Genes for glutathione synthesis from tomato: Structure, expression and role in cadmium tolerance

All organisms must balance the requirement for essential metals with the potential toxicity of these and other non-essential metals. In plants, phytochelatins and metallothioneins may provide mechanisms to keep metals such as zinc, copper and cadmium between beneficial and toxic levels. Phytochelatins are synthesized from glutathione, therefore glutathione biosynthesis is critical for production of these peptides. cDNAs from tomato encoding proteins with homology to $\gamma$-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2) were cloned and sequenced. The homology of tomato GSH1 and GSH2 at the protein level with corresponding Arabidopsis genes is 82% and 67%, respectively. The GSH2 protein contains a leucine zipper motif suggesting dimerization of the protein, consistent with observations that eukaryotic GSH2 proteins from dimers. Genomic DNA blot analysis indicated that the tomato GSH1 and GSH2 genes are present in low copy number. Further, RNA blot analysis indicated that the GSH1 and GSH2 2 genes are expressed constitutively in different tissues and are not induced by Cd or other stresses. Hybridization of GSH1 and GSH2 cDNAs to genomic DNA from unselected and selected Cd tolerant cell lines hybridized with the same pattern and intensity of hybridization, indicating that neither gene is amplified in tolerant cells. Amino acid substitutions in the $\gamma$-glutamylcysteine synthetase from wild type and Cd tolerant cell lines might contribute to altered activity of this enzyme in selected cells. Arabidopsis cad2 mutants, which are Cd sensitive and deficient in GSH, were transformed with the tomato GSH1 gene and this partially restored the wild type phenotype. Complementation of the cad2 mutant with tomato GSH1 cDNA under the control of the 35S promoter provides evidence that $\gamma$-GluCys synthetase is critical for Cd tolerance in plants. Cd tolerance, measured by root growth and biomass accumulation, was restored to some variable degree in these transformants. There was a negative correlation between the number of copies of the tomato GSH1 gene inserted in the transformants and the level of Cd tolerance. Overall, these results support the hypothesis that an increased capacity to synthesize glutathione, and therefore phytochelatins, might be used to increase the tolerance of plants to cadmium

Cognitive Patterns and Coping Mechanisms in the Context of Internet Use

Recent research indicates there are different cognitive patterns and coping mechanisms related to increased levels of Internet use and emotional distress in adolescents. This study aims to investigate the relationship between coping mechanisms, dysfunctional negative emotions, and Internet use. A total of 54 participants aged between 14 and 19 years old completed a questionnaire containing several measures and demographics information. We measured participants’ coping strategies, emotional distress, social and emotional loneliness, and their online behavior and Internet addiction using self-report questionnaires. In order to identify the relation between the investigated variables, we used correlation analysis and regression, and we tested one mediation model. The results showed that maladaptive coping strategies and Internet use were significant predictors of dysfunctional negative emotions. Moreover, passive wishful thinking, as a pattern of thinking, was associated with anxious and depressed feelings. The relation between Internet use and dysfunctional negative emotions was mediated by participants’ coping mechanisms. Therefore, we can conclude that the level of negative feelings is associated with the coping strategies used while showing an increased level of Internet addiction. Future studies should also consider different and multiple types of measurement other than self-reports, especially related to Internet addiction

Higher Desolvation Energy Reduces Molecular Recognition in Multi-Drug Resistant HIV-1 Protease

Designing HIV-1 protease inhibitors that overcome drug-resistance is still a challenging task. In this study, four clinical isolates of multi-drug resistant HIV-1 proteases that exhibit resistance to all the US FDA-approved HIV-1 protease inhibitors and also reduce the substrate recognition ability were examined. A multi-drug resistant HIV-1 protease isolate, MDR 769, was co-crystallized with the p2/NC substrate and the mutated CA/p2 substrate, CA/p2 P1’F. Both substrates display different levels of molecular recognition by the wild-type and multi-drug resistant HIV-1 protease. From the crystal structures, only limited differences can be identified between the wild-type and multi-drug resistant protease. Therefore, a wild-type HIV-1 protease and four multi-drug resistant HIV-1 proteases in complex with the two peptides were modeled based on the crystal structures and examined during a 10 ns-molecular dynamics simulation. The simulation results reveal that the multi-drug resistant HIV-1 proteases require higher desolvation energy to form complexes with the peptides. This result suggests that the desolvation of the HIV-1 protease active site is an important step of protease-ligand complex formation as well as drug resistance. Therefore, desolvation energy could be considered as a parameter in the evaluation of future HIV-1 protease inhibitor candidates

Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization

A properly established and maintained podocyte intercellular junction, or slit diaphragm, is a necessary component of the selective permeability barrier of the kidney glomerulus. The observation that mutation or deletion of the slit diaphragm transmembrane protein nephrin results in failure of podocyte foot process morphogenesis and concomitant proteinuria first suggested the hypothesis that nephrin serves as a component of a signaling complex that directly integrates podocyte junctional integrity with cytoskeletal dynamics. The observations made herein provide the first direct evidence to our knowledge for a phosphorylation-mediated signaling mechanism by which this integrative function is derived. Our data support the model that during podocyte intercellular junction formation, engagement of the nephrin ectodomain induces transient Fyn catalytic activity that results in nephrin phosphorylation on specific nephrin cytoplasmic domain tyrosine residues. We found that this nephrin phosphorylation event resulted in recruitment of the SH2–SH3 domain–containing adapter protein Nck and assembly of actin filaments in an Nck-dependent fashion. Considered in the context of the role of nephrin family proteins in other organisms and the integral relationship of actin dynamics and junction formation, these observations establish a function for nephrin in regulating actin cytoskeletal dynamics

Contribution of the 80s loop of HIV-1 protease to the multidrug-resistance mechanism: crystallographic study of MDR769 HIV-1 protease variants

Structural studies revealed a proline switch as a novel mechanism for the multidrug-resistant nature of multidrug-resistant clinical isolate 769 HIV-1 protease variants