Different Patterns of Akt and ERK Feedback Activation in Response to Rapamycin,Active-Site mTOR Inhibitors and Metformin in Pancreatic Cancer Cells

The mTOR pathway is aberrantly stimulated in many cancer cells, including pancreatic ductal adenocarcinoma (PDAC), and thus it is a potential target for therapy. However, the mTORC1/S6K axis also mediates negative feedback loops that attenuate signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feed-back loops unleashes over-activation of upstream pathways that potentially counterbalance the antiproliferative effects of mTOR inhibitors. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic cancer cells with either rapamycin or active-site mTOR inhibitors suppressed S6K and S6 phosphorylation induced by insulin and the GPCR agonist neurotensin. Rapamycin caused a striking increase in Akt phosphorylation at Ser⁴⁷³ while the active-site inhibitors of mTOR (KU63794 and PP242) completely abrogated Akt phosphorylation at this site. Conversely, active-site inhibitors of mTOR cause a marked increase in ERK activation whereas rapamycin did not have any stimulatory effect on ERK activation. The results imply that first and second generation of mTOR inhibitors promote over-activation of different pro-oncogenic pathways in PDAC cells, suggesting that suppression of feed-back loops should be a major consideration in the use of these inhibitors for PDAC therapy. In contrast, metformin abolished mTORC1 activation without over-stimulating Akt phosphorylation on Ser⁴⁷³ and prevented mitogen-stimulated ERK activation in PDAC cells. Metformin induced a more pronounced inhibition of proliferation than either KU63794 or rapamycin while, the active-site mTOR inhibitor was more effective than rapamycin. Thus, the effects of metformin on Akt and ERK activation are strikingly different from allosteric or active-site mTOR inhibitors in PDAC cells, though all these agents potently inhibited the mTORC1/S6K axis.     

Insulin-Receptor Substrate-2 (IRS-2) Is Required for Maintaining Glucokinase and Glucokinase Regulatory Protein Expression in Mouse Liver

Insulin receptor substrate (IRS) proteins play important roles in hepatic nutrient homeostasis. Since glucokinase (GK) and glucokinase regulatory protein (GKRP) function as key glucose sensors, we have investigated the expression of GK and GKRP in liver of Irs-2 deficient mice and Irs2(−/−) mice where Irs2 was reintroduced specifically into pancreatic β-cells [RIP-Irs-2/IRS-2(−/−)]. We observed that liver GK activity was significantly lower (p<0.0001) in IRS-2(−/−) mice. However, in RIP-Irs-2/IRS-2(−/−) mice, GK activity was similar to the values observed in wild-type animals. GK activity in hypothalamus was not altered in IRS-2(−/−) mice. GK and GKRP mRNA levels in liver of IRS-2(−/−) were significantly lower, whereas in RIP-Irs-2/IRS-2(−/−) mice, both GK and GKRP mRNAs levels were comparable to wild-type animals. At the protein level, the liver content of GK was reduced in IRS-2(−/−) mice as compared with controls, although GKRP levels were similar between these experimental models. Both GK and GKRP levels were lower in RIP-Irs-2/IRS-2(−/−) mice. These results suggest that IRS-2 signalling is important for maintaining the activity of liver GK. Moreover, the differences between liver and brain GK may be explained by the fact that expression of hepatic, but not brain, GK is controlled by insulin. GK activity was restored by the β-cell compensation in the RIP-Irs-2/IRS-2 mice. Interestingly, GK and GKRP protein expression remained low in RIP-Irs-2/IRS-2(−/−) mice, perhaps reflecting different mRNA half-lives or alterations in the process of translation and post-translational regulation.     

Lateralization in Escape Behaviour at Different Hierarchical Levels in a Gecko: Tarentola angustimentalis from Eastern Canary Islands

At the individual level, to be behaviourally lateralized avoids costly duplication of neural circuitry and decreases possible contradictory order from the two brain hemispheres. However, being prey behaviour lateralized at higher hierarchical levels could generate different negative implications, especially if predators are able to make predictions after multiple encounters. These conflicting pressures, namely between the advantages for individuals and the disadvantages for populations could be concealed if higher-level lateralization would arise from the combination of lateralized behaviours of individuals which are mutually dependent. Here, we investigated the lateralization patterns in the escape behaviour of the gecko Tarentola angustimentalis undergoing a predatory attack simulation in a “T” maze experiment. Results showed that gecko populations displayed different degrees of lateralization, with an overall dominance of right-biased individuals. This trend is similar to that observed in the Podarcis wall lizards, which share predators with Tarentola. In addition, different morphological parameters plausible to affect refuge selection were explored in order to link directional asymmetries at morphological level with lateralization during refuge selection.     

Bioaccumulation of Metals in Cultured Carp (Cyprinus carpio) from Lake Chapala,Mexico

Biological Trace Element Research - Fluoride accumulates and is toxic to bones. Clinical bone lesions occur in a phased manner, being less severe early in the natural course of skeletal fluorosis....     

Seed size,number and strategies in annual plants: a comparative functional analysis and synthesis

Background and AimsPlants depend fundamentally on establishment from seed. However, protocols in trait-based ecology currently estimate seed size but not seed number. This can be rectified. For annuals, seed number should simply be a positive function of vegetative biomass and a negative function of seed size.MethodsUsing published values of comparative seed number as the ‘gold standard’ and a large functional database, comparative seed yield and number per plant and per m² were predicted by multiple regression. Subsequently, ecological variation in each was explored for English and Spanish habitats, newly calculated C-S-R strategies and changed abundance in the British flora.Key ResultsAs predicted, comparative seed mass yield per plant was consistently a positive function of plant size and competitive ability, and largely independent of seed size. Regressions estimating comparative seed number included, additionally, seed size as a negative function. Relationships differed numerically between regions, habitats and C-S-R strategies. Moreover, some species differed in life history over their geographical range. Comparative seed yield per m² was positively correlated with FAO crop yield, and increasing British annuals produced numerous seeds. Nevertheless, predicted values must be viewed as comparative rather than absolute: they varied according to the ‘gold standard’ predictor used. Moreover, regressions estimating comparative seed yield per m² achieved low precision.ConclusionsFor the first time, estimates of comparative seed yield and number for >800 annuals and their predictor equations have been produced and the ecological importance of these regenerative traits has been illustrated. ‘Regenerative trait-based ecology’ remains in its infancy, with work needed on determinate vs. indeterminate flowering (‘bet-hedging’), C-S-R methodologies, phylogeny, comparative seed yield per m² and changing life history. Nevertheless, this has been a positive start and readers are invited to use estimates for >800 annuals, in the Supplementary data, to help advance ‘regenerative trait-based ecology’ to the next level.     

Amino Acid Transporters and Exchangers from the SLC1A Family: Structure,Mechanism and Roles in Physiology and Cancer

The Solute Carrier 1A (SLC1A) family includes two major mammalian transport systems—the alanine serine cysteine transporters (ASCT1-2) and the human glutamate transporters otherwise known as the excitatory amino acid transporters (EAAT1-5). The EAATs play a critical role in maintaining low synaptic concentrations of the major excitatory neurotransmitter glutamate, and hence they have been widely researched over a number of years. More recently, the neutral amino acid exchanger, ASCT2 has garnered attention for its important role in cancer biology and potential as a molecular target for cancer therapy. The nature of this role is still being explored, and several classes of ASCT2 inhibitors have been developed. However none have reached sufficient potency or selectivity for clinical use. Despite their distinct functions in biology, the members of the SLC1A family display structural and functional similarity. Since 2004, available structures of the archaeal homologues Glt_Ph and Glt_Tk have elucidated mechanisms of transport and inhibition common to the family. The recent determination of EAAT1 and ASCT2 structures may be of assistance in future efforts to design efficacious ASCT2 inhibitors. This review will focus on ASCT2, the present state of knowledge on its roles in tumour biology, and how structural biology is being used to progress the development of inhibitors.

     

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.     

Trophic niche partitioning of five skate species of genus Bathyraja in northern and central Patagonia,Argentina

Overexploitation of marine communities can lead to modifications in the structure of the food web and can force organisms like elasmobranchs to change their feeding habits. To evaluate the impact that fisheries have on food webs and on the interactions between species, it is necessary to describe and quantify the diet of the species involved and follow it through time. This study compares the diet of five skate species using the data obtained from the by-catch of the Argentine hake (Merluccius hubbsi) fishery in north and central Patagonia, Argentina. Diet composition was assessed by analysing the digestive tract contents and trophic overlapping between species of the genus Bathyraja: Bathyraja albomaculata, Bathyraja brachyurops, Bathyraja macloviana, Bathyraja magellanica and Bathyraja multispinis. A total of 184 stomachs were analysed. The diets of B. albomaculata and B. macloviana mainly comprised annelids, whereas that of B. brachyurops primarily comprised fish, including hake heads discarded by the fishery. The diets of B. magellanica and B. multispinis were largely based on crustaceans. Despite the morphological similarities and their shared preference for benthic habitats, no complete diet overlaps were found between the different species. These results suggest that these skate species have undergone a process of diet specialisation. This is a common feeding strategy that occurs to successfully eliminate competition when resources are limited, which corresponds to the conditions found in an environment being affected by the pressures of overfishing.     

Phenotypic and genetic differentiation between diadromous and landlocked puyen Galaxias maculatus

This study reports the phenotypic and genetic differences between individuals of puyen Galaxias maculatus from two sites in the same river basin in Tierra del Fuego National Park, southern South America. Individuals from the two sampling sites presented morphometric and genetic differences. The morphometric differences indicated that individuals from Laguna Negra (LN) were short and more robust and had large eyes, whereas those from Arroyo Negro (AN) were thin and elongated and had small eyes. Genetic differences showed that AN individuals had a greater genetic structuration and an older demographic history than LN individuals. The results of this study affirmed that the individuals from the two sampling sites belong to different populations with a high degree of isolation. The demographic history could indicate that the individuals of G. maculatus which migrated to northern areas during the last glaciation settled in the Beagle Channel after its formation. The LN population could have originated after the retreat of the glaciers, migrating from AN.     

Experimental evidence of habitat selection and territoriality in the Amazonian whip spider Heterophrynus longicornis (Arachnida, Amblypygi)

Animals that select and defend suitable habitats against conspecifics may be favored by maximizing prey encounter rate, gaining protection, or securing matings. However, the identification of habitat selection and territoriality may be hindered in observational studies in sedentary species with low-density populations, such as the whip spider Heterophrynus longicornis. To circumvent such difficulties, we adopted an experimental field approach to evaluate if H. longicornis selects and defends habitat in Central Amazon. To evaluate whether individuals perform habitat selection, we monitored the permanence of 29 experimentally released individuals in buttressed trees with a wide variation in diameter at breast height (DBH), with and without burrows at their bases. To evaluate whether individuals are territorial, we experimentally removed 21 individuals from their previously occupied trees and monitored the recolonization of these trees. If H. longicornis were territorial, we predicted that the recolonizers would be smaller than the removed individuals. We found 12 individuals in the trees where they were released on subsequent days, none of them in trees without burrows. The individual permanence was related to the presence of burrows, and not to DBH. There was recolonization by smaller males and females on ten trees from which the amblypygids were removed. In combination, the permanence of individuals in trees with burrows, the rapid recolonization of experimentally vacated trees by smaller individuals, and the preponderance of one individual per tree, suggest that both male and female H. longicornis perform habitat selection and also that after selecting a site, they defend it against conspecifics.