Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd^- phenotype), while other substitutions block kinase activation (Gcn^- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn^- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd^- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.     

Aqueous two-phase partition coefficients for Escherichia coli β-galactosidase fusions

The partition of native Escherichia coli -galactosidase and of two different fusion proteins comprised mainly of -galactosidase from E. coli was studied in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran. These fusions contain an amino-terminal segment from the E. coli outer membrane protein F (OmpF) and a linker peptide. Differences in the partition pattern could be observed for the three enzymes despite their similarity. Decreased polymer concentrations in the phase system increased the partition coefficient for all three -galactosidases.     

High concentrations of PEG as a possible uncoupler of the proton motive force: α-Amylase production with Bacillus amyloliquefaciens in aqueous two-phase systems and PEG solutions

Summary -Amylase production with Bacillus amyloliquefaciens was investigated in two different aqueous two-phase systems and in polyethylene glycol (PEG) 600 solutions of different concentrations. The cells did not partition totally to the bottom phases of the aqueous two-phase systems, and the enzyme production was repressed in both systems as well as in PEG 600 solutions. Concomitantly, the cultivation time was prolonged, indicating an increased maintenance metabolism. The surface properties of cells grown in 200 g/kg PEG 600 were investigated by phase partitioning and compared to the surface properties of Bacillus subtilis, which under these conditions showed increased -amylase production. The cells of B. amyloliquefaciens partitioned to the top phase in a PEG-dextran system, whereas the cells of B. subtilis partitioned to the bottom phase. The results are discussed in relation to water activity, oxygen transfer rate and PEG-induced changes of the surface properties of the cells. The possible role of PEG as an uncoupler of the proton motive force at high concentrations is also discussed.     

Structural and ecophysiological adaptations to forest gaps

To survive new microclimatic conditions of a forest gap environment, plant species must physiologically and structurally adjust. A morpho-anatomical, ultrastructural and ecophysiological study was performed at three different times in a forest gap that was created by illegal selective logging. The study followed the early successional Actinostemon verticillatus and the late-successional Metrodorea brevifolia, to elucidate the adaptive strategies of acclimation to gaps. Additionally, Schinus terebinthifolius was included in the study in order to test the plasticity of a pioneer species that grows on forest edges, where this species had higher values of leaf thickness, leaf mass area and succulence. M. brevifolia had succulent leaves, high leaf area and a thin cuticle. A. verticillatus presented the densest leaves and was the only species to show leaf morpho-anatomical plasticity. Ultrastructural and physiological differences were observed only in A. verticillatus and M. brevifolia leaves from the gap: increase in the stroma volume, oil droplets, plastoglobuli, photochemical and non-photochemical quenching. Photosynthetic efficiency showed that the early stages of gap formation are the most critical. Acclimation strategies of A. verticillatus suggest this species invests in the efficiency of photosynthesis by increasing its leaf thickness, leaf mass area and in water content maintenance by increasing the density of its leaves, at the expense of gas exchange, was compensated by a high density of stomata. M. brevifolia compensates for the higher cost of leaves and lower leaf plasticity with ultrastructural changes that are used to adjust the photosynthetic process, which promotes a shorter leaf payback time.     

On the reporting and review requirements of ISO 14044

The International Journal of Life Cycle Assessment -     

D1- and D2-like receptors differentially mediate the effects of dopaminergic transmission on cost–benefit evaluation and motivation in monkeys

It has been widely accepted that dopamine (DA) plays a major role in motivation, yet the specific contribution of DA signaling at D₁-like receptor (D₁R) and D₂-like receptor (D₂R) to cost–benefit trade-off remains unclear. Here, by combining pharmacological manipulation of DA receptors (DARs) and positron emission tomography (PET) imaging, we assessed the relationship between the degree of D₁R/D₂R blockade and changes in benefit- and cost-based motivation for goal-directed behavior of macaque monkeys. We found that the degree of blockade of either D₁R or D₂R was associated with a reduction of the positive impact of reward amount and increasing delay discounting. Workload discounting was selectively increased by D₂R antagonism. In addition, blocking both D₁R and D₂R had a synergistic effect on delay discounting but an antagonist effect on workload discounting. These results provide fundamental insight into the distinct mechanisms of DA action in the regulation of the benefit- and cost-based motivation, which have important implications for motivational alterations in both neurological and psychiatric disorders.

Using quantitatively controlled pharmacological manipulations, this study teases apart the role of D1- and D2-like dopamine receptors in motivation and goal-directed behavior in monkeys, revealing complementary roles of two dopamine receptor subtypes in the computation of the cost/benefit trade-off to guide action.     

Microtubule target for new antileishmanial drugs based on ethyl 3-haloacetamidobenzoates

A new family of antimicrotubule drugs named (3-haloacetamidobenzoyl) ureas and ethyl 3-haloacetamidobenzoates were found to be cytotoxic to the Leishmania parasite protozoa. While the benzoylureas were shown to strongly inhibit in vitro mammalian brain microtubule assembly, the ethyl ester derivatives were characterized as very poor inhibitors of this process. Ethyl 3-chloroacetamidobenzoate, MF29, was found to be the most efficient drug on the promastigote stage of three Leishmania species (IC₅₀: 0.3–1.8 μM). MF29 maintained its activity against the clinical relevant intracellular stage of L. mexicana with IC₅₀ value of 0.33 μM. It was the only compound that exhibits a high activity on all the Leishmania species tested. This compound appeared to alter parasite microtubule organisation as demonstrated by using antibodies directed against microtubule components and more precisely the class of microtubule decorated by the MAP2-like protein. It is interesting to notice that this MAP2-like protein was identified for the first time in a Leishmania parasite     

The Putative Thiosulfate Sulfurtransferases PspE and GlpE Contribute to Virulence of Salmonella Typhimurium in the Mouse Model of Systemic Disease

The phage-shock protein PspE and GlpE of the glycerol 3-phosphate regulon of Salmonella enterica serovar Typhimurium are predicted to belong to the class of thiosulfate sulfurtransferases, enzymes that traffic sulfur between molecules. In the present study we demonstrated that the two genes contribute to S. Typhimurium virulence, as a glpE and pspE double deletion strain showed significantly decreased virulence in a mouse model of systemic infection. However, challenge of cultured epithelial cells and macrophages did not reveal any virulence-associated phenotypes. We hypothesized that their contribution to virulence could be in sulfur metabolism or by contributing to resistance to nitric oxide, oxidative stress, or cyanide detoxification. In vitro studies demonstrated that glpE but not pspE was important for resistance to H₂O₂. Since the double mutant, which was the one affected in virulence, was not affected in this assay, we concluded that resistance to oxidative stress and the virulence phenotype was most likely not linked. The two genes did not contribute to nitric oxid stress, to synthesis of essential sulfur containing amino acids, nor to detoxification of cyanide. Currently, the precise mechanism by which they contribute to virulence remains elusive.