Structure,Dynamics, and Substrate-induced Conformational Changes of the Multidrug Transporter EmrE in Liposomes

EmrE, a member of the small multidrug transporters superfamily, extrudes positively charged hydrophobic compounds out of Escherichia coli cytoplasm in exchange for inward movement of protons down their electrochemical gradient. Although its transport mechanism has been thoroughly characterized, the structural basis of energy coupling and the conformational cycle mediating transport have yet to be elucidated. In this study, EmrE structure in liposomes and the substrate-induced conformational changes were investigated by systematic spin labeling and EPR analysis. Spin label mobilities and accessibilities describe a highly dynamic ligand-free (apo) conformation. Dipolar coupling between spin labels across the dimer reveals at least two spin label populations arising from different packing interfaces of the EmrE dimer. One population is consistent with antiparallel arrangement of the monomers, although the EPR parameters suggest deviations from the crystal structure of substrate-bound EmrE. Resolving these discrepancies requires an unusual disposition of TM3 relative to the membrane-water interface and a kink in its backbone that enables bending of its C-terminal part. Binding of the substrate tetraphenylphosphonium changes the environment of spin labels and their proximity in three transmembrane helices. The underlying conformational transition involves repacking of TM1, tilting of TM2, and changes in the backbone configurations of TM3 and the adjacent loop connecting it to TM4. A dynamic apo conformation is necessary for the polyspecificity of EmrE allowing the binding of structurally diverse substrates. The flexibility of TM3 may play a critical role in movement of substrates across the membrane.     

Fatty acid-induced angiogenesis in first trimester placental trophoblast cells: Possible roles of cellular fatty acid-binding proteins

Angiogenesis is involved in the growth of new blood vessels from the existing one. Consequently, angiogenesis plays an indispensable role in tissue growth and repair including early placentation processes. Besides angiogenic growth factors (vascular endothelial growth factor (VEGF), angiopoietin-like 4 (ANGPTL4), placental growth factor (PlGF), platelet derived growth factor (PDGF), fibroblast growth factors (FGF)), dietary fatty acids (c>16) also directly or indirectly modulate angiogenic processes in tumors and other cell systems. Usually n − 3 fatty acids inhibit whereas n − 6 fatty acids stimulate angiogenesis in tumors and other cells. Contrary to this, docosahexaenoic acid, 22:6n − 3 (DHA) and other fatty acids including conjugated linoleic acid stimulate angiogenesis in placental first trimester cells. In addition to the stimulation of expression of major angiogenic factors such as VEGF and ANGPTL4, fatty acids also stimulate expression of intracellular fatty acid-binding proteins (FABPs) FABP-4 and FABP-3 those are known to directly modulate angiogenesis. Emerging data indicate that FABPs may be involved in the angiogenesis process. This paper reviews the fatty acid mediated angiogenesis process and the involvement of their binding proteins in these processes.     

From molecular signal activation to locomotion: an integrated, multiscale analysis of cell motility on defined matrices

The adhesion, mechanics, and motility of eukaryotic cells are highly sensitive to the ligand density and stiffness of the extracellular matrix (ECM). This relationship bears profound implications for stem cell engineering, tumor invasion and metastasis. Yet, our quantitative understanding of how ECM biophysical properties, mechanotransductive signals, and assembly of contractile and adhesive structures collude to control these cell behaviors remains extremely limited. Here we present a novel multiscale model of cell migration on ECMs of defined biophysical properties that integrates local activation of biochemical signals with adhesion and force generation at the cell-ECM interface. We capture the mechanosensitivity of individual cellular components by dynamically coupling ECM properties to the activation of Rho and Rac GTPases in specific portions of the cell with actomyosin contractility, cell-ECM adhesion bond formation and rupture, and process extension and retraction. We show that our framework is capable of recreating key experimentally-observed features of the relationship between cell migration and ECM biophysical properties. In particular, our model predicts for the first time recently reported transitions from filopodial to "stick-slip" to gliding motility on ECMs of increasing stiffness, previously observed dependences of migration speed on ECM stiffness and ligand density, and high-resolution measurements of mechanosensitive protrusion dynamics during cell motility we newly obtained for this study. It also relates the biphasic dependence of cell migration speed on ECM stiffness to the tendency of the cell to polarize. By enabling the investigation of experimentally-inaccessible microscale relationships between mechanotransductive signaling, adhesion, and motility, our model offers new insight into how these factors interact with one another to produce complex migration patterns across a variety of ECM conditions.     

Apigenin induces apoptosis by targeting inhibitor of apoptosis proteins and Ku70–Bax interaction in prostate cancer

Dysfunction of the apoptotic pathway in prostate cancer cells confers apoptosis resistance towards various therapies. A novel strategy to overcome resistance is to directly target the apoptotic pathway in cancer cells. Apigenin, an anticancer agent, selectively toxic to cancer cells induces cell cycle arrest and apoptosis through mechanisms which are not fully explored. In the present study we provide novel insight into the mechanisms of apoptosis induction by apigenin. Treatment of androgen-refractory human prostate cancer PC-3 and DU145 cells with apigenin resulted in dose-dependent suppression of XIAP, c-IAP1, c-IAP2 and survivin protein levels. Apigenin treatment resulted in significant decrease in cell viability and apoptosis induction with the increase of cytochrome C in time-dependent manner. These effects of apigenin were accompanied by decrease in Bcl-xL and Bcl-2 and increase in the active form of Bax protein. The apigenin-mediated increase in Bax was due to dissociation of Bax from Ku70 which is essential for apoptotic activity of Bax. Apigenin treatment resulted in the inhibition of class I histone deacetylases and HDAC1 protein expression, thereby increasing the acetylation of Ku70 and the dissociation of Bax resulting in apoptosis of cancer cells. Furthermore, apigenin significantly reduced HDAC1 occupancy at the XIAP promoter, suggesting that histone deacetylation might be critical for XIAP downregulation. These results suggest that apigenin targets inhibitor of apoptosis proteins and Ku70–Bax interaction in the induction of apoptosis in prostate cancer cells and in athymic nude mouse xenograft model endorsing its in vivo efficacy.     

A model-based optimization framework for the inference on gene regulatory networks from DNA array data

MOTIVATION: Identification of the regulatory structures in genetic networks and the formulation of mechanistic models in the form of wiring diagrams is one of the significant objectives of expression profiling using DNA microarray technologies and it requires the development and application of identification frameworks. RESULTS: We have developed a novel optimization framework for identifying regulation in a genetic network using the S-system modeling formalism. We show that balance equations on both mRNA and protein species led to a formulation suitable for analyzing DNA-microarray data whereby protein concentrations have been eliminated and only mRNA relative concentrations are retained. Using this formulation, we examined if it is possible to infer a set of possible genetic regulatory networks consistent with observed mRNA expression patterns. Two origins of changes in mRNA expression patterns were considered. One derives from changes in the biophysical properties of the system that alter the molecular-interaction kinetics and/or message stability. The second is due to gene knock-outs. We reduced the identification problem to an optimization problem (of the so-called mixed-integer non-linear programming class) and we developed an algorithmic procedure for solving this optimization problem. Using simulated data generated by our mathematical model, we show that our method can actually find the regulatory network from which the data were generated. We also show that the number of possible alternate genetic regulatory networks depends on the size of the dataset (i.e. number of experiments), but this dependence is different for each of the two types of problems considered, and that a unique solution requires fewer datasets than previously estimated in the literature. This is the first method that also allows the identification of every possible regulatory network that could explain the data, when the number of experiments does not allow identification of unique regulatory structure.     

Novel role for the C terminus of Saccharomyces cerevisiae Rev1 in mediating protein-protein interactions

The Saccharomyces cerevisiae REV3/7-encoded polymerase zeta and Rev1 are central to the replicative bypass of DNA lesions, a process called translesion synthesis (TLS). While yeast polymerase zeta extends from distorted DNA structures, Rev1 predominantly incorporates C residues from across a template G and a variety of DNA lesions. Intriguingly, Rev1 catalytic activity does not appear to be required for TLS. Instead, yeast Rev1 is thought to participate in TLS by facilitating protein-protein interactions via an N-terminal BRCT motif. In addition, higher eukaryotic homologs of Rev1 possess a C terminus that interacts with other TLS polymerases. Due to a lack of sequence similarity, the yeast Rev1 C-terminal region, located after the polymerase domain, had initially been thought not to play a role in TLS. Here, we report that elevated levels of the yeast Rev1 C terminus confer a strong dominant-negative effect on viability and induced mutagenesis after DNA damage, highlighting the crucial role that the C terminus plays in DNA damage tolerance. We show that this phenotype requires REV7 and, using immunoprecipitations from crude extracts, demonstrate that, in addition to the polymerase-associated domain, the extreme Rev1 C terminus and the BRCT region of Rev1 mediate interactions with Rev7.     

Novel benzimidazole derivatives as selective CB2 agonists

The preparation and evaluation of a novel class of CB2 agonists based on a benzimidazole moiety are reported. They showed binding affinities up to 1nM towards the CB2 receptor with partial to full agonist potencies. They also demonstrated good to excellent selectivity (>1000-fold) over the CB1 receptor.     

An investigation of the foliar trichomes of Tetradenia riparia (Hochst.) Codd [Lamiaceae]: An important medicinal plant of Southern Africa

The morphology and distribution of leaf trichomes of Tetradenia riparia were studied using light and scanning microscopy. Three morphologically distinct types of trichomes were observed on T. riparia leaf surfaces: glandular capitate (short and long stalked), peltate and non-glandular. The glandular and non-glandular trichomes were present in abundance on both the adaxial and abaxial surfaces. Young leaves were densely covered with trichomes; however, the density of trichomes decreases progressively with leaf maturity. This suggests that the trichomes are established early in leaf differentiation and their density decreases with leaf development and age.