Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity

Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABA_ARs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABA_A autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca²⁺ photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl⁻]_i, autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30–150 GABA_A channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Na_v-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABA_A autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity.     

Role of G(i/o)-Src kinase-PI3K/Akt pathway and caveolin-1 in β₂-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery

Activation of the β₂-adrenoceptor (β₂-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β₂-AR-mediated eNOS activation, with special focus on G_i/o proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β₂-AR agonist procaterol was reduced by inhibitors of G_i/o proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser¹¹⁷⁷, which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr¹⁴, which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β₂-AR is coupled to a G_i/o-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser¹¹⁷⁷ leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr¹⁴, through a G_i/o-Src kinase pathway. Since pulmonary β₂-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.     

Contributing factors and variability of energy expenditure in non-obese, obese, and post-obese adolescents

Energy expenditure (EE) is a major determinant of energy balance and body composition. The objectives of this paper were to review the contributing factors of the main components of daily EE (DEE) and the inter-individual variability in these components in non-obese (NOb), obese (Ob), and post-obese (POb) adolescents. Body composition especially fat-free mass (FFM), is the major determinant of the basal metabolic rate which contributes 50-70% of DEE, whereas fat mass (FM) is a significant factor only in obese subjects. Physical activity is the second main variation factor of DEE, whereas growth, the thermic effect of food, and thermoregulation are generally of marginal importance. The energy costs and EE associated with various sedentary and physical activities were assessed in NOb, Ob and POb subjects both in standardised and in free-living conditions. The interindividual variability of DEE is high, even after adjustment for body composition, mainly because of great differences in time devoted to the various physical activities. DEE and EE associated with sleep and sedentary activities are significantly higher in Ob than in NOb, but not after adjustment for FFM. On the contrary, EE associated with physical activities is not significantly different between Ob and NOb adolescents, but 61% lower in Ob subjects after adjustment for body composition. Multidisciplinary weight-reduction programmes including moderate energy restriction and physical training result in great FM loss, maintenance of FFM, improvement of physical capacities, but reductions in organ and tissue metabolic rate and in EE associated with the various sedentary and physical activities, which may favour body weight regain in the less active POb subjects.     

DIMINUTO 1 affects the lignin profile and secondary cell wall formation in Arabidopsis

Brassinosteroids (BRs) play a crucial role in plant growth and development and DIMINUTO 1 (DIM1), a protein involved in BR biosynthesis, was previously identified as a cell elongation factor in Arabidopsis thaliana. Through promoter expression analysis, we showed that DIM1 was expressed in most of the tissue types in seedlings and sectioning of the inflorescence stem revealed that DIM1 predominantly localizes to the xylem vessels and in the interfascicular cambium. To investigate the role of DIM1 in cell wall formation, we generated loss-of-function and gain-of-function mutants. Disruption of the gene function caused a dwarf phenotype with up to 38 and 23% reductions in total lignin and cellulose, respectively. Metabolite analysis revealed a significant reduction in the levels of fructose, glucose and sucrose in the loss-of-function mutant compared to the wild type control. The loss-of-function mutant also had a lower S/G lignin monomer ratio relative to wild type, but no changes were detected in the gain-of-function mutant. Phloroglucinol and toluidine blue staining showed a size reduction of the vascular apparatus with smaller and disintegrated xylem vessels in the inflorescence stem of the loss-of-function mutant. Taken together, these data indicate a role for DIM1 in secondary cell wall formation. Moreover, this study demonstrated the potential role of BR hormones in modulating cell wall structure and composition.     

HTLV-1 Tax oncoprotein subverts the cellular DNA damage response via binding to DNA-dependent protein kinase

Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax.Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and gammaH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response.     

Manipulation of sinapine, choline and betaine accumulation in Arabidopsis seed: towards improving the nutritional value of the meal and enhancing the seedling performance under environmental stresses in oilseed crops.

Sinapoylcholine (sinapine) is the most abundant antinutritional phenolic compound in cruciferous seeds. The quaternary ammonium compounds, choline, betaine and N,N-dimethylglycine, reside along a biosynthetic pathway linked to the synthesis of membrane phospholipids and neurotransmitters with various biological functions. In chicken, choline intake is required for optimal egg-laying performance and a choline supplement in diet is positively correlated with weight gains. A key step in sinapine biosynthesis is catalyzed by sinapoylglucose: choline sinapoyltransferase (SCT; EC 2.3.1.91) to form an ester linkage with sinapoylglucose and choline. The objective of this work was to reduce the sinapine content and simultaneously enhance free choline levels in cruciferous seeds. We report here the characterization of an Arabidopsis T-DNA insertion mutant lacking SCT activity in the seed. The sct mutant seeds contain less than 1% of sinapine and a more than 2-fold increase in free choline compared with wild type. We further expressed a choline oxidase (COX; EC 1.1.3.17) gene from Arthrobacter pascens in the Arabidopsis sct mutant and wild-type background using a napin gene promoter to convert free choline into betaine, an effective stress-alleviating compound in plants. Betaine was not detected in WT or sct mutant seeds. The sct+COX seeds contain nearly 2-fold greater levels of betaine relative to WT+COX seeds, demonstrating a positive correlation between endogenous choline and betaine production. In contrast, stable comparable levels of free choline were detected between sct+COX and WT+COX plants suggesting choline homeostasis likely prevent high levels of betaine production in the seed of transgenic COX plants.     

The cinnamyl alcohol dehydrogenase gene family in Populus: phylogeny, organization, and expression

Background  

Lignin is a phenolic heteropolymer in secondary cell walls that plays a major role in the development of plants and their defense against pathogens. The biosynthesis of monolignols, which represent the main component of lignin involves many enzymes. The cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis as it catalyzes the final step in the synthesis of monolignols. The CAD gene family has been studied in Arabidopsis thaliana, Oryza sativa and partially in Populus. This is the first comprehensive study on the CAD gene family in woody plants including genome organization, gene structure, phylogeny across land plant lineages, and expression profiling in Populus.     

Anti-tubercular activity of ruthenium (II) complexes with polypyridines

A series of nine polypyridyl-ruthenium (II) complexes (N-ligands = 2,2'-bipyridines; 2,2'-6',2'-terpyridines, di-alkyloxy-2,2'-6,2-bipyridine-3,3'-di-carboxylates), were tested against Mycobacterium tuberculosis (MBT). The complex (11) showed remarkable activity against MBT as compared to other complexes, (1-10). The aquo ligand of complex (11), as opposed to other chloro and acetonitrile derivatives, appears to play a key role in the antitubercular potency of this new class of metal-based compounds.