-performance liquid chromatography

The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different total magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F₁ ATPase, have been followed by a chromatographic method consisting in the separation and determination of ADP. From the various series of curves, it is concluded that the experimental results (position of the maxima,K _m values) are better fitted by a mechanism involving the activation of the enzyme by magnesium ion and hydrolysis of free ATP, rather than by the classical mechanism, for which the enzyme hydrolyzes the MgATP complex and is inhibited by Mg²⁺. Although the equations giving the reaction rate are similar in the two cases, the calculated values ofK _m are widely different. The value obtained from the classical mechanism does not agree withK _D , the dissociation constant of the enzyme-substrate complex, measured by the Hummel and Dreyer method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzyme tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na⁺ is more easily explained as a competition between this ion and the activating Mg²⁺, than by the classical mechanism.     

Absence of the RGS9.Gbeta5 GTPase-activating complex in photoreceptors of the R9AP knockout mouse

Timely termination of the light response in retinal photoreceptors requires rapid inactivation of the G protein transducin. This is achieved through the stimulation of transducin GTPase activity by the complex of the ninth member of the regulator of G protein signaling protein family (RGS9) with type 5 G protein beta subunit (Gbeta5). RGS9.Gbeta5 is anchored to photoreceptor disc membranes by the transmembrane protein, R9AP. In this study, we analyzed visual signaling in the rods of R9AP knockout mice. We found that light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or Gbeta5 knockout rods. This effect was a consequence of the complete absence of any detectable RGS9 from the retinas of R9AP knockout mice. On the other hand, the level of RGS9 mRNA was not affected by the knockout. These data indicate that in photoreceptors R9AP determines the stability of the RGS9.Gbeta5 complex, and therefore all three proteins, RGS9, Gbeta5 , and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP.     

Neuron-glia interactions in the hypothalamus

The supraoptic (SON) and paraventricular (PVN) magnocellular nuclei of the hypothalamus undergo reversible anatomical remodeling under conditions of intense secretion of neurohypophysial hormones, such as lactation and chronic dehydration. This morphological plasticity is characterized by a pronounced reduction in astrocytic coverage of neurons, which results in an increased number and extent of directly juxtaposed somatic and dendritic surfaces. As a consequence, astrocyte-mediated clearance of glutamate from the extracellular space is altered, which causes an increased concentration and range of action of the excitatory amino acid in the extracellular space. This leads to a reduction of synaptic efficacy at excitatory and inhibitory inputs through the activation of presynaptic metabotropic glutamate receptors. By contrast, the action of glio transmitters released from astrocytes and acting on adjacent magnocellular neurons is limited during such anatomical remodeling. This includes glia derived ATP mediating potentiation of glutamatergic transmission, a process compromised by the neuronal-glial reorganization.Together, these studies on hypothalamic magnocellular nuclei provide new insights on the contribution of glial cells on neuronal activity.