Mitochondria and L-lactate metabolism

Although mitochondria have been the object of intensive study over many decades, some aspects of their metabolism remain to be fully elucidated, including the L-lactate metabolism. We review here the novel insights arisen from investigations on L-lactate metabolism in mammalian, plant and yeast mitochondria. The presence of L-lactate dehydrogenases inside mitochondria, where L-lactate enters in a carrier-mediated fashion, suggests that mitochondria play an important role in L-lactate metabolism. Functional studies have demonstrated the occurrence of several L-lactate carriers. Moreover, immunological investigations have proven the existence of monocarboxylate translocator isoforms in mitochondria.     

The subnanometer resolution structure of the glutamate synthase 1.2-MDa hexamer by cryoelectron microscopy and its oligomerization behavior in solution: functional implications

The three-dimensional structure of the hexameric (alphabeta)(6) 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex iron-sulfur flavoprotein. In the hexameric species, interprotomeric alpha-alpha and alpha-beta contacts are mediated by the C-terminal domain of the alpha subunit, which is based on a beta helical fold so far unique to glutamate synthases. The alphabeta protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the beta subunit, to the FMN on the alpha subunit, through the low potential [4Fe-4S](1+/2+) centers on the beta subunit and the [3Fe-4S](0/1+) cluster on the alpha subunit. The (alphabeta)(6) hexamer exhibits a concentration-dependent equilibrium with alphabeta monomers and (alphabeta)(2) dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP(+). Hexamerization seems to decrease the catalytic efficiency of the alphabeta protomer only 3-fold by increasing the K(m) values measured for l-Gln and 2-OG. However, it cannot be ruled out that the (alphabeta)(6) hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand.     

The Gi-coupled P2Y12 receptor regulates diacylglycerol-mediated signaling in human platelets

Stimulation of G(q)-coupled receptors activates phospholipase C and is supposed to promote both intracellular Ca(2+) mobilization and protein kinase C (PKC) activation. We found that ADP-induced phosphorylation of pleckstrin, the main platelet substrate for PKC, was completely inhibited not only by an antagonist of the G(q)-coupled P2Y1 receptor but also upon blockade of the G(i)-coupled P2Y12 receptor. The role of G(i) on PKC regulation required stimulation of phosphatidylinositol 3-kinase rather than inhibition of adenylyl cyclase. P2Y12 antagonists also inhibited pleckstrin phosphorylation, Rap1b activation, and platelet aggregation induced upon G(q) stimulation by the thromboxane A(2) analogue U46619. Importantly, activation of phospholipase C and intracellular Ca(2+) mobilization occurred normally. Phorbol 12-myristate 13-acetate overcame the inhibitory effect of P2Y12 receptor blockade on PKC activation but not on Rap1b activation and platelet aggregation. By contrast, inhibition of diacylglycerol kinase restored both PKC and Rap1b activity and caused platelet aggregation. Stimulation of P2Y12 receptor or direct inhibition of diacylglycerol kinase potentiated the effect of membrane-permeable sn-1,2-dioctanoylglycerol on platelet aggregation and pleckstrin phosphorylation, in association with inhibition of its phosphorylation to phosphatidic acid. These results reveal a novel and unexpected role of the G(i)-coupled P2Y12 receptor in the regulation of diacylglycerol-mediated events in activated platelets.     

Nanomechanics controls neuronal precursors adhesion and differentiation

The ability to control the differentiation of stem cells into specific neuronal types has a tremendous potential for the treatment of neurodegenerative diseases. In vitro neuronal differentiation can be guided by the interplay of biochemical and biophysical cues. Different strategies to increase the differentiation yield have been proposed, focusing everything on substrate topography, or, alternatively on substrate stiffness. Both strategies demonstrated an improvement of the cellular response. However it was often impossible to separate the topographical and the mechanical contributions. Here we investigate the role of the mechanical properties of nanostructured substrates, aiming at understanding the ultimate parameters which govern the stem cell differentiation. To this purpose a set of different substrates with controlled stiffness and with or without nanopatterning are used for stem cell differentiation. Our results show that the neuronal differentiation yield depends mainly on the substrate mechanical properties while the geometry plays a minor role. In particular nanostructured and flat polydimethylsiloxane (PDMS) substrates with comparable stiffness show the same neuronal yield. The improvement in the differentiation yield obtained through surface nanopatterning in the submicrometer scale could be explained as a consequence of a substrate softening effect. Finally we investigate by single cell force spectroscopy the neuronal precursor adhesion on the substrate immediately after seeding, as a possible critical step governing the neuronal differentiation efficiency. We observed that neuronal precursor adhesion depends on substrate stiffness but not on surface structure, and in particular it is higher on softer substrates. Our results suggest that cell–substrate adhesion forces and mechanical response are the key parameters to be considered for substrate design in neuronal regenerative medicine. Biotechnol. Bioeng. 2013; 110: 2301–2310. © 2013 Wiley Periodicals, Inc.     

γ-Aminobutyric Acid and Pentobarbital Enhance 2-[3H]Oxoquazepam Binding to Type I Benzodiazepine Recognition Sites in Rat and Human Brain

2-Oxoquazepam (2oxoquaz) is a novel benzodiazepine which shows preferential affinity for type I benzodiazepine recognition sites. In the present study, we analyzed the effect of gamma-aminobutyric acid (GABA), pentobarbital, and chloride ions on [3H]2oxoquaz and [3H]flunitrazepam ( [3H]FNT) binding to membrane preparations from rat and human brain. GABA stimulated [3H]-2oxoquaz and [3H]FNT binding in a concentration-dependent manner. The maximal enhancement produced by GABA on [3H]2oxoquaz binding was higher than that produced on [3H]FNT binding in both rat and human tissues. In the rat brain, the effect of GABA on [3H]2oxoquaz was similar throughout different brain areas, whereas the effect on [3H]FNT binding was lower in the cerebral cortex and hippocampus than in the cerebellum. Moreover, both [3H]2oxoquaz and [3H]FNT binding were stimulated by chloride ions and pentobarbital. The results are consistent with the hypothesis that type I benzodiazepine recognition sites are linked functionally to the GABA recognition site and the chloride ionophore.     

Ecological behavior of Quercus suber and Quercus ilex inferred by topographic wetness index (TWI)

The ecological behaviors of a network of pure evergreen oak stands (Quercus suber L. and Quercus ilex L.) in the Central-Western Mediterranean Basin were investigated toward climatic and edaphic factors implemented with the application of topographic wetness index (TWI). A Categorical Principal Component Analysis (Catpca) using climatic and soil physico-chemical parameters was performed on 23 cork oak and holm oak pure stands with the aim to understand better the effectiveness of TWI for characterizing soil ecology of the two species. Catpca pointed out that, although cork oak and holm oak are able to growth in similar Mediterranean conditions, they show different behaviors in terms of needs and tolerance to soil water content. TWI confirmed such results at local scale, allowing highlighting some interesting features of the species differential ecology. Although both species confirmed to be drought-tolerant, the heliophilous cork oak revealed to dominate the landscape on wettest soils with high TWI values—indicating the capacity to tolerate stresses due to periods of waterlogging—, while the shade-tolerant holm oak prevails for low-medium TWI values—drier and mesophilous sites. Despite the application of TWI to vegetation science and ecology is relatively recent, results are encouraging and suggest considering this user-friendly and synthetic index in ecological investigations and modeling.     

Breast cancer tissue slices as a model for evaluation of response to rapamycin

Rapamycin is a selective inhibitor of the mammalian target of rapamycin (mTOR), a regulator kinase that integrates growth factors signaling via the phosphoinositide-3-kinase pathway and that has emerged as a novel therapeutic modality in breast cancer (BC). We propose a pre-clinical “ex-vivo” personalized organotypic culture of BC that preserves the microenvironment to evaluate rapamycin-mediated gene expression changes. Freshly excised ductal invasive BC slices, 400 μm thick (n=30), were cultured in the presence or absence (control) of rapamycin (20 nM) for 24 h. Some slices were formalin-fixed for immunohistochemical determinations and some were processed for microarray analysis. Control slices in culture retained their tissue morphology and tissue viability (detected by BrdU uptake). The percentage of proliferating cells (assessed by Ki67) did not change up to 24 h of treatment. Immunohistochemical evaluation of p-AKT, p-mTOR, p-4EBP1 and p-S6K1 indicated that AKT/mTOR pathway activation was maintained during cultivation. For microarray analysis, slices were divided into two groups, according to the presence/absence of epidermal growth factor receptor-type 2 and analyzed separately. Limited overlap was seen among differentially expressed genes after treatment (P<0.01) in both groups suggesting different responses to rapamycin between these BC subtypes. Ontology analysis indicated that genes involved in biosynthetic processes were commonly reduced by rapamycin. Our network analysis suggested that concerted expression of these genes might distinguish controls from treated slices. Thus, breast carcinoma slices constitute a suitable physiological tool to evaluate the short-term effects of rapamycin on the gene profile of individual BC samples.     

SCL-Epred: a generalised de novo eukaryotic protein subcellular localisation predictor

Knowledge of the subcellular location of a protein provides valuable information about its function, possible interaction with other proteins and drug targetability, among other things. The experimental determination of a protein’s location in the cell is expensive, time consuming and open to human error. Fast and accurate predictors of subcellular location have an important role to play if the abundance of sequence data which is now available is to be fully exploited. In the post-genomic era, genomes in many diverse organisms are available. Many of these organisms are important in human and veterinary disease and fall outside of the well-studied plant, animal and fungi groups. We have developed a general eukaryotic subcellular localisation predictor (SCL-Epred) which predicts the location of eukaryotic proteins into three classes which are important, in particular, for determining the drug targetability of a protein—secreted proteins, membrane proteins and proteins that are neither secreted nor membrane. The algorithm powering SCL-Epred is a N-to-1 neural network and is trained on very large non-redundant sets of protein sequences. SCL-Epred performs well on training data achieving a Q of 86 % and a generalised correlation of 0.75 when tested in tenfold cross-validation on a set of 15,202 redundancy reduced protein sequences. The three class accuracy of SCL-Epred and LocTree2, and in particular a consensus predictor comprising both methods, surpasses that of other widely used predictors when benchmarked using a large redundancy reduced independent test set of 562 proteins. SCL-Epred is publicly available at http://distillf.ucd.ie/distill/.