Dual Neuroprotective and Neurotoxic Effects of Cannabinoid Drugs in Vitro

Either protective or toxic effects of cannabinoids on cell survival have been reported extensively in the literature; however, the factors that determine the direction of the effect are still obscured. In this study we have used the neuroblastoma cell line N18TG2 that expresses CB1 cannabinoid receptors to investigate several factors that may determine the consequences of exposure to cannabinoid agonists. Cells that were grown under optimal, stressful, or differentiating conditions were exposed to cannabinoid agonists and then assayed for cell viability by measuring MTT, LDH, and caspase-3 activity. Various cannabinoid agonists (CP 55,940, ∆9-THC, HU-210, and WIN 55,212-2) failed to affect cell viability when the cells were grown under optimal conditions. On the other hand, the same agonists significantly reduced cell viability when the cells were grown under stressful conditions (glucose- and serum-free medium), while enhancing the viability of cells grown in differentiation medium (0.5% serum and 1.5% DMSO). The toxic/protective profile was not dependent on the type or the concentration of the cannabinoid agonist that was applied. The cannabinoid agonist CP 55,940 similarly affected the non-neuronal HEK-293 cells that were grown under stressful conditions only when they expressed CB1 receptors. Our results shed light on the conflicting reports regarding the protective or toxic effects of cannabinoids in vitro and indicate that cannabinoids may activate different intracellular signaling mechanisms, depending on the state of the cell, thus leading to different physiological consequences.     

The role of ubiquitination in lysosomal trafficking of δ-opioid receptors

The δ-opioid receptor (DOR) undergoes ligand-induced downregulation by endosomal sorting complex required for transport (ESCRT)-dependent endocytic trafficking to lysosomes. In contrast to a number of other signaling receptors, the DOR can downregulate effectively when its ubiquitination is prevented. We explored the membrane trafficking basis of this behavior. First, we show that internalized DORs traverse the canonical multivesicular body (MVB) pathway and localize to intralumenal vesicles (ILVs). Second, we show that DOR ubiquitination stimulates, but is not essential for, receptor transfer to ILVs and proteolysis of the receptor endodomain. Third, we show that receptor ubiquitination plays no detectable role in the early sorting of internalized DORs out of the recycling pathway. Finally, we show that DORs undergo extensive proteolytic fragmentation in the ectodomain, even when receptor ubiquitination is prevented or ILV formation itself is blocked. Together, these results are sufficient to explain why DORs downregulate effectively in the absence of ubiquitination, and they place a discrete molecular sorting operation in the MVB pathway effectively upstream of the ESCRT. More generally, these findings support the hypothesis that mammalian cells can control the cytoplasmic accessibility of internalized signaling receptors independently from their ultimate trafficking fate.     

Determination of volatile substances in olives and their effect on reproduction of the olive fruit fly

Females of the olive fruit fly Bactrocera oleae lay their eggs in olives mainly using fruit volatile stimuli. Using GC-MS analysis, we determined the chemical composition of the volatile blend emitted from field-collected olive fruit of cv. Megaritiki, at different stages of maturity. GC-MS analysis demonstrated qualitative and quantitative differences in the headspace blend emitted by the olive fruit. Certain chemicals such as toluene, n-octane, α-pinene, limonene, ethyl hexanol, nonanal n-dodecane, decanal and n-tetradecane were detected in greater amounts, irrespective of the growth stage of the fruit. The flies’ exposure to a number of these chemicals, such as n-octane and α-pinene, as well as to a mixture consisting of n-octane, α-pinene, limonene, ethyl hexanol, nonanal, n-dodecane, decanal and n–tetradecane favoured successful mating and egg production. The results may contribute to the improvement of the mass rearing of the fly, through these findings, which illustrate the positive effect that certain fruit volatile chemicals have on the fly's reproduction and offer a better understanding of the relation between the fly and the host fruit.     

Structure and dimerization of the kinase domain from yeast Snf1, a member of the Snf1/AMPK protein family

The Snf1/AMPK kinases are intracellular energy sensors, and the AMPK pathway has been implicated in a variety of metabolic human disorders. Here we report the crystal structure of the kinase domain from yeast Snf1, revealing a bilobe kinase fold with greatest homology to cyclin-dependant kinase-2. Unexpectedly, the crystal structure also reveals a novel homodimer that we show also forms in solution, as demonstrated by equilibrium sedimentation, and in yeast cells, as shown by coimmunoprecipitation of differentially tagged intact Snf1. A mapping of sequence conservation suggests that dimer formation is a conserved feature of the Snf1/AMPK kinases. The conformation of the conserved alphaC helix, and the burial of the activation segment and substrate binding site within the dimer, suggests that it represents an inactive form of the kinase. Taken together, these studies suggest another layer of kinase regulation within the Snf1/AMPK family, and an avenue for development of AMPK-specific activating compounds.     

Metabolic Adaptations of Azospirillum brasilense to Oxygen Stress by Cell-to-Cell Clumping and Flocculation

The ability of bacteria to monitor their metabolism and adjust their behavior accordingly is critical to maintain competitiveness in the environment. The motile microaerophilic bacterium Azospirillum brasilense navigates oxygen gradients by aerotaxis in order to locate low oxygen concentrations that can support metabolism. When cells are exposed to elevated levels of oxygen in their surroundings, motile A. brasilense cells implement an alternative response to aerotaxis and form transient clumps by cell-to-cell interactions. Clumping was suggested to represent a behavior protecting motile cells from transiently elevated levels of aeration. Using the proteomics of wild-type and mutant strains affected in the extent of their clumping abilities, we show that cell-to-cell clumping represents a metabolic scavenging strategy that likely prepares the cells for further metabolic stresses. Analysis of mutants affected in carbon or nitrogen metabolism confirmed this assumption. The metabolic changes experienced as clumping progresses prime cells for flocculation, a morphological and metabolic shift of cells triggered under elevated-aeration conditions and nitrogen limitation. The analysis of various mutants during clumping and flocculation characterized an ordered set of changes in cell envelope properties accompanying the metabolic changes. These data also identify clumping and early flocculation to be behaviors compatible with the expression of nitrogen fixation genes, despite the elevated-aeration conditions. Cell-to-cell clumping may thus license diazotrophy to microaerophilic A. brasilense cells under elevated oxygen conditions and prime them for long-term survival via flocculation if metabolic stress persists.     

The consequences of parental age for development,body mass and resistance to stress in the red flour beetle

The performance of most animals deteriorates with age. Motivated by the inconsistency in the literature regarding the effect of parental age on offspring traits and performance, we studied how parental age affects offspring development time, body mass, and starvation and cold tolerance in the red flour beetle (Tribolium castaneum). Offspring of old parents pupated later and at a higher body mass, and there was a general positive correlation between body mass and starvation tolerance. Despite their higher body mass, offspring of old parents tolerated starvation less well than those of young parents, emphasizing the impaired performance of the former. However, parental age did not affect offspring thermal tolerance and offspring of old parents were not more sensitive to cold shock than those of young parents. We also examined how ageing affects body mass and cold tolerance in the parental generations. By contrast to the effect of ontogeny on thermal tolerance, which is better known, change in thermal tolerance with age is seldom studied and can take different shapes. Old beetles were more sensitive to cold shock than younger beetles. Similar to cold tolerance, body mass decreased with age. In summary, older beetles reflect a worse physiological condition than younger ones. Ageing leads to impaired cold tolerance, lower body mass, lower number of offspring reaching adulthood, and deteriorated performance of the offspring, expressed as a lower starvation tolerance and a longer development time of the offspring. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115 , 305–314.     

Charting the genetic interaction map of a cell

Genome sequencing projects have revealed a massive catalog of genes and astounding genetic diversity in a variety of organisms. We are now faced with the formidable challenge of assigning functions to thousands of genes, and how to use this information to understand how genes interact and coordinate cell function. Studies indicate that the majority of eukaryotic genes are dispensable, highlighting the extensive buffering of genomes against genetic and environmental perturbations. Such robustness poses a significant challenge to those seeking to understand the wiring diagram of the cell. Genome-scale screens for genetic interactions are an effective means to chart the network that underlies this functional redundancy. A complete atlas of genetic interactions offers the potential to assign functions to most genes identified by whole genome sequencing projects and to delineate a functional wiring diagram of the cell. Perhaps more importantly, mapping genetic networks on a large-scale will shed light on the general principles and rules governing genetic networks and provide valuable information regarding the important but elusive relationship between genotype and phenotype.