Image Analysis for the Quantitative Comparison of Stress Fibers and Focal Adhesions

Actin stress fibers (SFs) detect and transmit forces to the extracellular matrix through focal adhesions (FAs), and molecules in this pathway determine cellular behavior. Here, we designed two different computational tools to quantify actin SFs and the distribution of actin cytoskeletal proteins within a normalized cellular morphology. Moreover, a systematic cell response comparison between the control cells and those with impaired actin cytoskeleton polymerization was performed to demonstrate the reliability of the tools. Indeed, a variety of proteins that were present within the string beginning at the focal adhesions (vinculin) up to the actin SFs contraction (non-muscle myosin II (NMMII)) were analyzed. Finally, the software used allows for the quantification of the SFs based on the relative positions of FAs. Therefore, it provides a better insight into the cell mechanics and broadens the knowledge of the nature of SFs.     

The stress-activated protein kinases p38α/β and JNK1/2 cooperate with Chk1 to inhibit mitotic entry upon DNA replication arrest

Accurate DNA replication is crucial for the maintenance of genome integrity. To this aim, cells have evolved complex surveillance mechanisms to prevent mitotic entry in the presence of partially replicated DNA. ATR and Chk1 are key elements in the signal transduction pathways of DNA replication checkpoint; however, other kinases also make significant contributions. We show here that the stress kinases p38 and JNK are activated when DNA replication is blocked, and that their activity allows S/M, but not G₂/M, checkpoint maintenance when Chk1 is inhibited. Activation of both kinases by DNA replication inhibition is not mediated by the caffeine-sensitive kinases ATR or ATM. Phosphorylation of MKK3/6 and MKK4, p38 and JNK upstream kinases was also observed upon DNA replication inhibition. Using a genetic approach, we dissected the p38 pathway and showed that both p38α and p38β isoforms collaborate to inhibit mitotic entry. We further defined MKK3/6 and MK2/3 as the key upstream and downstream elements in the p38 signaling cascade after replication arrest. Accordingly, we found that the stress signaling pathways collaborate with Chk1 to keep cyclin B1/Cdk1 complexes inactive when DNA replication is inhibited, thereby preventing cell cycle progression when DNA replication is stalled. Our results show a complex response to replication stress, where multiple pathways are activated and fulfill overlapping roles to prevent mitotic entry with unreplicated DNA.     

2-Hydroxyoleic Acid Induces ER Stress and Autophagy in Various Human Glioma Cell Lines

Background

2-Hydroxyoleic acid is a synthetic fatty acid with potent anti-cancer activity which does not induce undesired side effects. However, the molecular and cellular mechanisms by which this compound selectively kills human glioma cancer cells without killing normal cells is not fully understood. The present study was designed to determine the molecular bases underlying the potency against 1321N1, SF-767 and U118 human glioma cell lines growth without affecting non cancer MRC-5 cells.

Methodology/Principal Findings

The cellular levels of endoplasmic reticulum (ER) stress, unfolded protein response (UPR) and autophagy markers were determined by quantitative RT-PCR and immunoblotting on 1321N1, SF-767 and U118 human glioma cells and non-tumor MRC-5 cells incubated in the presence or absence of 2OHOA or the ER stress/autophagy inducer, palmitate. The cellular response to these agents was evaluated by fluorescence microscopy, electron microscopy and flow cytometry. We have observed that 2OHOA treatments induced augments in the expression of important ER stress/UPR markers, such as phosphorylated eIF2α, IRE1α, CHOP, ATF4 and the spliced form of XBP1 in human glioma cells. Concomitantly, 2OHOA led to the arrest of 1321N1 cells in the G₂/M phase of the cell cycle, with down-regulation of cyclin B1 and Cdk1/Cdc2 proteins in the three glioma cell lines studied. Finally, 2OHOA induced autophagy in 1321N1, SF-767 and U118 cells, with the appearance of autophagic vesicles and the up-regulation of LC3BI, LC3BII and ATG7 in 1321N1 cells, increases of LC3BI, LC3BII and ATG5 in SF-767 cells and up-regulation of LC3BI and LC3BII in U118 cells. Importantly, 2OHOA failed to induce such changes in non-tumor MRC-5 cells.

Conclusion/Significance

The present results demonstrate that 2OHOA induces ER stress/UPR and autophagy in human glioma (1321N1, SF-767 and U118 cell lines) but not normal (MRC-5) cells, unraveling the molecular bases underlying the efficacy and lack of toxicity of this compound.     

Habitat specialization and phylogenetic structure of tree species in a coastal Brazilian white-sand forest

Aims The coastal Brazilian rainforest on white-sand (restinga) ranks among the most fragmented forest types in the tropics, owing to both the patchy distribution of sandy soils and widespread coastal development activities. Here we study the environmental and evolutionary determinants of a forest tree assemblage at a single restinga forest in Southeastern Brazil. We also explore the ability of competing hypotheses to explain the maintenance of species diversity in this forest type, which includes contrasting extremes of edaphic conditions associated with flooding stress.Methods The study was conducted in a white-sand forest permanent plot of 10.24 ha on the coastal plain of Southeastern Brazil. This plot was divided into 256 quadrats of 20×20 m, which were classified into two main edaphic habitats (flooded and drained). Trees with a diameter ≥1cm at breast height were identified. We assembled DNA sequence data for each of the 116 morphospecies recognized using two chloroplast markers (rbcL and matK). A phylogenetic tree was obtained using the maximum likelihood method, and a phylogenetic distance matrix was produced from an ultrametric tree. We analyzed similarity in floristic composition and structure between habitats and related them to cross-plot distances using permutation procedures. Null model torus shift simulations were performed to obtain a statistical significance level for habitat association for each species. The phylogenetic structure for the two habitats and for each 20×20 m quadrat was calculated using the mean phylogenetic distance weighted by species abundance and checked for significance using the standardized effect size generated by 5000 randomizations of phylogenetic tip labels.Important findings Our results indicate that partitioning among edaphic habitats is important for explaining species distributions and coexistence in restinga forests. Species distributions within the plot were found to be non-random: there was greater floristic similarity within than between habitats, and>40% of the more abundant species were positively or negatively associated with at least one habitat. Patterns of habitat association were not independent of phylogenetic relatedness: the community was overdispersed with respect to space and habitat type. Closely related species tended to occur in different habitats, while neighboring trees tended to belong to more distantly related species. We conclude that habitat specialization is important for the coexistence of species in restinga forests and that habitat heterogeneity is therefore an essential factor in explaining the maintenance of diversity of this unique but fragile and threatened type of forest.     

Response of the grapevine rootstock Richter 110 to inoculation with native and selected arbuscular mycorrhizal fungi and growth performance in a replant vineyard

Two indigenous arbuscular mycorrhizal (AM) fungi from the Mediterranean wine growing area in the Northeast of Spain were isolated and classified as Glomus intraradices Schenck & Smith. Both native fungi were found to increase the growth of the vine rootstock 110 Richter under greenhouse conditions compared with G. intraradices (BEG 72) and a phosphorus (P) fertilization treatment. The effectivity of field inoculation of Cabernet Sauvignon plants grafted on Richter 110 with the former native fungi and with G. intraradices BEG 72 in a replant vineyard severely infested by the root-rot fungus Armillaria mellea (Vahl ex Fr.) Kummer was assessed. The native fungi were not effective at enhancing plant development, and only G. intraradices BEG 72, resulted in a positive response. Field inoculation with this selected fungus increased plant shoot dry weight at the end of the first growing season.     

Selenium deficiency in cultured adrenocortical cells: restoration of glutathione peroxidase and resistance to hydroperoxides on addition of selenium

Cultured bovine adrenocortical cells were previously shown to be functionally deficient in selenium and vitamin E when grown in medium supplemented with fetal bovine serum. In the present experiments, the lack of significant bioavailable amounts of selenium in the medium was demonstrated by the finding of only low levels of glutathione peroxidase in the cultured cells (0.008 U/mg protein compared with 0.045 U/mg protein in fresh adrenocortical tissue). When 20 nM selenium as selenite was added to the cultured adrenocortical cells, glutathione peroxidase activity increased continuously over 72 h, with a total increase of about eightfold over this period. Over the same time-course, the highest concentration of cumene hydroperoxide tolerated by the cells without cell death increased progressively from 10 microM to 50 microM. Addition of 1 microM alpha-tocopherol also increased the amount of cumene hydroperoxide tolerated to 50 microM. Cell death was measured by cloning efficiency after removal of cumene hydroperoxide. Addition of either selenium or alpha-tocopherol had little effect on the growth rate of the cells over six passages, even when residual vitamin E was removed from the serum by extraction with ether and residual low molecular weight selenium compounds were removed by dialysis. It is concluded that combined deficiency of selenium and vitamin E, at least in the presence of other components of fetal bovine serum, has little effect on the ability of the cells to survive under normal conditions, as evidenced by continued long-term proliferation. However, the low levels of glutathione peroxidase resulting from selenium deficiency cause an increase susceptibility to peroxide-mediated toxicity. The combined deficiency of selenium and vitamin E impairs the ability of cells to survive under adverse conditions, as well as altering mitochondrial functions, as previously demonstrated.     

Lettuce production and antioxidant capacity are differentially modified by salt stress and light intensity under ambient and elevated CO2

As a consequence of the increasing importance of vegetables in the human diet, there is an interest in enhancing both the productivity and quality of vegetables. A number of factors, including plant genotype and environmental growing conditions, can impact the production and quality of vegetables. The objective of this study was to determine whether elevated CO₂, salinity, or high light treatments assayed individually, or salinity or high light in combination with elevated CO₂, increased biomass production and antioxidant capacity in two lettuce cultivars. Elevated CO₂ and its combination with salinity or high light increased biomass production in both cultivars, while high light treatment alone increased production in green-leaf lettuce but not in red-leaf lettuce. On the other hand, elevated CO₂ and its combination with salinity or high light increased the antioxidant capacity of both cultivars, while high light treatment alone increased the antioxidant capacity of red-leaf lettuce, but not of green-leaf lettuce.