Regulation of Ras Exchange Factors and Cellular Localization of Ras Activation by Lipid Messengers in T Cells

PubMed Central

Jun, Jesse E.; Rubio, Ignacio; Roose, Jeroen P.

2013-01-01

The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs) catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and Son of Sevenless (SOS)-family GEFs. Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood. One large group of biomolecules critically involved in the control of RasGEFs functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR) stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells. PMID:24027568

Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion

PubMed Central

Rennoll-Bankert, Kristen E.; Rahman, M. Sayeedur; Gillespie, Joseph J.; Guillotte, Mark L.; Kaur, Simran J.; Lehman, Stephanie S.; Beier-Sexton, Magda; Azad, Abdu F.

2015-01-01

Bacterial Sec7-domain-containing proteins (RalF) are known only from species of Legionella and Rickettsia, which have facultative and obligate intracellular lifestyles, respectively. L. pneumophila RalF, a type IV secretion system (T4SS) effector, is a guanine nucleotide exchange factor (GEF) of ADP-ribosylation factors (Arfs), activating and recruiting host Arf1 to the Legionella-containing vacuole. In contrast, previous in vitro studies showed R. prowazekii (Typhus Group) RalF is a functional Arf-GEF that localizes to the host plasma membrane and interacts with the actin cytoskeleton via a unique C-terminal domain. As RalF is differentially encoded across Rickettsia species (e.g., pseudogenized in all Spotted Fever Group species), it may function in lineage-specific biology and pathogenicity. Herein, we demonstrate RalF of R. typhi (Typhus Group) interacts with the Rickettsia T4SS coupling protein (RvhD4) via its proximal C-terminal sequence. RalF is expressed early during infection, with its inactivation via antibody blocking significantly reducing R. typhi host cell invasion. For R. typhi and R. felis (Transitional Group), RalF ectopic expression revealed subcellular localization with the host plasma membrane and actin cytoskeleton. Remarkably, R. bellii (Ancestral Group) RalF showed perinuclear localization reminiscent of ectopically expressed Legionella RalF, for which it shares several structural features. For R. typhi, RalF co-localization with Arf6 and PI(4,5)P2 at entry foci on the host plasma membrane was determined to be critical for invasion. Thus, we propose recruitment of PI(4,5)P2 at entry foci, mediated by RalF activation of Arf6, initiates actin remodeling and ultimately facilitates bacterial invasion. Collectively, our characterization of RalF as an invasin suggests that, despite carrying a similar Arf-GEF unknown from other bacteria, different intracellular lifestyles across Rickettsia and Legionella species have driven divergent roles for Ral

The Amino-Terminus of Vav1 Regulates TCR-Induced SLP-76 Microclusters via the Calponin Homology Domain and Non-Catalytic Surfaces within the GEF Module

PubMed Central

Sylvain, Nicholas R.; Nguyen, Ken; Bunnell, Stephen C.

2013-01-01

The guanine nucleotide exchange factor (GEF) Vav1 synergizes with the adapter SLP-76 to support T cell development and activation. Here, we demonstrate that Vav1 controls the stability and movement T cell receptor-induced SLP-76 microclusters. The SH2 domain enables the recruitment of Vav1 into SLP-76 microclusters, whereas the SH3 domains of Vav1 cooperate to enhance microcluster stability and function. Although the amino-terminus of Vav1 is essential for downstream signaling, it possesses novel scaffolding functions that are unaffected by the inactivation of the Vav1 GEF or by the constitutive GEF activation that accompanies the mutation of the regulatory tyrosine residues 142, 160, and 174. In contrast, GEF-inactivating point mutations predicted to perturb the structural integrity of the Vav1 GEF abolish these scaffolding functions. Paradoxically, the excision of catalytic Dbl-homology (DH) / pleckstrin homology (PH) cassette produces a relatively mild scaffolding defect, indicating that the L213A and L278Q point mutations antagonize scaffolding functions mediated by adjacent domains. A deletion mutant lacking the CH domain potently inhibits calcium responses, but also exhibits mild scaffolding defects. We conclude multiple GEF-independent scaffolding functions contained within the amino-terminus of Vav1 contribute to T cell activation by acting synergistically to increase the stability and functionality of SLP-76 microclusters. PMID:21386095

The Tea4-PP1 landmark promotes local growth by dual Cdc42 GEF recruitment and GAP exclusion.

PubMed

Kokkoris, Kyriakos; Gallo Castro, Daniela; Martin, Sophie G

2014-05-01

Cell polarization relies on small GTPases, such as Cdc42, which can break symmetry through self-organizing principles, and landmarks that define the axis of polarity. In fission yeast, microtubules deliver the Tea1-Tea4 complex to mark cell poles for growth, but how this complex activates Cdc42 is unknown. Here, we show that ectopic targeting of Tea4 to cell sides promotes the local activation of Cdc42 and cell growth. This activity requires that Tea4 binds the type I phosphatase (PP1) catalytic subunit Dis2 or Sds21, and ectopic targeting of either catalytic subunit is similarly instructive for growth. The Cdc42 guanine-nucleotide-exchange factor Gef1 and the GTPase-activating protein Rga4 are required for Tea4-PP1-dependent ectopic growth. Gef1 is recruited to ectopic Tea4 and Dis2 locations to promote Cdc42 activation. By contrast, Rga4 is locally excluded by Tea4, and its forced colocalization with Tea4 blocks ectopic growth, indicating that Rga4 must be present, but at sites distinct from Tea4. Thus, a Tea4-PP1 landmark promotes local Cdc42 activation and growth both through Cdc42 GEF recruitment and by creating a local trough in a Cdc42 GAP.

Global Environment Facility (GEF): An Overview

DTIC Science & Technology

2010-05-17

e.g., choosing solar energy technology over coal technology meets the same national development goal of power generation but is more costly. GEF grants...mechanism for a specific convention. Through an association with regional agreements, it targets trans-boundary water systems, such as river basins with...Caribbean corridor in Costa Rica. Prevention and Management of Marine Pollution in the East Asian Seas in and around Indonesia GEF Grant: $8,025,000

Dynamic Control of Excitatory Synapse Development by a Rac1 GEF/GAP Regulatory Complex

PubMed Central

Um, Kyongmi; Niu, Sanyong; Duman, Joseph G.; Cheng, Jinxuan; Tu, Yen-Kuei; Schwechter, Brandon; Liu, Feng; Hiles, Laura; Narayanan, Anjana; Ash, Ryan T.; Mulherkar, Shalaka; Alpadi, Kannan; Smirnakis, Stelios M.; Tolias, Kimberley F.

2014-01-01

SUMMARY The small GTPase Rac1 orchestrates actin-dependent remodeling essential for numerous cellular processes including synapse development. While precise spatiotemporal regulation of Rac1 is necessary for its function, little is known about the mechanisms that enable Rac1 activators (GEFs) and inhibitors (GAPs) to act in concert to regulate Rac1 signaling. Here we identify a regulatory complex composed of a Rac-GEF (Tiam1) and a Rac-GAP (Bcr) that cooperate to control excitatory synapse development. Disruption of Bcr function within this complex increases Rac1 activity and dendritic spine remodeling, resulting in excessive synaptic growth that is rescued by Tiam1 inhibition. Notably, EphB receptors utilize the Tiam1-Bcr complex to control synaptogenesis. Following EphB activation, Tiam1 induces Rac1-dependent spine formation, whereas Bcr prevents Rac1-mediated receptor internalization, promoting spine growth over retraction. The finding that a Rac-specific GEF/GAP complex is required to maintain optimal levels of Rac1 signaling provides an important insight into the regulation of small GTPases. PMID:24960694

Neurodevelopmental disease-associated de novo mutations and rare sequence variants affect TRIO GDP/GTP exchange factor activity.

PubMed

Katrancha, Sara M; Wu, Yi; Zhu, Minsheng; Eipper, Betty A; Koleske, Anthony J; Mains, Richard E

2017-12-01

Bipolar disorder, schizophrenia, autism and intellectual disability are complex neurodevelopmental disorders, debilitating millions of people. Therapeutic progress is limited by poor understanding of underlying molecular pathways. Using a targeted search, we identified an enrichment of de novo mutations in the gene encoding the 330-kDa triple functional domain (TRIO) protein associated with neurodevelopmental disorders. By generating multiple TRIO antibodies, we show that the smaller TRIO9 isoform is the major brain protein product, and its levels decrease after birth. TRIO9 contains two guanine nucleotide exchange factor (GEF) domains with distinct specificities: GEF1 activates both Rac1 and RhoG; GEF2 activates RhoA. To understand the impact of disease-associated de novo mutations and other rare sequence variants on TRIO function, we utilized two FRET-based biosensors: a Rac1 biosensor to study mutations in TRIO (T)GEF1, and a RhoA biosensor to study mutations in TGEF2. We discovered that one autism-associated de novo mutation in TGEF1 (K1431M), at the TGEF1/Rac1 interface, markedly decreased its overall activity toward Rac1. A schizophrenia-associated rare sequence variant in TGEF1 (F1538Intron) was substantially less active, normalized to protein level and expressed poorly. Overall, mutations in TGEF1 decreased GEF1 activity toward Rac1. One bipolar disorder-associated rare variant (M2145T) in TGEF2 impaired inhibition by the TGEF2 pleckstrin-homology domain, resulting in dramatically increased TGEF2 activity. Overall, genetic damage to both TGEF domains altered TRIO catalytic activity, decreasing TGEF1 activity and increasing TGEF2 activity. Importantly, both GEF changes are expected to decrease neurite outgrowth, perhaps consistent with their association with neurodevelopmental disorders. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Specific retention of the protostome-specific PsGEF may parallel with the evolution of mushroom bodies in insect and lophotrochozoan brains.

PubMed

Higuchi, Nozomu; Kohno, Keigo; Kadowaki, Tatsuhiko

2009-05-07

Gene gain and subsequent retention or loss during evolution may be one of the underlying mechanisms involved in generating the diversity of metazoan nervous systems. However, the causal relationships acting therein have not been studied extensively. We identified the gene PsGEF (protostome-specific GEF), which is present in all the sequenced genomes of insects and limpet but absent in those of sea anemones, deuterostomes, and nematodes. In Drosophila melanogaster, PsGEF encodes a short version of a protein with the C2 and PDZ domains, as well as a long version with the C2, PDZ, and RhoGEF domains through alternative splicing. Intriguingly, the exons encoding the RhoGEF domain are specifically deleted in the Daphnia pulex genome, suggesting that Daphnia PsGEF contains only the C2 and PDZ domains. Thus, the distribution of PsGEF containing the C2, PDZ, and RhoGEF domains among metazoans appears to coincide with the presence of mushroom bodies. Mushroom bodies are prominent neuropils involved in the processing of multiple sensory inputs as well as associative learning in the insect, platyhelminth, and annelid brains. In the adult Drosophila brain, PsGEF is expressed in mushroom bodies, antennal lobe, and optic lobe, where it is necessary for the correct axon branch formation of alpha/beta neurons in mushroom bodies. PsGEF genetically interacts with Rac1 but not other Rho family members, and the RhoGEF domain of PsGEF induces actin polymerization in the membrane, thus resulting in the membrane ruffling that is observed in cultured cells with activated forms of Rac. The specific acquisition of PsGEF by the last common ancestor of protostomes followed by its retention or loss in specific animal species during evolution demonstrates that there are some structural and/or functional features common between insect and lophotrochozoan nervous systems (for example, mushroom bodies), which are absent in all deuterostomes and cnidarians. PsGEF is therefore one of genes

AKAP13 Rho-GEF and PKD-Binding Domain Deficient Mice Develop Normally but Have an Abnormal Response to β-Adrenergic-Induced Cardiac Hypertrophy

PubMed Central

Spindler, Matthew J.; Burmeister, Brian T.; Huang, Yu; Hsiao, Edward C.; Salomonis, Nathan; Scott, Mark J.; Srivastava, Deepak; Carnegie, Graeme K.; Conklin, Bruce R.

2013-01-01

Background A-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate G-protein signaling events to regulate development, physiology, and disease. One family member, AKAP13, encodes for multiple protein isoforms that contain binding sites for protein kinase A (PKA) and D (PKD) and an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. In mice, AKAP13 is required for development as null embryos die by embryonic day 10.5 with cardiovascular phenotypes. Additionally, the AKAP13 Rho-GEF and PKD-binding domains mediate cardiomyocyte hypertrophy in cell culture. However, the requirements for the Rho-GEF and PKD-binding domains during development and cardiac hypertrophy are unknown. Methodology/Principal Findings To determine if these AKAP13 protein domains are required for development, we used gene-trap events to create mutant mice that lacked the Rho-GEF and/or the protein kinase D-binding domains. Surprisingly, heterozygous matings produced mutant mice at Mendelian ratios that had normal viability and fertility. The adult mutant mice also had normal cardiac structure and electrocardiograms. To determine the role of these domains during β-adrenergic-induced cardiac hypertrophy, we stressed the mice with isoproterenol. We found that heart size was increased similarly in mice lacking the Rho-GEF and PKD-binding domains and wild-type controls. However, the mutant hearts had abnormal cardiac contractility as measured by fractional shortening and ejection fraction. Conclusions These results indicate that the Rho-GEF and PKD-binding domains of AKAP13 are not required for mouse development, normal cardiac architecture, or β-adrenergic-induced cardiac hypertrophic remodeling. However, these domains regulate aspects of β-adrenergic-induced cardiac hypertrophy. PMID:23658642

Function of the nucleotide exchange activity of vav1 in T cell development and activation.

PubMed

Saveliev, Alexander; Vanes, Lesley; Ksionda, Olga; Rapley, Jonathan; Smerdon, Stephen J; Rittinger, Katrin; Tybulewicz, Victor L J

2009-12-15

The guanine nucleotide exchange factor (GEF) Vav1 is essential for transducing T cell antigen receptor (TCR) signals and therefore plays a critical role in the development and activation of T cells. It has been presumed that the GEF activity of Vav1 is important for its function; however, there has been no direct demonstration of this. Here, we generated mice expressing enzymatically inactive, but normally folded, Vav1 protein. Analysis of these mice showed that the GEF activity of Vav1 was necessary for the selection of thymocytes and for the optimal activation of T cells, including signal transduction to Rac1, Akt, and integrins. In contrast, the GEF activity of Vav1 was not required for TCR-induced calcium flux, activation of extracellular signal-regulated kinase and protein kinase D1, and cell polarization. Thus, in T cells, the GEF activity of Vav1 is essential for some, but not all, of its functions.

Function of the Nucleotide Exchange Activity of Vav1 in T cell Development and Activation*

PubMed Central

Saveliev, Alexander; Vanes, Lesley; Ksionda, Olga; Rapley, Jonathan; Smerdon, Stephen J.; Rittinger, Katrin; Tybulewicz, Victor L. J.

2012-01-01

The guanine nucleotide exchange factor (GEF) Vav1 is essential for transducing T cell antigen receptor (TCR) signals and therefore plays a critical role in the development and activation of T cells. It has been presumed that the GEF activity of Vav1 is important for its function; however, there has been no direct demonstration of this. Here, we generated mice expressing enzymatically inactive, but normally folded, Vav1 protein. Analysis of these mice showed that the GEF activity of Vav1 was necessary for the selection of thymocytes and for the optimal activation of T cells, including signal transduction to Rac1, Akt, and integrins. In contrast, the GEF activity of Vav1 was not required for TCR-induced calcium flux, activation of extracellular signal–regulated kinase (ERK) and protein kinase D1 (PKD1), and cell polarization. Thus, in T cells, the GEF activity of Vav1 is essential for some, but not all, of its functions. PMID:20009105

The Evolutionary Landscape of Dbl-Like RhoGEF Families: Adapting Eukaryotic Cells to Environmental Signals

PubMed Central

Blangy, Anne

2017-01-01

Abstract The dynamics of cell morphology in eukaryotes is largely controlled by small GTPases of the Rho family. Rho GTPases are activated by guanine nucleotide exchange factors (RhoGEFs), of which diffuse B-cell lymphoma (Dbl)-like members form the largest family. Here, we surveyed Dbl-like sequences from 175 eukaryotic genomes and illuminate how the Dbl family evolved in all eukaryotic supergroups. By combining probabilistic phylogenetic approaches and functional domain analysis, we show that the human Dbl-like family is made of 71 members, structured into 20 subfamilies. The 71 members were already present in ancestral jawed vertebrates, but several members were subsequently lost in specific clades, up to 12% in birds. The jawed vertebrate repertoire was established from two rounds of duplications that occurred between tunicates, cyclostomes, and jawed vertebrates. Duplicated members showed distinct tissue distributions, conserved at least in Amniotes. All 20 subfamilies have members in Deuterostomes and Protostomes. Nineteen subfamilies are present in Porifera, the first phylum that diverged in Metazoa, 14 in Choanoflagellida and Filasterea, single-celled organisms closely related to Metazoa and three in Fungi, the sister clade to Metazoa. Other eukaryotic supergroups show an extraordinary variability of Dbl-like repertoires as a result of repeated and independent gain and loss events. Last, we observed that in Metazoa, the number of Dbl-like RhoGEFs varies in proportion of cell signaling complexity. Overall, our analysis supports the conclusion that Dbl-like RhoGEFs were present at the origin of eukaryotes and evolved as highly adaptive cell signaling mediators. PMID:28541439

Activated RhoA Is a Positive Feedback Regulator of the Lbc Family of Rho Guanine Nucleotide Exchange Factor Proteins*

PubMed Central

Medina, Frank; Carter, Angela M.; Dada, Olugbenga; Gutowski, Stephen; Hadas, Jana; Chen, Zhe; Sternweis, Paul C.

2013-01-01

The monomeric Rho GTPases are essential for cellular regulation including cell architecture and movement. A direct mechanism for hormonal regulation of the RhoA-type GTPases is their modulation by the G12 and G13 proteins via RH (RGS homology) containing RhoGEFs. In addition to the interaction of the G protein α subunits with the RH domain, activated RhoA also binds to the pleckstrin homology (PH) domain of PDZRhoGEF. The latter interaction is now extended to all seven members of the homologous Lbc family of RhoGEFs which includes the RH-RhoGEFs. This is evinced by direct measurements of binding or through effects on selected signaling pathways in cells. Overexpression of these PH domains alone can block RhoA-dependent signaling in cells to various extents. Whereas activated RhoA does not modulate the intrinsic activity of the RhoGEFs, activated RhoA associated with phospholipid vesicles can facilitate increased activity of soluble RhoGEFs on vesicle-delimited substrate (RhoA-GDP). This demonstrates feasibility of the hypothesis that binding of activated RhoA to the PH domains acts as a positive feedback mechanism. This is supported by cellular studies in which mutation of this binding site on PH strongly attenuates the stimulation of RhoA observed by overexpression of five of the RhoGEF DH-PH domains. This mutation is even more dramatic in the context of full-length p115RhoGEF. The utilization of this mechanism by multiple RhoGEFs suggests that this regulatory paradigm may be a common feature in the broader family of RhoGEFs. PMID:23493395

RHGF-2 Is an Essential Rho-1 Specific RhoGEF that binds to the Multi-PDZ Domain Scaffold Protein MPZ-1 in Caenorhabditis elegans

PubMed Central

Lin, Li; Tran, Thuy; Hu, Shuang; Cramer, Todd; Komuniecki, Richard; Steven, Robert M.

2012-01-01

RhoGEF proteins activate the Rho family of small GTPases and thus play a key role in regulating fundamental cellular processes such as cell morphology and polarity, cell cycle progression and gene transcription. We identified a Caenorhabditis elegans RhoGEF protein, RHGF-2, as a binding partner of the C. elegans multi-PDZ domain scaffold protein MPZ-1 (MUPP1 in mammals). RHGF-2 exhibits significant identity to the mammalian RhoGEFs PLEKHG5/Tech/Syx and contains a class I C-terminal PDZ binding motif (SDV) that interacts most strongly to MPZ-1 PDZ domain eight. RHGF-2 RhoGEF activity is specific to the C. elegans RhoA homolog RHO-1 as determined by direct binding, GDP/GTP exchange and serum response element-driven reporter activity. rhgf-2 is an essential gene since rhgf-2 deletion mutants do not elongate during embryogenesis and hatch as short immobile animals that arrest development. Interestingly, the expression of a functional rhgf-2::gfp transgene appears to be exclusively neuronal and rhgf-2 overexpression results in loopy movement with exaggerated body bends. Transient expression of RHGF-2 in N1E-115 neuroblastoma cells prevents neurite outgrowth similar to constitutive RhoA activation in these cells. Together, these observations indicate neuronally expressed RHGF-2 is an essential RHO-1 specific RhoGEF that binds most strongly to MPZ-1 PDZ domain eight and is required for wild-type C. elegans morphology and growth. PMID:22363657

Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malaby, Andrew W.; Das, Sanchaita; Chakravarthy, Srinivas

Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negative-stain electron microscopy were used to investigate the structural organization andmore » conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.« less

Viral Activation of MK2-hsp27-p115RhoGEF-RhoA Signaling Axis Causes Cytoskeletal Rearrangements, P-body Disruption and ARE-mRNA Stabilization

PubMed Central

Corcoran, Jennifer A.; Johnston, Benjamin P.; McCormick, Craig

2015-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of several AIDS-related cancers, including the endothelial cell (EC) neoplasm Kaposi's sarcoma (KS). KSHV-infected ECs secrete abundant host-derived pro-inflammatory molecules and angiogenic factors that contribute to tumorigenesis. The precise contributions of viral gene products to this secretory phenotype remain to be elucidated, but there is emerging evidence for post-transcriptional regulation. The Kaposin B (KapB) protein is thought to contribute to the secretory phenotype in infected cells by binding and activating the stress-responsive kinase MK2, thereby selectively blocking decay of AU-rich mRNAs (ARE-mRNAs) encoding pro-inflammatory cytokines and angiogenic factors. Processing bodies (PBs) are cytoplasmic ribonucleoprotein foci in which ARE-mRNAs normally undergo rapid 5′ to 3′ decay. Here, we demonstrate that PB dispersion is a feature of latent KSHV infection, which is dependent on kaposin protein expression. KapB is sufficient to disperse PBs, and KapB-mediated ARE-mRNA stabilization could be partially reversed by treatments that restore PBs. Using a combination of genetic and chemical approaches we provide evidence that KapB-mediated PB dispersion is dependent on activation of a non-canonical Rho-GTPase signaling axis involving MK2, hsp27, p115RhoGEF and RhoA. PB dispersion in latently infected cells is likewise dependent on p115RhoGEF. In addition to PB dispersion, KapB-mediated RhoA activation in primary ECs caused actin stress fiber formation, increased cell motility and angiogenesis; these effects were dependent on the activity of the RhoA substrate kinases ROCK1/2. By contrast, KapB-mediated PB dispersion occurred in a ROCK1/2-independent manner. Taken together, these observations position KapB as a key contributor to viral reprogramming of ECs, capable of eliciting many of the phenotypes characteristic of KS tumor cells, and strongly contributing to the post

Improving GEFS Weather Forecasts for Indian Monsoon with Statistical Downscaling

NASA Astrophysics Data System (ADS)

Agrawal, Ankita; Salvi, Kaustubh; Ghosh, Subimal

2014-05-01

Weather forecast has always been a challenging research problem, yet of a paramount importance as it serves the role of 'key input' in formulating modus operandi for immediate future. Short range rainfall forecasts influence a wide range of entities, right from agricultural industry to a common man. Accurate forecasts actually help in minimizing the possible damage by implementing pre-decided plan of action and hence it is necessary to gauge the quality of forecasts which might vary with the complexity of weather state and regional parameters. Indian Summer Monsoon Rainfall (ISMR) is one such perfect arena to check the quality of weather forecast not only because of the level of intricacy in spatial and temporal patterns associated with it, but also the amount of damage it can cause (because of poor forecasts) to the Indian economy by affecting agriculture Industry. The present study is undertaken with the rationales of assessing, the ability of Global Ensemble Forecast System (GEFS) in predicting ISMR over central India and the skill of statistical downscaling technique in adding value to the predictions by taking them closer to evidentiary target dataset. GEFS is a global numerical weather prediction system providing the forecast results of different climate variables at a fine resolution (0.5 degree and 1 degree). GEFS shows good skills in predicting different climatic variables but fails miserably over rainfall predictions for Indian summer monsoon rainfall, which is evident from a very low to negative correlation values between predicted and observed rainfall. Towards the fulfilment of second rationale, the statistical relationship is established between the reasonably well predicted climate variables (GEFS) and observed rainfall. The GEFS predictors are treated with multicollinearity and dimensionality reduction techniques, such as principal component analysis (PCA) and least absolute shrinkage and selection operator (LASSO). Statistical relationship is

A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

PubMed

Reichman, Melvin; Schabdach, Amanda; Kumar, Meera; Zielinski, Tom; Donover, Preston S; Laury-Kleintop, Lisa D; Lowery, Robert G

2015-12-01

Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs. © 2015 Society for Laboratory Automation and Screening.

Synthesis and properties of Rb2GeF6:Mn4+ red-emitting phosphors

NASA Astrophysics Data System (ADS)

Sakurai, Shono; Nakamura, Toshihiro; Adachi, Sadao

2018-02-01

Rb2GeF6:Mn4+ red-emitting phosphors were synthesized by coprecipitation and their structural and optical properties were investigated by laser microscopy observation, X-ray diffraction (XRD) analysis, photoluminescence (PL) analysis, PL excitation (PLE) spectroscopy, and PL decay measurement. Single-crystalline ingots in the form of a hexagonal pyramid were prepared with a basal plane diameter of ˜2 mm. The XRD analysis suggested that Rb2GeF6 crystallizes in the hexagonal structure (C6v4 = P63mc) with a = 0.5955 nm and c = 0.9672 nm. The phosphor exhibited the strong Mn4+-related zero-phonon line (ZPL) emission peak typically observed in host crystals with piezoelectrically active lattices such as a hexagonal lattice. The quantum efficiencies of the bulk ingot and powdered samples were 87 and 74%, respectively, with nearly the same luminescence decay time of ˜6 ms. The exact ZPL energies and related crystal-field and Racah parameters were obtained from the PL and PLE spectra by Franck-Condon analysis. Temperature-dependent PL intensities were analyzed from T = 20 to 500 K using a thermal quenching model by considering Bose-Einstein phonon statistics. A comparative discussion on the phosphor properties of Rb2GeF6:Mn4+ and Rb2MF6:Mn4+ with M = Si and Ti was also given.

Acetylation of the RhoA GEF Net1A controls its subcellular localization and activity

PubMed Central

Song, Eun Hyeon; Oh, Wonkyung; Ulu, Arzu; Carr, Heather S.; Zuo, Yan; Frost, Jeffrey A.

2015-01-01

ABSTRACT Net1 isoform A (Net1A) is a RhoA GEF that is required for cell motility and invasion in multiple cancers. Nuclear localization of Net1A negatively regulates its activity, and we have recently shown that Rac1 stimulates Net1A relocalization to the plasma membrane to promote RhoA activation and cytoskeletal reorganization. However, mechanisms controlling the subcellular localization of Net1A are not well understood. Here, we show that Net1A contains two nuclear localization signal (NLS) sequences within its N-terminus and that residues surrounding the second NLS sequence are acetylated. Treatment of cells with deacetylase inhibitors or expression of active Rac1 promotes Net1A acetylation. Deacetylase inhibition is sufficient for Net1A relocalization outside the nucleus, and replacement of the N-terminal acetylation sites with arginine residues prevents cytoplasmic accumulation of Net1A caused by deacetylase inhibition or EGF stimulation. By contrast, replacement of these sites with glutamine residues is sufficient for Net1A relocalization, RhoA activation and downstream signaling. Moreover, the N-terminal acetylation sites are required for rescue of F-actin accumulation and focal adhesion maturation in Net1 knockout MEFs. These data indicate that Net1A acetylation regulates its subcellular localization to impact on RhoA activity and actin cytoskeletal organization. PMID:25588829

A dual role for the RhoGEF Ephexin5 in regulation of dendritic spine outgrowth

PubMed Central

Hamilton, AM; Lambert, JT; Parajuli, LK; Vivas, O; Park, DK; Stein, IS; Jahncke, JN; Greenberg, ME; Margolis, SS; Zito, K

2017-01-01

The outgrowth of new dendritic spines is closely linked to the formation of new synapses, and is thought to be a vital component of the experience-dependent circuit plasticity that supports learning. Here, we examined the role of the RhoGEF Ephexin5 in driving activity-dependent spine outgrowth. We found that reducing Ephexin5 levels increased spine outgrowth, and increasing Ephexin5 levels decreased spine outgrowth in a GEF-dependent manner, suggesting that Ephexin5 acts as an inhibitor of spine outgrowth. Notably, we found that increased neural activity led to a proteasome-dependent reduction in the levels of Ephexin5 in neuronal dendrites, which could facilitate the enhanced spine outgrowth observed following increased neural activity. Surprisingly, we also found that Ephexin5-GFP levels were elevated on the dendrite at sites of future new spines, prior to new spine outgrowth. Moreover, lowering neuronal Ephexin5 levels inhibited new spine outgrowth in response to both global increases in neural activity and local glutamatergic stimulation of the dendrite, suggesting that Ephexin5 is necessary for activity-dependent spine outgrowth. Our data support a model in which Ephexin5 serves a dual role in spinogenesis, acting both as a brake on overall spine outgrowth and as a necessary component in the site-specific formation of new spines. PMID:28185854

A dual role for the RhoGEF Ephexin5 in regulation of dendritic spine outgrowth.

PubMed

Hamilton, A M; Lambert, J T; Parajuli, L K; Vivas, O; Park, D K; Stein, I S; Jahncke, J N; Greenberg, M E; Margolis, S S; Zito, K

2017-04-01

The outgrowth of new dendritic spines is closely linked to the formation of new synapses, and is thought to be a vital component of the experience-dependent circuit plasticity that supports learning. Here, we examined the role of the RhoGEF Ephexin5 in driving activity-dependent spine outgrowth. We found that reducing Ephexin5 levels increased spine outgrowth, and increasing Ephexin5 levels decreased spine outgrowth in a GEF-dependent manner, suggesting that Ephexin5 acts as an inhibitor of spine outgrowth. Notably, we found that increased neural activity led to a proteasome-dependent reduction in the levels of Ephexin5 in neuronal dendrites, which could facilitate the enhanced spine outgrowth observed following increased neural activity. Surprisingly, we also found that Ephexin5-GFP levels were elevated on the dendrite at sites of future new spines, prior to new spine outgrowth. Moreover, lowering neuronal Ephexin5 levels inhibited new spine outgrowth in response to both global increases in neural activity and local glutamatergic stimulation of the dendrite, suggesting that Ephexin5 is necessary for activity-dependent spine outgrowth. Our data support a model in which Ephexin5 serves a dual role in spinogenesis, acting both as a brake on overall spine outgrowth and as a necessary component in the site-specific formation of new spines. Copyright © 2017 Elsevier Inc. All rights reserved.

General Description of Fission Observables: GEF Model Code

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmidt, K.-H.; Jurado, B., E-mail: jurado@cenbg.in2p3.fr; Amouroux, C.

2016-01-15

The GEF (“GEneral description of Fission observables”) model code is documented. It describes the observables for spontaneous fission, neutron-induced fission and, more generally, for fission of a compound nucleus from any other entrance channel, with given excitation energy and angular momentum. The GEF model is applicable for a wide range of isotopes from Z = 80 to Z = 112 and beyond, up to excitation energies of about 100 MeV. The results of the GEF model are compared with fission barriers, fission probabilities, fission-fragment mass- and nuclide distributions, isomeric ratios, total kinetic energies, and prompt-neutron and prompt-gamma yields and energymore » spectra from neutron-induced and spontaneous fission. Derived properties of delayed neutrons and decay heat are also considered. The GEF model is based on a general approach to nuclear fission that explains a great part of the complex appearance of fission observables on the basis of fundamental laws of physics and general properties of microscopic systems and mathematical objects. The topographic theorem is used to estimate the fission-barrier heights from theoretical macroscopic saddle-point and ground-state masses and experimental ground-state masses. Motivated by the theoretically predicted early localisation of nucleonic wave functions in a necked-in shape, the properties of the relevant fragment shells are extracted. These are used to determine the depths and the widths of the fission valleys corresponding to the different fission channels and to describe the fission-fragment distributions and deformations at scission by a statistical approach. A modified composite nuclear-level-density formula is proposed. It respects some features in the superfluid regime that are in accordance with new experimental findings and with theoretical expectations. These are a constant-temperature behaviour that is consistent with a considerably increased heat capacity and an increased pairing condensation energy

The Impact of STTP on the GEFS Forecast of Week-2 and Beyond in the Presence of Stochastic Physics

NASA Astrophysics Data System (ADS)

Hou, D.

2015-12-01

The Stochastic Total Tendency Perturbation (STTP) scheme was designed to represent the model related uncertainties not considered in the numerical model itself and the physics based stochastic schemes. It has been applied in NCEP's Global Ensemble Forecast System (GEFS) since 2010, showing significant positive impacts on the forecast with improved spread-error ratio and probabilistic forecast skills. The scheme is robust and it went well with the resolution increases and model improvements in 2012 and 2015 with minimum changes. Recently, a set of stochastic physics schemes are coded in the Global Forecast System model and tested in the GEFS package. With these schemes turned on and STTP off, the forecast performance is comparable or even superior to the operational GEFS, in which STTP is the only contributor to the model related uncertainties. This is true especially in week one. However, over the second week and beyond, both the experimental and the operational GEFS has insufficient spread, especially over the warmer seasons. This is a major challenge when the GEFS is extended to sub-seasonal (week 4-6) time scales. The impact of STTP on the GEFS forecast in the presence of stochastic physics is investigated by turning both the stochastic physics schemes and STTP on and carefully tuning their amplitudes. Analysis will be focused on the forecast of extended range, especially week 2. Its impacts on week 3-4 will also be addressed.

Son of sevenless directly links the Robo receptor to rac activation to control axon repulsion at the midline.

PubMed

Yang, Long; Bashaw, Greg J

2006-11-22

Son of sevenless (Sos) is a dual specificity guanine nucleotide exchange factor (GEF) that regulates both Ras and Rho family GTPases and thus is uniquely poised to integrate signals that affect both gene expression and cytoskeletal reorganization. Here, using genetics, biochemistry, and cell biology, we demonstrate that Sos is recruited to the plasma membrane, where it forms a ternary complex with the Roundabout receptor and the SH3-SH2 adaptor protein Dreadlocks (Dock) to regulate Rac-dependent cytoskeletal rearrangement in response to the Slit ligand. Intriguingly, the Ras and Rac-GEF activities of Sos can be uncoupled during Robo-mediated axon repulsion; Sos axon guidance function depends on its Rac-GEF activity, but not its Ras-GEF activity. These results provide in vivo evidence that the Ras and RhoGEF domains of Sos are separable signaling modules and support a model in which Robo recruits Sos to the membrane via Dock to activate Rac during midline repulsion.

Robuste Verzweigungserkennung von Gefäßen in CTA-Datensätzen zur modellbasierten Extraktion der Centerline

NASA Astrophysics Data System (ADS)

Beck, Thomas; Fritz, Dominik; Biermann, Christina; Dillmann, Rüdiger

Bei der Befundung und Visualisierung von Blutgefäßen ist deren Centerline von zentraler Bedeutung. Die Unterscheidung zwischen unverzweigten Abschnitten des Gefäßes und Verzweigungsbereichen ermöglicht den Einsatz spezialisierter und sehr effizienter Algorithmen zur modellbasierten Extraktion der Centerline. In diesem Artikel wird ein robustes Verfahren zur Verzweigungserkennung vorgestellt. Das Verfahren beruht auf einem Front-Propagation-Ansatz mit dynamisch angepassten Schwellwerten und einer anschließenden Clusteranalyse. Die vorgestellte Methode zur Verzweigungserkennung wurde als Komponente einer Architektur zur Extraktion der Centerline auf handannotierten Datensätzen getestet. Erste Ergebnisse sind sehr vielversprechend und ermöglichen auch bei pathologischen Gefäßen eine robuste Detektion von Gefäßverzweigungen.

TGF-β regulates LARG and GEF-H1 during EMT to affect stiffening response to force and cell invasion

PubMed Central

Osborne, Lukas D.; Li, George Z.; How, Tam; O'Brien, E. Tim; Blobe, Gerard C.; Superfine, Richard; Mythreye, Karthikeyan

2014-01-01

Recent studies implicate a role for cell mechanics in cancer progression. The epithelial-to-mesenchymal transition (EMT) regulates the detachment of cancer cells from the epithelium and facilitates their invasion into stromal tissue. Although classic EMT hallmarks include loss of cell–cell adhesions, morphology changes, and increased invasion capacity, little is known about the associated mechanical changes. Previously, force application on integrins has been shown to initiate cytoskeletal rearrangements that result in increased cell stiffness and a stiffening response. Here we demonstrate that transforming growth factor β (TGF-β)–induced EMT results in decreased stiffness and loss of the normal stiffening response to force applied on integrins. We find that suppression of the RhoA guanine nucleotide exchange factors (GEFs) LARG and GEF-H1 through TGF-β/ALK5–enhanced proteasomal degradation mediates these changes in cell mechanics and affects EMT-associated invasion. Taken together, our results reveal a functional connection between attenuated stiffness and stiffening response and the increased invasion capacity acquired after TGF-β–induced EMT. PMID:25143398

ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling*

PubMed Central

Kanamarlapudi, Venkateswarlu; Thompson, Aiysha; Kelly, Eamonn; López Bernal, Andrés

2012-01-01

The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a Gs-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated that ADP-ribosylation factor (ARF)6 and its GTP-exchange factor (GEF) cytohesin 2 regulate LHCGR internalization in follicular membranes. However, the mechanisms by which ARF6 and cytohesin 2 regulate LHCGR internalization remain incompletely understood. Here we investigated the role of the ARF6 signaling pathway in the internalization of heterologously expressed human LHCGR (HLHCGR) in intact cells using a combination of pharmacological inhibitors, siRNA and the expression of mutant proteins. We found that human CG (HCG)-induced HLHCGR internalization, cAMP accumulation and ARF6 activation were inhibited by Gallein (βγ inhibitor), Wortmannin (PI 3-kinase inhibitor), SecinH3 (cytohesin ARF GEF inhibitor), QS11 (an ARF GAP inhibitor), an ARF6 inhibitory peptide and ARF6 siRNA. However, Dynasore (dynamin inhibitor), the dominant negative mutants of NM23-H1 (dynamin activator) and clathrin, and PBP10 (PtdIns 4,5-P2-binding peptide) inhibited agonist-induced HLHCGR and cAMP accumulation but not ARF6 activation. These results indicate that heterotrimeric G-protein, phosphatidylinositol (PI) 3-kinase (PI3K), cytohesin ARF GEF and ARF GAP function upstream of ARF6 whereas dynamin and clathrin act downstream of ARF6 in the regulation of HCG-induced HLHCGR internalization and signaling. In conclusion, we have identified the components and molecular details of the ARF6 signaling pathway required for agonist-induced HLHCGR internalization. PMID:22523074

Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin

PubMed Central

Lin, Changsheng; Ear, Jason; Midde, Krishna; Lopez-Sanchez, Inmaculada; Aznar, Nicolas; Garcia-Marcos, Mikel; Kufareva, Irina; Abagyan, Ruben; Ghosh, Pradipta

2014-01-01

A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein–protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein–protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV—Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs. PMID:25187647

Kin5 Knockdown in Tetrahymena thermophila Using RNAi Blocks Cargo Transport of Gef1

PubMed Central

Awan, Aashir; Bell, Aaron J.; Satir, Peter

2009-01-01

A critical process that builds and maintains the eukaryotic cilium is intraflagellar transport (IFT). This process utilizes members of the kinesin-2 superfamily to transport cargo into the cilium (anterograde transport) and a dynein motor for the retrograde traffic. Using a novel RNAi knockdown method, we have analyzed the function of the homodimeric IFT kinesin-2, Kin5, in Tetrahymena ciliary transport. In RNAi transformants, Kin5 was severely downregulated and disappeared from the cilia, but cilia did not resorb, although tip structure was affected. After deciliation of the knockdown cell, cilia regrew and cells swam, which suggested that Kin5 is not responsible for the trafficking of axonemal precursors to build the cilium, but could be transporting molecules that act in ciliary signal transduction, such as guanine nucleotide exchange proteins (GEFs). Gef1 is a Tetrahymena ciliary protein, and current coimmunoprecipitation and immunofluorescence studies showed that it is absent in regrowing cilia of the knockdown cells lacking ciliary Kin5. We suggest that one important cargo of Kin5 is Gef1 and knockdown of Kin5 results in cell lethality. PMID:19290045

Diverse roles of guanine nucleotide exchange factors in regulating collective cell migration

PubMed Central

Tseng, Yun-Yu; Rabadán, M. Angeles; Krishna, Shefali; Hall, Alan

2017-01-01

Efficient collective migration depends on a balance between contractility and cytoskeletal rearrangements, adhesion, and mechanical cell–cell communication, all controlled by GTPases of the RHO family. By comprehensive screening of guanine nucleotide exchange factors (GEFs) in human bronchial epithelial cell monolayers, we identified GEFs that are required for collective migration at large, such as SOS1 and β-PIX, and RHOA GEFs that are implicated in intercellular communication. Down-regulation of the latter GEFs differentially enhanced front-to-back propagation of guidance cues through the monolayer and was mirrored by down-regulation of RHOA expression and myosin II activity. Phenotype-based clustering of knockdown behaviors identified RHOA-ARHGEF18 and ARHGEF3-ARHGEF28-ARHGEF11 clusters, indicating that the latter may signal through other RHO-family GTPases. Indeed, knockdown of RHOC produced an intermediate between the two phenotypes. We conclude that for effective collective migration, the RHOA-GEFs → RHOA/C → actomyosin pathways must be optimally tuned to compromise between generation of motility forces and restriction of intercellular communication. PMID:28512143

A non-canonical role for Rgnef in promoting integrin-stimulated focal adhesion kinase activation

PubMed Central

Miller, Nichol L. G.; Lawson, Christine; Kleinschmidt, Elizabeth G.; Tancioni, Isabelle; Uryu, Sean; Schlaepfer, David D.

2013-01-01

Summary Rgnef (also known as p190RhoGEF or ARHGEF28) is a Rho guanine-nucleotide-exchange factor (GEF) that binds focal adhesion kinase (FAK). FAK is recruited to adhesions and activated by integrin receptors binding to matrix proteins, such as fibronectin (FN). Canonical models place Rgnef downstream of integrin–FAK signaling in regulating Rho GTPase activity and cell movement. Herein, we establish a new, upstream role for Rgnef in enhancing FAK localization to early peripheral adhesions and promoting FAK activation upon FN binding. Rgnef-null mouse embryo fibroblasts (MEFs) exhibit defects in adhesion formation, levels of FAK phosphotyrosine (pY)-397 and FAK localization to peripheral adhesions upon re-plating on FN. Rgnef re-expression rescues these defects, but requires Rgnef–FAK binding. A mutation in the Rgnef pleckstrin homology (PH) domain inhibits adhesion formation, FAK localization, and FAK-Y397 and paxillin-Y118 phosphorylation without disrupting the Rgnef–FAK interaction. A GEF-inactive Rgnef mutant rescues FAK-Y397 phosphorylation and early adhesion localization, but not paxillin-Y118 phosphorylation. This suggests that, downstream of FN binding, paxillin-pY118 requires Rgnef GEF activity through a mechanism distinct from adhesion formation and FAK activation. These results support a scaffolding role for Rgnef in FAK localization and activation at early adhesions in a PH-domain-dependent but GEF-activity-independent manner. PMID:24006257

Identification of a negative regulatory region for the exchange activity and characterization of T332I mutant of Rho guanine nucleotide exchange factor 10 (ARHGEF10).

PubMed

Chaya, Taro; Shibata, Satoshi; Tokuhara, Yasunori; Yamaguchi, Wataru; Matsumoto, Hiroshi; Kawahara, Ichiro; Kogo, Mikihiko; Ohoka, Yoshiharu; Inagaki, Shinobu

2011-08-26

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1-332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant.

Identification of a Negative Regulatory Region for the Exchange Activity and Characterization of T332I Mutant of Rho Guanine Nucleotide Exchange Factor 10 (ARHGEF10)*

PubMed Central

Chaya, Taro; Shibata, Satoshi; Tokuhara, Yasunori; Yamaguchi, Wataru; Matsumoto, Hiroshi; Kawahara, Ichiro; Kogo, Mikihiko; Ohoka, Yoshiharu; Inagaki, Shinobu

2011-01-01

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1–332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant. PMID:21719701

Trio combines with dock to regulate Pak activity during photoreceptor axon pathfinding in Drosophila.

PubMed

Newsome, T P; Schmidt, S; Dietzl, G; Keleman, K; Asling, B; Debant, A; Dickson, B J

2000-04-28

Correct pathfinding by Drosophila photoreceptor axons requires recruitment of p21-activated kinase (Pak) to the membrane by the SH2-SH3 adaptor Dock. Here, we identify the guanine nucleotide exchange factor (GEF) Trio as another essential component in photoreceptor axon guidance. Regulated exchange activity of one of the two Trio GEF domains is critical for accurate pathfinding. This GEF domain activates Rac, which in turn activates Pak. Mutations in trio result in projection defects similar to those observed in both Pak and dock mutants, and trio interacts genetically with Rac, Pak, and dock. These data define a signaling pathway from Trio to Rac to Pak that links guidance receptors to the growth cone cytoskeleton. We propose that distinct signals transduced via Trio and Dock act combinatorially to activate Pak in spatially restricted domains within the growth cone, thereby controlling the direction of axon extension.

Gamma and x-ray irradiation effects on different Ge and Ge/F doped optical fibers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alessi, A., E-mail: antonino.alessi@univ-st-etienne.fr; Girard, S.; Di Francesca, D.

2015-08-28

We performed electron paramagnetic resonance (EPR) measurements on γ and X ray irradiated Ge doped and Ge/F co-doped optical fibers. We considered three different drawing conditions (speed and tension), and for each type of drawing, we studied Ge and Ge/F doped samples having Ge doping level above 4% by weight. The EPR data recorded for the γ ray irradiated fibers confirm that all the samples exhibit a very close radiation response regardless of the drawing conditions corresponding to values used for the production of specialty fibers. Furthermore, as for the X irradiated materials, in the γ ray irradiated F co-dopedmore » fibers, we observed that the Ge(1) and the Ge(2) defects generation is unchanged, whereas it was enhanced for the E'Ge. In the various fibers, the comparison of the γ and X-ray induced concentrations of these kinds of Ge related defects indicates that the two irradiations induce similar effects regardless of the different employed dose rates and sources. Confocal microscopy luminescence results show that the starting content of the Germanium Lone Pair Center (GLPC) is neither strongly affected by the Ge content nor by the drawing conditions, and we consider the similarity of the GLPC content as key factor in determining many of the above reported similarities.« less

A Novel GABRG2 Mutation, p.R136*, in a family with GEFS+ and extended phenotypes

PubMed Central

Shen, Wangzhen; Pickrell, William O.; Cushion, Thomas D.; Davies, Jeffrey S.; Baer, Kristin; Mullins, Jonathan G.L.; Hammond, Carrie L.; Chung, Seo-Kyung; Thomas, Rhys H.; White, Cathy; Smith, Phil E.M.

2014-01-01

Genetic mutations in voltage-gated and ligand-gated ion channel genes have been identified in a small number of Mendelian families with genetic generalised epilepsies (GGEs). They are commonly associated with febrile seizures (FS), childhood absence epilepsy (CAE) and particularly with generalised or genetic epilepsy with febrile seizures plus (GEFS+). In clinical practice, despite efforts to categorise epilepsy and epilepsy families into syndromic diagnoses, many generalised epilepsies remain unclassified with a presumed genetic basis. During the systematic collection of epilepsy families, we assembled a cohort of families with evidence of GEFS+ and screened for variations in the γ2 subunit of the γ-aminobutyric acid (GABA) type A receptor gene (GABRG2). We detected a novel GABRG2(p.R136*) premature translation termination codon in one index-case from a two-generation nuclear family, presenting with an unclassified GGE, a borderline GEFS+ phenotype with learning difficulties and autism spectrum disorder (ASD). The GABRG2(p.R136*) mutation segregates with the febrile seizure component of this family's GGE and is absent in 190 healthy control samples. In vitro expression assays demonstrated that γ2(p.R136*) subunits were produced, but had reduced cell-surface and total expression. When γ2(p.R136*) subunits were co-expressed with α1 and β2 subunits in HEK 293T cells, GABA–evoked currents were reduced. Furthermore, γ2(p.R136*) subunits were highly-expressed in intracellular aggregations surrounding the nucleus and endoplasmic reticulum (ER), suggesting compromised receptor trafficking. A novel GABRG2(p.R136*) mutation extends the spectrum of GABRG2 mutations identified in GEFS+ and GGE phenotypes, causes GABAA receptor dysfunction, and represents a putative epilepsy mechanism. PMID:24407264

In vitro guanine nucleotide exchange activity of DHR-2/DOCKER/CZH2 domains.

PubMed

Côté, Jean-François; Vuori, Kristiina

2006-01-01

Rho family GTPases regulate a large variety of biological processes, including the reorganization of the actin cytoskeleton. Like other members of the Ras superfamily of small GTP-binding proteins, Rho GTPases cycle between a GDP-bound (inactive) and a GTP-bound (active) state, and, when active, the GTPases relay extracellular signals to a large number of downstream effectors. Guanine nucleotide exchange factors (GEFs) promote the exchange of GDP for GTP on Rho GTPases, thereby activating them. Most Rho-GEFs mediate their effects through their signature domain known as the Dbl Homology-Pleckstrin Homology (DH-PH) module. Recently, we and others identified a family of evolutionarily conserved, DOCK180-related proteins that also display GEF activity toward Rho GTPases. The DOCK180-family of proteins lacks the canonical DH-PH module. Instead, they rely on a novel domain, termed DHR-2, DOCKER, or CZH2, to exchange GDP for GTP on Rho targets. In this chapter, the experimental approach that we used to uncover the exchange activity of the DHR-2 domain of DOCK180-related proteins will be described.

Mechanisms of CDC-42 activation during contact-induced cell polarization

PubMed Central

Chan, Emily; Nance, Jeremy

2013-01-01

Summary Polarization of early embryos provides a foundation to execute essential patterning and morphogenetic events. In Caenorhabditis elegans, cell contacts polarize early embryos along their radial axis by excluding the cortical polarity protein PAR-6 from sites of cell contact, thereby restricting PAR-6 to contact-free cell surfaces. Radial polarization requires the cortically enriched Rho GTPase CDC-42, which in its active form recruits PAR-6 through direct binding. The Rho GTPase activating protein (RhoGAP) PAC-1, which localizes specifically to cell contacts, triggers radial polarization by inactivating CDC-42 at these sites. The mechanisms responsible for activating CDC-42 at contact-free surfaces are unknown. Here, in an overexpression screen of Rho guanine nucleotide exchange factors (RhoGEFs), which can activate Rho GTPases, we identify CGEF-1 and ECT-2 as RhoGEFs that act through CDC-42 to recruit PAR-6 to the cortex. We show that ECT-2 and CGEF-1 localize to the cell surface and that removing their activity causes a reduction in levels of cortical PAR-6. Through a structure–function analysis, we show that the tandem DH-PH domains of CGEF-1 and ECT-2 are sufficient for GEF activity, but that regions outside of these domains target each protein to the cell surface. Finally, we provide evidence suggesting that the N-terminal region of ECT-2 may direct its in vivo preference for CDC-42 over another known target, the Rho GTPase RHO-1. We propose that radial polarization results from a competition between RhoGEFs, which activate CDC-42 throughout the cortex, and the RhoGAP PAC-1, which inactivates CDC-42 at cell contacts. PMID:23424200

Mechanisms of CDC-42 activation during contact-induced cell polarization.

PubMed

Chan, Emily; Nance, Jeremy

2013-04-01

Polarization of early embryos provides a foundation to execute essential patterning and morphogenetic events. In Caenorhabditis elegans, cell contacts polarize early embryos along their radial axis by excluding the cortical polarity protein PAR-6 from sites of cell contact, thereby restricting PAR-6 to contact-free cell surfaces. Radial polarization requires the cortically enriched Rho GTPase CDC-42, which in its active form recruits PAR-6 through direct binding. The Rho GTPase activating protein (RhoGAP) PAC-1, which localizes specifically to cell contacts, triggers radial polarization by inactivating CDC-42 at these sites. The mechanisms responsible for activating CDC-42 at contact-free surfaces are unknown. Here, in an overexpression screen of Rho guanine nucleotide exchange factors (RhoGEFs), which can activate Rho GTPases, we identify CGEF-1 and ECT-2 as RhoGEFs that act through CDC-42 to recruit PAR-6 to the cortex. We show that ECT-2 and CGEF-1 localize to the cell surface and that removing their activity causes a reduction in levels of cortical PAR-6. Through a structure-function analysis, we show that the tandem DH-PH domains of CGEF-1 and ECT-2 are sufficient for GEF activity, but that regions outside of these domains target each protein to the cell surface. Finally, we provide evidence suggesting that the N-terminal region of ECT-2 may direct its in vivo preference for CDC-42 over another known target, the Rho GTPase RHO-1. We propose that radial polarization results from a competition between RhoGEFs, which activate CDC-42 throughout the cortex, and the RhoGAP PAC-1, which inactivates CDC-42 at cell contacts.

A non-typical sequence of phase transitions in (NH4)3GeF7: optical and structural characterization.

PubMed

Mel'nikova, S V; Molokeev, M S; Laptash, N M; Misyul, S V

2016-03-28

Single crystals of germanium double salt (NH4)3GeF7 = (NH4)2GeF6·NH4F = (NH4)3[GeF6]F were grown and studied by the methods of polarization optics and X-ray diffraction. The birefringence Δn = (no - ne), the rotation angle of the optical indicatrix ϕ(T) and unit cell parameters were measured in the temperature range 100-400 K. Three structural phase transitions were found at the temperatures: T1↓ = 279.2 K (T1↑ = 279.4 K), T2↑ = 270 K (T2↓ = 268.9 K), T3↓ = 218 K (T3↑ = 227 K). An unusual sequence of symmetry transformations with temperature change was established: P4/mbm (Z = 2) (G1) ↔ Pbam (Z = 4) (G2) ↔ P21/c (Z = 4) (G3) ↔ Pa3[combining macron] (Z = 8) (G4). The crystal structures of different phases were determined. The experimental data were additionally interpreted by a group-theoretical analysis of the complete condensate of order parameters taking into account the critical and noncritical atomic displacements. Strengthening of the N-HF hydrogen bonds can be a driving force of the observed phase transitions.

GIV/Girdin activates Gαi and inhibits Gαs via the same motif

PubMed Central

Gupta, Vijay; Bhandari, Deepali; Leyme, Anthony; Aznar, Nicolas; Midde, Krishna K.; Lo, I-Chung; Ear, Jason; Niesman, Ingrid; López-Sánchez, Inmaculada; Blanco-Canosa, Juan Bautista; von Zastrow, Mark; Garcia-Marcos, Mikel; Farquhar, Marilyn G.; Ghosh, Pradipta

2016-01-01

We previously showed that guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV), a guanine-nucleotide exchange factor (GEF), transactivates Gα activity-inhibiting polypeptide 1 (Gαi) proteins in response to growth factors, such as EGF, using a short C-terminal motif. Subsequent work demonstrated that GIV also binds Gαs and that inactive Gαs promotes maturation of endosomes and shuts down mitogenic MAPK–ERK1/2 signals from endosomes. However, the mechanism and consequences of dual coupling of GIV to two G proteins, Gαi and Gαs, remained unknown. Here we report that GIV is a bifunctional modulator of G proteins; it serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs using the same motif that allows it to serve as a GEF for Gαi. Upon EGF stimulation, GIV modulates Gαi and Gαs sequentially: first, a key phosphomodification favors the assembly of GIV–Gαi complexes and activates GIV’s GEF function; then a second phosphomodification terminates GIV’s GEF function, triggers the assembly of GIV–Gαs complexes, and activates GIV’s GDI function. By comparing WT and GIV mutants, we demonstrate that GIV inhibits Gαs activity in cells responding to EGF. Consequently, the cAMP→PKA→cAMP response element-binding protein signaling axis is inhibited, the transit time of EGF receptor through early endosomes are accelerated, mitogenic MAPK–ERK1/2 signals are rapidly terminated, and proliferation is suppressed. These insights define a paradigm in G-protein signaling in which a pleiotropically acting modulator uses the same motif both to activate and to inhibit G proteins. Our findings also illuminate how such modulation of two opposing Gα proteins integrates downstream signals and cellular responses. PMID:27621449

Multiple roles of the Rho GEF ephexin1 in synapse remodeling

PubMed Central

Shi, Lei; Fu, Amy KY

2010-01-01

Synapse remodeling, which involves changes in the synaptic structure and their molecular composition, is required for the maturation and refinement of neural circuits. Although synapse remodeling is known to be tightly dependent on the assembly of local actin cytoskeleton, how actin directs the structural changes of synapse and targeting of synaptic proteins are not fully understood. Recently, we identified ephexin1, a Rho guanine nucleotide exchange factor (GEF) that regulates actin dynamics, to play an essential role in the maturation and functioning of the mammalian neuromuscular junction (NMJ). We showed that ephexin1 regulates the synaptic organization of the neurotransmitter receptor acetylcholine receptor (AChR) clusters through RhoA-dependent actin reorganization. Interestingly, ephexin1 has been implicated in the regulation of postsynaptic structure as well as the presynaptic vesicle release at various types of synapses. Our findings thus establish a novel function of ephexin1 in synapse remodeling through regulating the synaptic targeting of neurotransmitter receptors, revealing a versatile role of ephexin1 at synapses. PMID:21331259

The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs.

PubMed

Levine, Timothy P; Daniels, Rachel D; Gatta, Alberto T; Wong, Louise H; Hayes, Matthew J

2013-02-15

Fronto-temporal dementia (FTD) and amyotrophic lateral sclerosis (ALS, also called motor neuron disease, MND) are severe neurodegenerative diseases that show considerable overlap at the clinical and cellular level. The most common single mutation in families with FTD or ALS has recently been mapped to a non-coding repeat expansion in the uncharacterized gene C9ORF72. Although a plausible mechanism for disease is that aberrant C9ORF72 mRNA poisons splicing, it is important to determine the cellular function of C9ORF72, about which nothing is known. Sensitive homology searches showed that C9ORF72 is a full-length distant homologue of proteins related to Differentially Expressed in Normal and Neoplasia (DENN), which is a GDP/GTP exchange factor (GEF) that activates Rab-GTPases. Our results suggest that C9ORF72 is likely to regulate membrane traffic in conjunction with Rab-GTPase switches, and we propose to name the gene and its product DENN-like 72 (DENNL72).

Site-specific regulation of the GEF Cdc24p by the scaffold protein Far1p during yeast mating

PubMed Central

Wiget, Philippe; Shimada, Yukiko; Butty, Anne-Christine; Bi, Efrei; Peter, Matthias

2004-01-01

Receptor-mediated cell polarization via heterotrimeric G-proteins induces cytoskeletal rearrangements in a variety of organisms. In yeast, Far1p is required for orienting cell growth towards the mating partner by linking activated Gβγ to the guanine-nucleotide exchange factor (GEF) Cdc24p, which activates the Rho-type GTPase Cdc42p. Here we investigated the role of Far1p in the regulation of Cdc24p in vivo. Using time-lapse microscopy of mating cells and artificial membrane targeting of Far1p, we show that Far1p is necessary and sufficient to recruit Cdc24p to the plasma membrane. Wild-type Far1p contains a PH-like domain, which is required for its membrane localization in vivo. Interestingly, expression of membrane-targeted Far1p causes toxicity, most likely by activating Cdc42p uniformly at the cell cortex. The ability of full-length Far1p to function as an activator of Cdc24p in vivo requires its interaction with Cdc24p and Gβγ. Our results imply that Gβγ not only targets Far1p to the correct site but may also trigger a conformational change in Far1p that is required for its ability to activate Cdc24p in vivo. PMID:14988725

Machineries regulating the activity of the small GTPase Arf6 in cancer cells are potential targets for developing innovative anti-cancer drugs.

PubMed

Yamauchi, Yohei; Miura, Yuki; Kanaho, Yasunori

2017-01-01

The Small GTPase ADP-ribosylation factor 6 (Arf6) functions as the molecular switch in cellular signaling pathways by cycling between GDP-bound inactive and GTP-bound active form, which is precisely regulated by two regulators, guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Numerous studies have shown that these machineries play critical roles in tumor angiogenesis/growth and cancer cell invasion/metastasis through regulating the cycling of Arf6. Here, we summarize accumulating knowledge for involvement of Arf6 GEFs/GAPs and small molecule inhibitors of Arf6 signaling/cycling in cancer progression, and discuss possible strategies for developing innovative anti-cancer drugs targeting Arf6 signaling/cycling. Copyright © 2016 Elsevier Ltd. All rights reserved.

Phosphorylation-dependent Regulation of Connecdenn/DENND1 Guanine Nucleotide Exchange Factors*

PubMed Central

Kulasekaran, Gopinath; Nossova, Nadya; Marat, Andrea L.; Lund, Ingrid; Cremer, Christopher; Ioannou, Maria S.; McPherson, Peter S.

2015-01-01

Connecdenn 1/2 are DENN (differentially expressed in normal and neoplastic cells) domain-bearing proteins that function as GEFs (guanine nucleotide exchange factors) for the small GTPase Rab35. Disruption of connecdenn/Rab35 function leads to defects in the recycling of multiple cargo proteins from endosomes with altered cell function, yet the regulation of connecdenn GEF activity is unexplored. We now demonstrate that connecdenn 1/2 are autoinhibited such that the purified, full-length proteins have significantly less Rab35 binding and GEF activity than the isolated DENN domain. Both proteins are phosphorylated with prominent phosphorylation sites between residues 500 and 600 of connecdenn 1. A large scale proteomics screen revealed that connecdenn 1 is phosphorylated at residues Ser-536 and Ser-538 in an Akt-dependent manner in response to insulin stimulation of adipocytes. Interestingly, we find that an Akt inhibitor reduces connecdenn 1 interaction with Rab35 after insulin treatment of adipocytes. Remarkably, a peptide flanking Ser-536/Ser-538 binds the DENN domain of connecdenn 1, whereas a phosphomimetic peptide does not. Moreover, connecdenn 1 interacts with 14-3-3 proteins, and this interaction is also disrupted by Akt inhibition and by mutation of Ser-536/Ser-538. We propose that Akt phosphorylation of connecdenn 1 downstream of insulin activation regulates connecdenn 1 function through an intramolecular interaction. PMID:26055712

The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor β-Stimulated Aortic Endothelial Cells ▿

PubMed Central

Daubon, Thomas; Buccione, Roberto; Génot, Elisabeth

2011-01-01

Podosomes are dynamic actin-rich adhesion plasma membrane microdomains endowed with extracellular matrix-degrading activities. In aortic endothelial cells, podosomes are induced by transforming growth factor β (TGF-β), but how this occurs is largely unknown. It is thought that, in endothelial cells, podosomes play a role in vessel remodeling and/or in breaching anatomical barriers. We demonstrate here that, in bovine aortic endothelial cells, that the Cdc42-specific guanine exchange factor (GEF) Fgd1 is expressed and regulated by TGF-β to induce Cdc42-dependent podosome assembly. Within 15 min of TGF-β stimulation, Fgd1, but none of the other tested Cdc42 GEFs, undergoes tyrosine phosphorylation, associates with Cdc42, and translocates to the subcortical cytoskeleton via a cortactin-dependent mechanism. Small interfering RNA-mediated Fgd1 knockdown inhibits TGF-β-induced Cdc42 activation. Fgd1 depletion also reduces podosome formation and associated matrix degradation and these defects are rescued by reexpression of Fgd1. Although overexpression of Fgd1 does not promote podosome formation per se, it enhances TGF-β-induced matrix degradation. Our results identify Fgd1 as a TGF-β-regulated GEF and, as such, the first GEF to be involved in the process of cytokine-induced podosome formation. Our findings reveal the involvement of Fgd1 in endothelial cell biology and open up new avenues to study its role in vascular pathophysiology. PMID:21911474

Discovery of Aminopiperidine Indoles That Activate the Guanine Nucleotide Exchange Factor SOS1 and Modulate RAS Signaling.

PubMed

Abbott, Jason R; Hodges, Timothy R; Daniels, R Nathan; Patel, Pratiq A; Kennedy, Jack Phillip; Howes, Jennifer E; Akan, Denis T; Burns, Michael C; Sai, Jiqing; Sobolik, Tammy; Beesetty, Yugandhar; Lee, Taekyu; Rossanese, Olivia W; Phan, Jason; Waterson, Alex G; Fesik, Stephen W

2018-06-01

Deregulated RAS activity, often the result of mutation, is implicated in approximately 30% of all human cancers. Despite this statistic, no clinically successful treatment for RAS-driven tumors has yet been developed. One approach for modulating RAS activity is to target and affect the activity of proteins that interact with RAS, such as the guanine nucleotide exchange factor (GEF) son of sevenless homologue 1 (SOS1). Here, we report on structure-activity relationships (SAR) in an indole series of compounds. Using structure-based design, we systematically explored substitution patterns on the indole nucleus, the pendant amino acid moiety, and the linker unit that connects these two fragments. Best-in-class compounds activate the nucleotide exchange process at sub-micromolar concentrations in vitro, increase levels of active RAS-GTP in HeLa cells, and elicit signaling changes in the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway, resulting in a decrease in pERK1/2 T202/Y204 protein levels at higher compound concentrations.

The RhoGAP activity of CYK-4/MgcRacGAP functions non-canonically by promoting RhoA activation during cytokinesis

PubMed Central

Zhang, Donglei; Glotzer, Michael

2015-01-01

Cytokinesis requires activation of the GTPase RhoA. ECT-2, the exchange factor responsible for RhoA activation, is regulated to ensure spatiotemporal control of contractile ring assembly. Centralspindlin, composed of the Rho family GTPase-activating protein (RhoGAP) MgcRacGAP/CYK-4 and the kinesin MKLP1/ZEN-4, is known to activate ECT-2, but the underlying mechanism is not understood. We report that ECT-2-mediated RhoA activation depends on the ability of CYK-4 to localize to the plasma membrane, bind RhoA, and promote GTP hydrolysis by RhoA. Defects resulting from loss of CYK-4 RhoGAP activity can be rescued by activating mutations in ECT-2 or depletion of RGA-3/4, which functions as a conventional RhoGAP for RhoA. Consistent with CYK-4 RhoGAP activity contributing to GEF activation, the catalytic domains of CYK-4 and ECT-2 directly interact. Thus, counterintuitively, CYK-4 RhoGAP activity promotes RhoA activation. We propose that the most active form of the cytokinetic RhoGEF involves complex formation between ECT-2, centralspindlin and RhoA. DOI: http://dx.doi.org/10.7554/eLife.08898.001 PMID:26252513

Novel Role for p21-activated Kinase 2 in Thrombin-induced Monocyte Migration*

PubMed Central

Gadepalli, Ravisekhar; Kotla, Sivareddy; Heckle, Mark R.; Verma, Shailendra K.; Singh, Nikhlesh K.; Rao, Gadiparthi N.

2013-01-01

To understand the role of thrombin in inflammation, we tested its effects on migration of THP-1 cells, a human monocytic cell line. Thrombin induced THP-1 cell migration in a dose-dependent manner. Thrombin induced tyrosine phosphorylation of Pyk2, Gab1, and p115 RhoGEF, leading to Rac1- and RhoA-dependent Pak2 activation. Downstream to Pyk2, Gab1 formed a complex with p115 RhoGEF involving their pleckstrin homology domains. Furthermore, inhibition or depletion of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, or Pak2 levels substantially attenuated thrombin-induced THP-1 cell F-actin cytoskeletal remodeling and migration. Inhibition or depletion of PAR1 also blocked thrombin-induced activation of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2, resulting in diminished THP-1 cell F-actin cytoskeletal remodeling and migration. Similarly, depletion of Gα12 negated thrombin-induced Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2 activation, leading to attenuation of THP-1 cell F-actin cytoskeletal remodeling and migration. These novel observations reveal that thrombin induces monocyte/macrophage migration via PAR1-Gα12-dependent Pyk2-mediated Gab1 and p115 RhoGEF interactions, leading to Rac1- and RhoA-targeted Pak2 activation. Thus, these findings provide mechanistic evidence for the role of thrombin and its receptor PAR1 in inflammation. PMID:24025335

The integrity of the plant Golgi apparatus depends on cell growth-controlled activity of GNL1.

PubMed

Du, Wenyan; Tamura, Kentaro; Stefano, Giovanni; Brandizzi, Federica

2013-05-01

Membrane traffic and organelle integrity in the plant secretory pathway depend on ARF-GTPases, which are activated by guanine-nucleotide exchange factors (ARF-GEFs). While maintenance of conserved roles, evolution of unique functions as well as tissue-specific roles have been shown for a handful of plant ARF-GEFs, a fundamental yet unanswered question concerns the extent to which their function overlaps during cell growth. To address this, we have characterized pao, a novel allele of GNOM-like 1 (GNL1), a brefeldin A (BFA)-insensitive ARF-GEF, isolated through a confocal microscopy-based forward genetics screen of the Golgi in Arabidopsis thaliana. Specifically, we have analyzed the dependence of the integrity of trafficking routes and secretory organelles on GNL1 availability during expansion stages of cotyledon epidermal cells, an exquisite model system for vegetative cell growth analyses in intact tissues. We show that Golgi traffic is influenced largely by GNL1 availability at early stages of cotyledon cell expansion but by BFA-sensitive GEFs when cell growth terminates. These data reveal an unanticipated level of complexity in the biology of GNL1 by showing that its cellular roles are correlated with cell growth. These results also indicate that the cell growth stage is an important element weighting into functional analyses of the cellular roles of ARF-GEFs.

An Assessment of the Subseasonal Forecast Performance in the Extended Global Ensemble Forecast System (GEFS)

NASA Astrophysics Data System (ADS)

Sinsky, E.; Zhu, Y.; Li, W.; Guan, H.; Melhauser, C.

2017-12-01

Optimal forecast quality is crucial for the preservation of life and property. Improving monthly forecast performance over both the tropics and extra-tropics requires attention to various physical aspects such as the representation of the underlying SST, model physics and the representation of the model physics uncertainty for an ensemble forecast system. This work focuses on the impact of stochastic physics, SST and the convection scheme on forecast performance for the sub-seasonal scale over the tropics and extra-tropics with emphasis on the Madden-Julian Oscillation (MJO). A 2-year period is evaluated using the National Centers for Environmental Prediction (NCEP) Global Ensemble Forecast System (GEFS). Three experiments with different configurations than the operational GEFS were performed to illustrate the impact of the stochastic physics, SST and convection scheme. These experiments are compared against a control experiment (CTL) which consists of the operational GEFS but its integration is extended from 16 to 35 days. The three configurations are: 1) SPs, which uses a Stochastically Perturbed Physics Tendencies (SPPT), Stochastic Perturbed Humidity (SHUM) and Stochastic Kinetic Energy Backscatter (SKEB); 2) SPs+SST_bc, which uses a combination of SPs and a bias-corrected forecast SST from the NCEP Climate Forecast System Version 2 (CFSv2); and 3) SPs+SST_bc+SA_CV, which combines SPs, a bias-corrected forecast SST and a scale aware convection scheme. When comparing to the CTL experiment, SPs shows substantial improvement. The MJO skill has improved by about 4 lead days during the 2-year period. Improvement is also seen over the extra-tropics due to the updated stochastic physics, where there is a 3.1% and a 4.2% improvement during weeks 3 and 4 over the northern hemisphere and southern hemisphere, respectively. Improvement is also seen when the bias-corrected CFSv2 SST is combined with SPs. Additionally, forecast performance enhances when the scale aware

The Arf GEF GBF1 and Arf4 synergize with the sensory receptor cargo, rhodopsin, to regulate ciliary membrane trafficking.

PubMed

Wang, Jing; Fresquez, Theresa; Kandachar, Vasundhara; Deretic, Dusanka

2017-12-01

The small GTPase Arf4 and the Arf GTPase-activating protein (GAP) ASAP1 cooperatively sequester sensory receptor cargo into transport carriers targeted to primary cilia, but the input that drives Arf4 activation in this process remains unknown. Here, we show, by using frog retinas and recombinant human proteins, that during the carrier biogenesis from the photoreceptor Golgi/ trans -Golgi network (TGN) a functional complex is formed between Arf4, the Arf guanine nucleotide exchange factor (GEF) GBF1 and the light-sensing receptor, rhodopsin. Rhodopsin and Arf4 bind the regulatory N-terminal dimerization and cyclophillin-binding (DCB)-homology upstream of Sec7 (HUS) domain of GBF1. The complex is sensitive to Golgicide A (GCA), a selective inhibitor of GBF1 that accordingly blocks rhodopsin delivery to the cilia, without disrupting the photoreceptor Golgi. The emergence of newly synthesized rhodopsin in the endomembrane system is essential for GBF1-Arf4 complex formation in vivo Notably, GBF1 interacts with the Arf GAP ASAP1 in a GCA-resistant manner. Our findings indicate that converging signals on GBF1 from the influx of cargo into the Golgi/TGN and the feedback from Arf4, combined with input from ASAP1, control Arf4 activation during sensory membrane trafficking to primary cilia. © 2017. Published by The Company of Biologists Ltd.

RhoA GTPase inhibition organizes contraction during epithelial morphogenesis

PubMed Central

Mason, Frank M.; Xie, Shicong; Vasquez, Claudia G.; Tworoger, Michael

2016-01-01

During morphogenesis, contraction of the actomyosin cytoskeleton within individual cells drives cell shape changes that fold tissues. Coordination of cytoskeletal contractility is mediated by regulating RhoA GTPase activity. Guanine nucleotide exchange factors (GEFs) activate and GTPase-activating proteins (GAPs) inhibit RhoA activity. Most studies of tissue folding, including apical constriction, have focused on how RhoA is activated by GEFs to promote cell contractility, with little investigation as to how GAPs may be important. Here, we identify a critical role for a RhoA GAP, Cumberland GAP (C-GAP), which coordinates with a RhoA GEF, RhoGEF2, to organize spatiotemporal contractility during Drosophila melanogaster apical constriction. C-GAP spatially restricts RhoA pathway activity to a central position in the apical cortex. RhoGEF2 pulses precede myosin, and C-GAP is required for pulsation, suggesting that contractile pulses result from RhoA activity cycling. Finally, C-GAP expression level influences the transition from reversible to irreversible cell shape change, which defines the onset of tissue shape change. Our data demonstrate that RhoA activity cycling and modulating the ratio of RhoGEF2 to C-GAP are required for tissue folding. PMID:27551058

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

PubMed Central

Jun, Jesse E.; Yang, Ming; Chen, Hang; Chakraborty, Arup K.

2013-01-01

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation. PMID:23589333

Open Ocean Assessments for Management in the GEF Transboundary Waters Assessment Project (TWAP)

NASA Astrophysics Data System (ADS)

Fischer, A. S.; Alverson, K. D.

2010-12-01

A methodology for a thematic and scientifically-credible assessment of Open Ocean waters as a part of the Global Environment Facility (GEF) Transboundary Waters Assessment Project (TWAP) has been developed in the last 18 months by the Intergovernmental Oceanographic Commission of UNESCO, and is presented for feedback and comment. While developed to help the GEF International Waters focal area target investment to manage looming environmental threats in interlinked freshwater and marine systems (a very focused decision support system), the assessment methodology could contribute to other assessment and management efforts in the UN system and elsewhere. Building on a conceptual framework that describes the relationships between human systems and open ocean natural systems, and on mapping of the human impact on the marine environment, the assessment will evaluate and make projections on a thematic basis, identifying key metrics, indices, and indicators. These themes will include the threats on key ecosystem services of climate change through sea level rise, changed stratification, warming, and ocean acidification; vulnerabilities of ecosystems, habitats, and living marine resources; the impact and sustainability of fisheries; and pollution. Global-level governance arrangements will also be evaluated, with an eye to identifying scope for improved global-level management. The assessment will build on sustained ocean observing systems, model projections, and an assessment of scientific literature, as well as tools for combining knowledge to support identification of priority concerns and in developing scenarios for management. It will include an assessment of key research and observing needs as one way to deal with the scientific uncertainty inherent in such an exercise, and to better link policy and science agendas.

Identification of a Novel, Putative Rho-specific GDP/GTP Exchange Factor and a RhoA-binding Protein: Control of Neuronal Morphology

PubMed Central

Gebbink, Martijn F.B.G.; Kranenburg, Onno; Poland, Mieke; van Horck, Francis P.G.; Houssa, Brahim; Moolenaar, Wouter H.

1997-01-01

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein–coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116Rip, that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116Rip stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116Rip fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein–coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116Rip inhibits RhoA-stimulated contractility and promotes neurite outgrowth. PMID:9199174

A BAC transgenic mouse model reveals neuron subtype-specific effects of a Generalized Epilepsy with Febrile Seizures Plus (GEFS+) mutation

PubMed Central

Tang, Bin; Dutt, Karoni; Papale, Ligia; Rusconi, Raffaella; Shankar, Anupama; Hunter, Jessica; Tufik, Sergio; Yu, Frank H.; Catterall, William A.; Mantegazza, Massimo; Goldin, Alan L.; Escayg, Andrew

2009-01-01

Mutations in the voltage-gated sodium channel SCN1A are responsible for a number of seizure disorders including Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and Severe Myoclonic Epilepsy of Infancy (SMEI). To determine the effects of SCN1A mutations on channel function in vivo, we generated a bacterial artificial chromosome (BAC) transgenic mouse model that expresses the human SCN1A GEFS+ mutation, R1648H. Mice with the R1648H mutation exhibit a more severe response to the proconvulsant kainic acid compared with mice expressing a control Scn1a transgene. Electrophysiological analysis of dissociated neurons from mice with the R1648H mutation reveal delayed recovery from inactivation and increased use-dependent inactivation only in inhibitory bipolar neurons, as well as a hyperpolarizing shift in the voltage dependence of inactivation only in excitatory pyramidal neurons. These results demonstrate that the effects of SCN1A mutations are cell type-dependent and that the R1648H mutation specifically leads to a reduction in interneuron excitability. PMID:19409490

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation*

PubMed Central

Kistler, Samantha; George, Samuel D.; Kuhlmann, Nora; Garvey, Leslie; Huynh, Minh; Bagni, Rachel K.; Lammers, Michael; Der, Channing J.; Campbell, Sharon L.

2017-01-01

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity. PMID:28154176

The tight-adhesion proteins TadGEF of Bradyrhizobium diazoefficiens USDA 110 are involved in cell adhesion and infectivity on soybean roots.

PubMed

Mongiardini, Elías J; Parisi, Gustavo D; Quelas, Juan I; Lodeiro, Aníbal R

2016-01-01

Adhesion of symbiotic bacteria to host plants is an essential early step of the infection process that leads to the beneficial interaction. In the Bradyrhizobium diazoefficiens-soybean symbiosis few molecular determinants of adhesion are known. Here we identified the tight-adhesion gene products TadGEF in the open-reading frames blr3941-blr3943 of the B. diazoefficiens USDA 110 complete genomic sequence. Predicted structure of TadG indicates a transmembrane domain and two extracytosolic domains, from which the C-terminal has an integrin fold. TadE and TadF are also predicted as bearing transmembrane segments. Mutants in tadG or the small cluster tadGEF were impaired in adhesion to soybean roots, and the root infection was delayed. However, nodule histology was not compromised by the mutations, indicating that these effects were restricted to the earliest contact of the B. diazoefficiens and root surfaces. Knowledge of preinfection determinants is important for development of inoculants that are applied to soybean crops worldwide. Copyright © 2015 Elsevier GmbH. All rights reserved.

Cdc42 and the Guanine Nucleotide Exchange Factors Ect2 and Trio Mediate Fn14-Induced Migration and Invasion of Glioblastoma Cells

PubMed Central

Fortin, Shannon P.; Ennis, Matthew J.; Schumacher, Cassie A.; Zylstra-Diegel, Cassandra R.; Williams, Bart O.; Ross, Julianna T.D.; Winkles, Jeffrey A.; Loftus, Joseph C.; Symons, Marc H.; Tran, Nhan L.

2012-01-01

Malignant glioblastomas are characterized by their ability to infiltrate into normal brain. We previously reported that binding of the multifunctional cytokine TNF-like weak inducer of apoptosis (TWEAK) to its receptor fibroblast growth factor–inducible 14 (Fn14) induces glioblastoma cell invasion via Rac1 activation. Here, we show that Cdc42 plays an essential role in Fn14-mediated activation of Rac1. TWEAK-treated glioma cells display an increased activation of Cdc42, and depletion of Cdc42 using siRNA abolishes TWEAK-induced Rac1 activation and abrogates glioma cell migration and invasion. In contrast, Rac1 depletion does not affect Cdc42 activation by Fn14, showing that Cdc42 mediates TWEAK-stimulated Rac1 activation. Furthermore, we identified two guanine nucleotide exchange factors (GEF), Ect2 and Trio, involved in TWEAK-induced activation of Cdc42 and Rac1, respectively. Depletion of Ect2 abrogates both TWEAK-induced Cdc42 and Rac1 activation, as well as subsequent TWEAK-Fn14–directed glioma cell migration and invasion. In contrast, Trio depletion inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Finally, inappropriate expression of Fn14 or Ect2 in mouse astrocytes in vivo using an RCAS vector system for glial-specific gene transfer in G-tva transgenic mice induces astrocyte migration within the brain, corroborating the in vitro importance of the TWEAK-Fn14 signaling cascade in glioblastoma invasion. Our results suggest that the TWEAK-Fn14 signaling axis stimulates glioma cell migration and invasion through two GEF-GTPase signaling units, Ect2-Cdc42 and Trio-Rac1. Components of the Fn14-Rho GEF-Rho GTPase signaling pathway present innovative drug targets for glioma therapy. PMID:22571869

The C-terminus of the oncoprotein TGAT is necessary for plasma membrane association and efficient RhoA-mediated signaling.

PubMed

van Unen, J; Botman, D; Yin, T; Wu, Y I; Hink, M A; Gadella, T W J; Postma, M; Goedhart, J

2018-06-07

Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, adhesion and proliferation. Alternative splicing of the RhoGEF Trio produces TGAT. The RhoGEF TGAT is an oncoprotein with constitutive RhoGEF activity. We investigated whether the subcellular location of TGAT is critical for its RhoGEF activity. Since plasma membrane associated RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was examined. To this end, we developed a highly sensitive image analysis method to quantitatively measure plasma membrane association. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest. The analysis revealed that wild-type TGAT is partially co-localized with the plasma membrane. Strikingly, cysteine TGAT-mutants lacking one or more putative palmitoylation sites in the C-tail, still showed membrane association. In contrast, a truncated variant, lacking the last 15 amino acids, TGAT Δ15 , lost membrane association. We show that membrane localization of TGAT was responsible for high RhoGEF activity by using a RhoA FRET-sensor and by determining F-actin levels. Mutants of TGAT that still maintained membrane association showed similar activity as wild-type TGAT. In contrast, the activity was abrogated for the cytoplasmic TGAT Δ15 variant. Synthetic recruitment of TGAT Δ15 to membranes confirmed that TGAT effectively activates RhoA at the plasma membrane. Together, these results show that membrane association of TGAT is critical for its activity.

Rapid kinetic BRET measurements to monitor G protein activation by GPCR and non-GPCR proteins.

PubMed

Maziarz, Marcin; Garcia-Marcos, Mikel

2017-01-01

Heterotrimeric G proteins are central hubs of signal transduction whose activity is controlled by G protein-coupled receptors (GPCRs) as well as by a complex network of regulatory proteins. Recently, bioluminescence resonance energy transfer (BRET)-based assays have been used to monitor real-time activation of heterotrimeric G proteins in cells. Here we describe the use of a previously established BRET assay to monitor G protein activation upon GPCR stimulation and its adaptation to measure G protein activation by non-GPCR proteins, such as by cytoplasmic guanine nucleotide exchange factors (GEFs) like GIV/Girdin. The BRET assay monitors the release of free Gβγ from Gα-Gβγ heterotrimers as a readout of G protein activation, which is readily observable upon agonist stimulation of GPCRs. To control the signal input for non-GPCR activators, we describe the use of a chemically induced dimerization strategy to promote rapid membrane translocation of proteins containing the Gα-binding and -activating (GBA) motif found in some nonreceptor GEFs. The assay described here allows the kinetic measurement of G protein activation with subsecond temporal resolution and to compare the levels of activation induced by GPCR agonists vs those induced by the membrane recruitment of nonreceptor G protein signaling activators. © 2017 Elsevier Inc. All rights reserved.

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation.

PubMed

Yin, Guowei; Kistler, Samantha; George, Samuel D; Kuhlmann, Nora; Garvey, Leslie; Huynh, Minh; Bagni, Rachel K; Lammers, Michael; Der, Channing J; Campbell, Sharon L

2017-03-17

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling

PubMed Central

Sandri, Chiara; Caccavari, Francesca; Valdembri, Donatella; Camillo, Chiara; Veltel, Stefan; Santambrogio, Martina; Lanzetti, Letizia; Bussolino, Federico; Ivaska, Johanna; Serini, Guido

2012-01-01

During developmental and tumor angiogenesis, semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases. R-Ras is mainly expressed in vascular cells, where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms. We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and -angiogenic activity of R-Ras. Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia. Upon binding, GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF) to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active β1 integrins and then causes the translocation of R-Ras to early endosomes. Here, the R-Ras/RIN2/Rab5 signaling module activates Rac1-dependent cell adhesion via TIAM1, a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate. In conclusion, the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active β1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Rac1. PMID:22825554

Dissociation of Rac1(GDP)·RhoGDI Complexes by the Cooperative Action of Anionic Liposomes Containing Phosphatidylinositol 3,4,5-Trisphosphate, Rac Guanine Nucleotide Exchange Factor, and GTP*

PubMed Central

Ugolev, Yelena; Berdichevsky, Yevgeny; Weinbaum, Carolyn; Pick, Edgar

2008-01-01

Rac plays a pivotal role in the assembly of the superoxide-generating NADPH oxidase of phagocytes. In resting cells, Rac is found in the cytosol in complex with Rho GDP dissociation inhibitor (RhoGDI). NADPH oxidase assembly involves dissociation of the Rac·RhoGDI complex and translocation of Rac to the membrane. We reported that liposomes containing high concentrations of monovalent anionic phospholipids cause Rac·RhoGDI complex dissociation (Ugolev, Y., Molshanski-Mor, S., Weinbaum, C., and Pick, E. (2006) J. Biol. Chem.281 ,19204 -1921916702219). We now designed an in vitro model mimicking membrane phospholipid remodeling during phagocyte stimulation in vivo. We showed that liposomes of “resting cell membrane” composition (less than 20 mol % monovalent anionic phospholipids), supplemented with 1 mol % of polyvalent anionic phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in conjunction with constitutively active forms of the guanine nucleotide exchange factors (GEFs) for Rac, Trio, or Tiam1 and a non-hydrolyzable GTP analogue, cause dissociation of Rac1(GDP)·RhoGDI complexes, GDP to GTP exchange on Rac1, and binding of Rac1(GTP) to the liposomes. Complexes were not dissociated in the absence of GEF and GTP, and optimal dissociation required the presence of PtdIns(3,4,5)P3 in the liposomes. Dissociation of Rac1(GDP)·RhoGDI complexes was correlated with the affinity of particular GEF constructs, via the N-terminal pleckstrin homology domain, for PtdIns(3,4,5)P3 and involved GEF-mediated GDP to GTP exchange on Rac1. Phagocyte membranes enriched in PtdIns(3,4,5)P3 responded by NADPH oxidase activation upon exposure in vitro to Rac1(GDP)·RhoGDI complexes, p67phox, GTP, and Rac GEF constructs with affinity for PtdIns(3,4,5)P3 at a level superior to that of native membranes. PMID:18505730

Small-GTPase-associated signaling by the guanine nucleotide exchange factors CpDock180 and CpCdc24, the GTPase effector CpSte20, and the scaffold protein CpBem1 in Claviceps purpurea.

PubMed

Herrmann, Andrea; Tillmann, Britta A M; Schürmann, Janine; Bölker, Michael; Tudzynski, Paul

2014-04-01

Monomeric GTPases of the Rho subfamily are important mediators of polar growth and NADPH (Nox) signaling in a variety of organisms. These pathways influence the ability of Claviceps purpurea to infect host plants. GTPase regulators contribute to the nucleotide loading cycle that is essential for proper functionality of the GTPases. Scaffold proteins gather GTPase complexes to facilitate proper function. The guanine nucleotide exchange factors (GEFs) CpCdc24 and CpDock180 activate GTPase signaling by triggering nucleotide exchange of the GTPases. Here we show that CpCdc24 harbors nucleotide exchange activity for both Rac and Cdc42 homologues. The GEFs partly share the cellular distribution of the GTPases and interact with the putative upstream GTPase CpRas1. Interaction studies show the formation of higher-order protein complexes, mediated by the scaffold protein CpBem1. Besides the GTPases and GEFs, these complexes also contain the GTPase effectors CpSte20 and CpCla4, as well as the regulatory protein CpNoxR. Functional characterizations suggest a role of CpCdc24 mainly in polarity, whereas CpDock180 is involved in stress tolerance mechanisms. These findings indicate the dynamic formation of small GTPase complexes and improve the model for GTPase-associated signaling in C. purpurea.

Vav1-mediated scaffolding interactions stabilize SLP-76 microclusters and contribute to antigen-dependent T cell responses.

PubMed

Sylvain, Nicholas R; Nguyen, Ken; Bunnell, Stephen C

2011-03-08

The guanine nucleotide exchange factor (GEF) Vav1 synergizes with the adaptor protein SLP-76 (Src homology 2 domain--containing leukocyte phosphoprotein of 76 kD) to support T cell development and activation. In response to ligation of the T cell receptor (TCR), SLP-76 is assembled into microclusters that provide an essential platform for the signaling events that drive T cell activation. We found that Vav1 selectively entered SLP-76 microclusters, rather than TCR microclusters, influencing their stability and function. The carboxyl terminus of Vav1, which consists of Src homology domains, was both necessary and sufficient for the entry of Vav1 into SLP-76 microclusters; however, this fragment of Vav1 was insufficient to stabilize the microclusters, and it potently suppressed T cell activation. This indicated that the amino terminus of Vav1, which has the GEF domain, also contributed to the integrity of SLP-76 microclusters and thereby to T cell activation. These microcluster-stabilizing functions were independent of the GEF activity in the amino terminus of Vav1 and were unaffected if the GEF function of Vav1 was either inactivated or constitutively activated by mutation. In contrast, Vav1 deletion mutants lacking either the calponin homology domain or the catalytic core of the GEF exhibited mild scaffolding defects, but they differentially affected TCR-dependent calcium ion (Ca²+) responses. We conclude that multiple GEF-independent scaffolding functions distributed throughout the amino terminus of Vav1 contribute to the activation of T cells by acting synergistically to increase the stability and function of SLP-76 microclusters.

Agonist-induced Ca2+ Sensitization in Smooth Muscle

PubMed Central

Artamonov, Mykhaylo V.; Momotani, Ko; Stevenson, Andra; Trentham, David R.; Derewenda, Urszula; Derewenda, Zygmunt S.; Read, Paul W.; Gutkind, J. Silvio; Somlyo, Avril V.

2013-01-01

Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca2+]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca2+ sensitization. Gα12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca2+-sensitized force are not well understood. Using permeabilized blood vessels from PRG(−/−) mice and a new method to silence LARG in organ-cultured blood vessels, we show that both RhoGEFs are activated by the physiologically and pathophysiologically important thromboxane A2 and endothelin-1 receptors. The co-activation is the result of direct and independent activation of both RhoGEFs as well as their co-recruitment due to heterodimerization. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca2+-sensitized force. We used photolysis of caged phenylephrine, caged guanosine 5′-O-(thiotriphosphate) (GTPγS) in solution, and caged GTPγS or caged GTP loaded on the RhoA·RhoGDI complex to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca2+ transient and phasic force components and the onset of Ca2+-sensitized force. PMID:24106280

Stress- and Rho-activated ZO-1–associated nucleic acid binding protein binding to p21 mRNA mediates stabilization, translation, and cell survival

PubMed Central

Nie, Mei; Balda, Maria S.; Matter, Karl

2012-01-01

A central component of the cellular stress response is p21WAF1/CIP1, which regulates cell proliferation, survival, and differentiation. Inflammation and cell stress often up-regulate p21 posttranscriptionally by regulatory mechanisms that are poorly understood. ZO-1–associated nucleic acid binding protein (ZONAB)/DbpA is a Y-box transcription factor that is regulated by components of intercellular junctions that are affected by cytokines and tissue damage. We therefore asked whether ZONAB activation is part of the cellular stress response. Here, we demonstrate that ZONAB promotes cell survival in response to proinflammatory, hyperosmotic, and cytotoxic stress and that stress-induced ZONAB activation involves the Rho regulator GEF-H1. Unexpectedly, stress-induced ZONAB activation does not stimulate ZONAB’s activity as a transcription factor but leads to the posttranscriptional up-regulation of p21 protein and mRNA. Up-regulation is mediated by ZONAB binding to specific sites in the 3′-untranslated region of the p21 mRNA, resulting in mRNA stabilization and enhanced translation. Binding of ZONAB to mRNA is activated by GEF-H1 via Rho stimulation and also mediates Ras-induced p21 expression. We thus identify a unique type of stress and Rho signaling activated pathway that drives mRNA stabilization and translation and links the cellular stress response to p21 expression and cell survival. PMID:22711822

Anomalous behaviour of thermodynamic properties at successive phase transitions in (NH4)3GeF7

NASA Astrophysics Data System (ADS)

Bogdanov, Evgeniy V.; Kartashev, Andrey V.; Pogoreltsev, Evgeniy I.; Gorev, Mikhail V.; Laptash, Natalia M.; Flerov, Igor N.

2017-12-01

Heat capacity, thermal dilatation, susceptibility to hydrostatic pressure and dielectric properties associated with succession of three phase transitions below room temperature in double fluoride salt (NH4)3GeF7 were studied. A possible transformation into the parent Pm-3m cubic phase was not observed up to the decomposition of compound. Nonferroelectric nature of structural distortions was confirmed. The DTA under pressure studies revealed a high temperature stability of two phases: P4/mbm and Pbam. The entropies of the phase transitions agree well with the model of structural distortions. Analysis of the thermal properties associated with the individual phase transitions in the framework of thermodynamic equations has shown a high reliability of the data obtained.

Reduced cell number in the hindgut epithelium disrupts hindgut left-right asymmetry in a mutant of pebble, encoding a RhoGEF, in Drosophila embryos.

PubMed

Nakamura, Mitsutoshi; Matsumoto, Kenjiroo; Iwamoto, Yuta; Muguruma, Takeshi; Nakazawa, Naotaka; Hatori, Ryo; Taniguchi, Kiichiro; Maeda, Reo; Matsuno, Kenji

2013-02-01

Animals often show left-right (LR) asymmetry in their body structures. In some vertebrates, the mechanisms underlying LR symmetry breaking and the subsequent signals responsible for LR asymmetric development are well understood. However, in invertebrates, the molecular bases of these processes are largely unknown. Therefore, we have been studying the genetic pathway of LR asymmetric development in Drosophila. The embryonic gut is the first organ that shows directional LR asymmetry during Drosophila development. We performed a genetic screen to identify mutations affecting LR asymmetric development of the embryonic gut. From this screen, we isolated pebble (pbl), which encodes a homolog of a mammalian RhoGEF, Ect2. The laterality of the hindgut was randomized in embryos homozygous for a null mutant of pbl. Pbl is a multi-functional protein required for cytokinesis and the epithelial-to-mesenchymal transition in Drosophila. Consistent with Pbl's role in cytokinesis, we found reduced numbers of cells in the hindgut epithelium in pbl homozygous embryos. The specific expression of pbl in the hindgut epithelium, but not in other tissues, rescued the LR defects and reduced cell number in embryonic pbl homozygotes. Embryos homozygous for string (stg), a mutant that reduces cell number through a different mechanism, also showed LR defects of the hindgut. However, the reduction in cell number in the pbl mutants was not accompanied by defects in the specification of hindgut epithelial tissues or their integrity. Based on these results, we speculate that the reduction in cell number may be one reason for the LR asymmetry defect of the pbl hindgut, although we cannot exclude contributions from other functions of Pbl, including regulation of the actin cytoskeleton through its RhoGEF activity. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Rabex-5 ubiquitin ligase activity restricts Ras signaling to establish pathway homeostasis in Drosophila.

PubMed

Yan, Hua; Jahanshahi, Maryam; Horvath, Elizabeth A; Liu, Hsiu-Yu; Pfleger, Cathie M

2010-08-10

The Ras signaling pathway allows cells to translate external cues into diverse biological responses. Depending on context and the threshold reached, Ras signaling can promote growth, proliferation, differentiation, or cell survival. Failure to maintain precise control of Ras can have adverse physiological consequences. Indeed, excess Ras signaling disrupts developmental patterning and causes developmental disorders [1, 2], and in mature tissues, it can lead to cancer [3-5]. We identify Rabex-5 as a new component of Ras signaling crucial for achieving proper pathway outputs in multiple contexts in vivo. We show that Drosophila Rabex-5 restricts Ras signaling to establish organism size, wing vein pattern, and eye versus antennal fate. Rabex-5 has both Rab5 guanine nucleotide exchange factor (GEF) activity that regulates endocytic trafficking [6] and ubiquitin ligase activity [7, 8]. Surprisingly, overexpression studies demonstrate that Rabex-5 ubiquitin ligase activity, not its Rab5 GEF activity, is required to restrict wing vein specification and to suppress the eye phenotypes of oncogenic Ras expression. Furthermore, genetic interaction experiments indicate that Rabex-5 acts at the step of Ras, and tissue culture studies show that Rabex-5 promotes Ras ubiquitination. Together, these findings reveal a new mechanism for attenuating Ras signaling in vivo and suggest an important role for Rabex-5-mediated Ras ubiquitination in pathway homeostasis. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

ApoER2 and Reelin are expressed in regenerating peripheral nerve and regulate Schwann cell migration by activating the Rac1 GEF protein, Tiam1.

PubMed

Pasten, Consuelo; Cerda, Joaquín; Jausoro, Ignacio; Court, Felipe A; Cáceres, Alfredo; Marzolo, Maria-Paz

2015-11-01

ApoER2 and its ligand Reelin participate in neuronal migration during development. Upon receptor binding, Reelin induces the proteolytic processing of ApoER2 as well as the activation of signaling pathway, including small Rho GTPases. Besides its presence in the central nervous system (CNS), Reelin is also secreted by Schwann cells (SCs), the glial cells of the peripheral nervous system (PNS). Reelin deficient mice (reeler) show decreased axonal regeneration in the PNS; however neither the presence of ApoER2 nor the role of the Reelin signaling pathway in the PNS have been evaluated. Interestingly SC migration occurs during PNS development and during injury-induced regeneration and involves activation of small Rho GTPases. Thus, Reelin-ApoER2 might regulate SC migration during axon regeneration in the PNS. Here we demonstrate the presence of ApoER2 in PNS. After sciatic nerve injury Reelin was induced and its receptor ApoER2 was proteolytically processed. In vitro, SCs express both Reelin and ApoER2 and Reelin induces SC migration. To elucidate the molecular mechanism underlying Reelin-dependent SC migration, we examined the involvement of Rac1, a conspicuous small GTPase family member. FRET experiments revealed that Reelin activates Rac1 at the leading edge of SCs. In addition, Tiam1, a major Rac1-specific GEF was required for Reelin-induced SC migration. Moreover, Reelin-induced SC migration was decreased after suppression of the polarity protein PAR3, consistent with its association to Tiam1. Even more interesting, we demonstrated that PAR3 binds preferentially to the full-length cytoplasmic tail of ApoER2 corresponding to the splice-variant containing the exon 19 that encodes a proline-rich insert and that ApoER2 was required for SC migration. Our study reveals a novel function for Reelin/ApoER2 in PNS, inducing cell migration of SCs, a process relevant for PNS development and regeneration. Copyright © 2015 Elsevier Inc. All rights reserved.

Assessment of the Activation State of Rho Family GTP-Binding Proteins in Breast Cancer Cells and Specimens

DTIC Science & Technology

2004-08-01

AF038388 Vav3 GEF for Rho and Rac Proto-oncogene product NM020505 hPEM -2 GEF for Cdc42 Predominantly expressed in brain AB007884 GEF-H1 GEF for Rac and Rho...fence tions, and desmosomes play a fundamental role in maintain- function). A functional tight junction is crucial to maintain ing the polarized phenotype

GIV/Girdin transmits signals from multiple receptors by triggering trimeric G protein activation.

PubMed

Garcia-Marcos, Mikel; Ghosh, Pradipta; Farquhar, Marilyn G

2015-03-13

Activation of trimeric G proteins has been traditionally viewed as the exclusive job of G protein-coupled receptors (GPCRs). This view has been challenged by the discovery of non-receptor activators of trimeric G proteins. Among them, GIV (a.k.a. Girdin) is the first for which a guanine nucleotide exchange factor (GEF) activity has been unequivocally associated with a well defined motif. Here we discuss how GIV assembles alternative signaling pathways by sensing cues from various classes of surface receptors and relaying them via G protein activation. We also describe the dysregulation of this mechanism in disease and how its targeting holds promise for novel therapeutics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cell wall integrity modulates RHO1 activity via the exchange factor ROM2.

PubMed Central

Bickle, M; Delley, P A; Schmidt, A; Hall, M N

1998-01-01

The essential phosphatidylinositol kinase homologue TOR2 of Saccharomyces cerevisiae controls the actin cytoskeleton by activating a GTPase switch consisting of RHO1 (GTPase), ROM2 (GEF) and SAC7 (GAP). We have identified two mutations, rot1-1 and rot2-1, that suppress the loss of TOR2 and are synthetic-lethal. The wild-type ROT1 and ROT2 genes and a multicopy suppressor, BIG1, were isolated by their ability to rescue the rot1-1 rot2-1 double mutant. ROT2 encodes glucosidase II, and ROT1 and BIG1 encode novel proteins. We present evidence that cell wall defects activate RHO1. First, rot1, rot2, big1, cwh41, gas1 and fks1 mutations all confer cell wall defects and suppress tor2(ts). Second, destabilizing the cell wall by supplementing the growth medium with 0.005% SDS also suppresses a tor2(ts) mutation. Third, disturbing the cell wall with SDS or a rot1, rot2, big1, cwh41, gas1 or fks1 mutation increases GDP/GTP exchange activity toward RHO1. These results suggest that cell wall defects suppress a tor2 mutation by activating RHO1 independently of TOR2, thereby inducing TOR2-independent polarization of the actin cytoskeleton and cell wall synthesis. Activation of RHO1, a subunit of the cell wall synthesis enzyme glucan synthase, by a cell wall alteration would ensure that cell wall synthesis occurs only when and where needed. The mechanism of RHO1 activation by a cell wall alteration is via the exchange factor ROM2 and could be analogous to signalling by integrin receptors in mammalian cells. PMID:9545237

Structure of Shigella IpgB2 in Complex with Human RhoA

PubMed Central

Klink, Björn U.; Barden, Stephan; Heidler, Thomas V.; Borchers, Christina; Ladwein, Markus; Stradal, Theresia E. B.; Rottner, Klemens; Heinz, Dirk W.

2010-01-01

A common theme in bacterial pathogenesis is the manipulation of eukaryotic cells by targeting the cytoskeleton. This is in most cases achieved either by modifying actin, or indirectly via activation of key regulators controlling actin dynamics such as Rho-GTPases. A novel group of bacterial virulence factors termed the WXXXE family has emerged as guanine nucleotide exchange factors (GEFs) for these GTPases. The precise mechanism of nucleotide exchange, however, has remained unclear. Here we report the structure of the WXXXE-protein IpgB2 from Shigella flexneri and its complex with human RhoA. We unambiguously identify IpgB2 as a bacterial RhoA-GEF and dissect the molecular mechanism of GDP release, an essential prerequisite for GTP binding. Our observations uncover that IpgB2 induces conformational changes on RhoA mimicking DbI- but not DOCK family GEFs. We also show that dissociation of the GDP·Mg2+ complex is preceded by the displacement of the metal ion to the α-phosphate of the nucleotide, diminishing its affinity to the GTPase. These data refine our understanding of the mode of action not only of WXXXE GEFs but also of mammalian GEFs of the DH/PH family. PMID:20363740

Dbl oncogene expression in MCF-10 A epithelial cells disrupts mammary acinar architecture, induces EMT and angiogenic factor secretion

PubMed Central

Vanni, Cristina; Ognibene, Marzia; Finetti, Federica; Mancini, Patrizia; Cabodi, Sara; Segalerba, Daniela; Torrisi, Maria Rosaria; Donnini, Sandra; Bosco, Maria Carla; Varesio, Luigi; Eva, Alessandra

2015-01-01

The proteins of the Dbl family are guanine nucleotide exchange factors (GEFs) of Rho GTPases and are known to be involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders, neoplastic transformation, and tumor metastasis. We have previously demonstrated that expression of Dbl oncogene in lens epithelial cells modulates genes encoding proteins involved in epithelial-mesenchymal-transition (EMT) and induces angiogenesis in the lens. Our present study was undertaken to investigate the role of Dbl oncogene in epithelial cells transformation, providing new insights into carcinoma progression.To assess how Dbl oncogene can modulate EMT, cell migration, morphogenesis, and expression of pro-apoptotic and angiogenic factors we utilized bi- and 3-dimensional cultures of MCF-10 A cells. We show that upon Dbl expression MCF-10 A cells undergo EMT. In addition, we found that Dbl overexpression sustains Cdc42 and Rac activation inducing morphological alterations, characterized by the presence of lamellipodia and conferring a high migratory capacity to the cells. Moreover, Dbl expressing MCF-10 A cells form altered 3D structures and can induce angiogenesis by producing proangiogenic factors such as CCL2. These results support a role for Dbl oncogene in epithelial cell differentiation and transformation and suggest the relevance of GEF deregulation in tumor onset and progression. PMID:25723869

The G Protein α Chaperone Ric-8 as a Potential Therapeutic Target

PubMed Central

Papasergi, Makaía M.; Patel, Bharti R.

2015-01-01

Resistance to inhibitors of cholinesterase (Ric-8)A and Ric-8B are essential genes that encode positive regulators of heterotrimeric G protein α subunits. Controversy persists surrounding the precise way(s) that Ric-8 proteins affect G protein biology and signaling. Ric-8 proteins chaperone nucleotide-free Gα-subunit states during biosynthetic protein folding prior to G protein heterotrimer assembly. In organisms spanning the evolutionary window of Ric-8 expression, experimental perturbation of Ric-8 genes results in reduced functional abundances of G proteins because G protein α subunits are misfolded and degraded rapidly. Ric-8 proteins also act as Gα-subunit guanine nucleotide exchange factors (GEFs) in vitro. However, Ric-8 GEF activity could strictly be an in vitro phenomenon stemming from the ability of Ric-8 to induce partial Gα unfolding, thereby enhancing GDP release. Ric-8 GEF activity clearly differs from the GEF activity of G protein–coupled receptors (GPCRs). G protein βγ is inhibitory to Ric-8 action but obligate for receptors. It remains an open question whether Ric-8 has dual functions in cells and regulates G proteins as both a molecular chaperone and GEF. Clearly, Ric-8 has a profound influence on heterotrimeric G protein function. For this reason, we propose that Ric-8 proteins are as yet untested therapeutic targets in which pharmacological inhibition of the Ric-8/Gα protein–protein interface could serve to attenuate the effects of disease-causing G proteins (constitutively active mutants) and/or GPCR signaling. This minireview will chronicle the understanding of Ric-8 function, provide a comparative discussion of the Ric-8 molecular chaperoning and GEF activities, and support the case for why Ric-8 proteins should be considered potential targets for development of new therapies. PMID:25319541

Activation of multiple signaling pathways causes developmental defects in mice with a Noonan syndrome–associated Sos1 mutation

PubMed Central

Chen, Peng-Chieh; Wakimoto, Hiroko; Conner, David; Araki, Toshiyuki; Yuan, Tao; Roberts, Amy; Seidman, Christine E.; Bronson, Roderick; Neel, Benjamin G.; Seidman, Jonathan G.; Kucherlapati, Raju

2010-01-01

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, unique facial features, and congenital heart disease. About 10%–15% of individuals with NS have mutations in son of sevenless 1 (SOS1), which encodes a RAS and RAC guanine nucleotide exchange factor (GEF). To understand the role of SOS1 in the pathogenesis of NS, we generated mice with the NS-associated Sos1E846K gain-of-function mutation. Both heterozygous and homozygous mutant mice showed many NS-associated phenotypes, including growth delay, distinctive facial dysmorphia, hematologic abnormalities, and cardiac defects. We found that the Ras/MAPK pathway as well as Rac and Stat3 were activated in the mutant hearts. These data provide in vivo molecular and cellular evidence that Sos1 is a GEF for Rac under physiological conditions and suggest that Rac and Stat3 activation might contribute to NS phenotypes. Furthermore, prenatal administration of a MEK inhibitor ameliorated the embryonic lethality, cardiac defects, and NS features of the homozygous mutant mice, demonstrating that this signaling pathway might represent a promising therapeutic target for NS. PMID:21041952

Evidence for the Involvement of Lfc and Tctex-1 in Axon Formation

PubMed Central

Conde, Cecilia; Arias, Cristina; Robin, Maria; Li, Aiqun; Saito, Masaki; Chuang, Jen-Zen; Nairn, Angus C.; Sung, Ching-Hwa; Cáceres, Alfredo

2013-01-01

RhoA and Rac play key and opposite roles during neuronal polarization. We now show that Lfc, a guanosine nucleotide exchange factor (GEF), localizes to the Golgi apparatus and growth cones of developing neurons and negatively regulates neurite sprouting and axon formation through a Rho signaling pathway. Tctex-1, a dynein light chain implicated in axon outgrowth by modulating actin dynamics and Rac activity, colocalizes and physically interacts with Lfc, thus inhibiting its GEF activity, decreasing Rho-GTP levels, and functionally antagonizing Lfc during neurite formation. PMID:20463241

Heregulin/ErbB3 Signaling Enhances CXCR4-Driven Rac1 Activation and Breast Cancer Cell Motility via Hypoxia-Inducible Factor 1α

PubMed Central

Lopez-Haber, Cynthia; Barrio-Real, Laura; Casado-Medrano, Victoria

2016-01-01

The growth factor heregulin (HRG), a ligand of ErbB3 and ErbB4 receptors, contributes to breast cancer development and the promotion of metastatic disease, and its expression in breast tumors has been associated with poor clinical outcome and resistance to therapy. In this study, we found that breast cancer cells exposed to sustained HRG treatment show markedly enhanced Rac1 activation and migratory activity in response to the CXCR4 ligand SDF-1/CXCL12, effects mediated by P-Rex1, a Rac-guanine nucleotide exchange factor (GEF) aberrantly expressed in breast cancer. Notably, HRG treatment upregulates surface expression levels of CXCR4, a G protein-coupled receptor (GPCR) implicated in breast cancer metastasis and an indicator of poor prognosis in breast cancer patients. A detailed mechanistic analysis revealed that CXCR4 upregulation and sensitization of the Rac response/motility by HRG are mediated by the transcription factor hypoxia-inducible factor 1α (HIF-1α) via ErbB3 and independently of ErbB4. HRG caused prominent induction in the nuclear expression of HIF-1α, which transcriptionally activates the CXCR4 gene via binding to a responsive element located in positions −1376 to −1372 in the CXCR4 promoter, as revealed by mutagenesis analysis and chromatin immunoprecipitation (ChIP). Our results uncovered a novel function for ErbB3 in enhancing breast cancer cell motility and sensitization of the P-Rex1/Rac1 pathway through HIF-1α-mediated transcriptional induction of CXCR4. PMID:27185877

[Subgroup Analysis of the Non-interventional REASON Study: PFS and OS According to Age, Smoking History, Gender, and Histology in NSCLC Patients Treated with Gefitinib or Chemotherapy].

PubMed

Schuette, W; Eberhardt, W E E; Waller, C; Schirmacher, P; Dietel, M; Zirrgiebel, U; Radke, S; Thomas, M

2016-09-01

Assessment of several clinical factors on progression-free (PFS) and overall survival (OS) in NSCLC patients (pts.) (stage IV) with mutated epidermal growth factor receptor (EGFRm+) treated with gefitinib (gef) or with chemotherapy (CT) under real-world conditions. 285 EGFRm+ pts. of the non-interventional REASON study treated with gef (n = 206) or CT (n = 79) as first-line therapy or with gef (n = 213) or CT (n = 61) in any line throughout the course of therapy were analyzed according to age, gender, smoking history and histology. Compared with CT, patients treated with gef showed prolongation of PFS and OS in all subgroups. PFS was significantly increased in women and non-smokers. OS was significantly increased in women, non-smokers, (ex)-smokers, patients with adenocarcinoma and elderly patients when treated with gef compared to CT. Female gender turned out to be an independent positive predictive factor for OS in patients treated with gef (HRmale: 1.74, p = 0.0009). A clinical benefit of gef was shown for all analyzed clinical subgroups of EGFRm+ pts. This was confirmed for the female gender in a multivariate analysis. © Georg Thieme Verlag KG Stuttgart · New York.

Heat induced temperature dysregulation and seizures in Dravet Syndrome/GEFS+ Gabrg2+/Q390X mice.

PubMed

Warner, Timothy A; Liu, Zhong; Macdonald, Robert L; Kang, Jing-Qiong

2017-08-01

It has been established that febrile seizures and its extended syndromes like generalized epilepsy with febrile seizures (FS) plus (GEFS+) and Dravet syndrome have been associated with mutations especially in SCN1A and GABRG2 genes. In patients, the onset of FS is likely due to the combined effect of temperature and inflammation in genetically vulnerable individuals because fever is often associated with infection. Much effort has been spent to understand the mechanisms underlying fever induction of seizures. In addition to the role of cytokines in FS, previous studies in Scn1a +/- knockout mice, a model of Dravet syndrome, indicated that temperature elevation alone could result in seizure generation, and the effect of elevated temperature inducing seizures was age-dependent. Here, we report the thermal effect in a different mouse model of Dravet syndrome, the Gabrg2 +/Q390X knockin mouse. We demonstrated age-dependent dysregulated temperature control and that temperature elevation produced myoclonic jerks, generalized tonic clonic seizures (GTCSs) and heightened anxiety-like symptoms in Gabrg2 +/Q390X mice. The study indicated that regardless of other inflammatory factors, brief heat alone increased brain excitability and induced multiple types of seizures in Gabrg2 +/Q390X mice, suggesting that mutations like GABRG2(Q390X) may alter brain thermal regulation and precipitate seizures during temperature elevations. Copyright © 2017 Elsevier B.V. All rights reserved.

PSD-Zip70 Deficiency Causes Prefrontal Hypofunction Associated with Glutamatergic Synapse Maturation Defects by Dysregulation of Rap2 Activity.

PubMed

Mayanagi, Taira; Yasuda, Hiroki; Sobue, Kenji

2015-10-21

Dysregulation of synapse formation and plasticity is closely related to the pathophysiology of psychiatric and neurodevelopmental disorders. The prefrontal cortex (PFC) is particularly important for executive functions such as working memory, cognition, and emotional control, which are impaired in the disorders. PSD-Zip70 (Lzts1/FEZ1) is a postsynaptic density (PSD) protein predominantly expressed in the frontal cortex, olfactory bulb, striatum, and hippocampus. Here we found that PSD-Zip70 knock-out (PSD-Zip70KO) mice exhibit working memory and cognitive defects, and enhanced anxiety-like behaviors. These abnormal behaviors are caused by impaired glutamatergic synapse transmission accompanied by tiny-headed immature dendritic spines in the PFC, due to aberrant Rap2 activation, which has roles in synapse formation and plasticity. PSD-Zip70 modulates the Rap2 activity by interacting with SPAR (spine-associated RapGAP) and PDZ-GEF1 (RapGEF) in the postsynapse. Furthermore, suppression of the aberrant Rap2 activation in the PFC rescued the behavioral defects in PSD-Zip70KO mice. Our data demonstrate a critical role for PSD-Zip70 in Rap2-dependent spine synapse development in the PFC and underscore the importance of this regulation in PFC-dependent behaviors. PSD-Zip70 deficiency causes behavioral defects in working memory and cognition, and enhanced anxiety due to prefrontal hypofunction. This study revealed that PSD-Zip70 plays essential roles in glutamatergic synapse maturation via modulation of the Rap2 activity in the PFC. PSD-Zip70 interacts with both SPAR (spine-associated RapGAP) and PDZ-GEF1 (RapGEF) and modulates the Rap2 activity in postsynaptic sites. Our results provide a novel Rap2-specific regulatory mechanism in synaptic maturation involving PSD-Zip70. Copyright © 2015 the authors 0270-6474/15/3514327-14$15.00/0.

Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase.

PubMed

Zheng, R; Iwase, A; Shen, R; Goodman, O B; Sugimoto, N; Takuwa, Y; Lerner, D J; Nanus, D M

2006-09-28

The neuropeptides bombesin and endothelin-1 stimulate prostate cancer (PC) cell migration and invasion (J Clin Invest, 2000; 106: 1399-1407). The intracellular signaling pathways that direct this cell movement are not well delineated. The monomeric GTPase RhoA is required for migration in several cell types including neutrophils, monocytes and fibroblasts. We demonstrate that bombesin-stimulated PC cell migration occurs via the heterotrimeric G-protein-coupled receptors (G-protein) G alpha 13 subunit leading to activation of RhoA, and Rho-associated coiled-coil forming protein kinase (ROCK). Using siRNA to suppress expression of the three known G-protein alpha-subunit-associated RhoA guanine nucleotide exchange factors (GEFs), we also show that two of these RhoA GEFs, PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), link bombesin receptors to RhoA in a non-redundant manner in PC cells. We next show that focal adhesion kinase, which activates PDZ-RhoGEF and LARG, is required for bombesin-stimulated RhoA activation. Neutral endopeptidase (NEP) is expressed on normal prostate epithelium whereas loss of NEP expression contributes to PC progression. We also demonstrate that NEP inhibits neuropeptide activation of RhoA. Together, these results establish a contiguous signaling pathway from the bombesin receptor to ROCK in PC cells, and they implicate NEP as a major regulator of neuropeptide-stimulated RhoA in these cells. This work also identifies members of this signaling pathway as potential targets for rational pharmacologic manipulation of neuropeptide-stimulated migration of PC cells.

Assessment of the Activation of Rho Family GTP-Binding Proteins in Breast Cancer Cells and Specimens

DTIC Science & Technology

2001-08-01

lymphopenia Vav2 GEF for Rho, Rac, and Cdc42 proto-oncogene product; NM009500 Vav3 GEF for Rho and Rac proto-oncogene product; NM020505 hPEM -2 GEF for...junctions, and desmosomes play a fundamental role in maintaining the polarized phenotype and vectorial transport functions of epithelial cells. The tight

C3G promotes a selective release of angiogenic factors from activated mouse platelets to regulate angiogenesis and tumor metastasis.

PubMed

Martín-Granado, Víctor; Ortiz-Rivero, Sara; Carmona, Rita; Gutiérrez-Herrero, Sara; Barrera, Mario; San-Segundo, Laura; Sequera, Celia; Perdiguero, Pedro; Lozano, Francisco; Martín-Herrero, Francisco; González-Porras, José Ramón; Muñoz-Chápuli, Ramón; Porras, Almudena; Guerrero, Carmen

2017-12-19

Previous observations indicated that C3G (RAPGEF1) promotes α-granule release, evidenced by the increase in P-selectin exposure on the platelet surface following its activation. The goal of the present study is to further characterize the potential function of C3G as a modulator of the platelet releasate and its implication in the regulation of angiogenesis. Proteomic analysis revealed a decreased secretion of anti-angiogenic factors from activated transgenic C3G and C3G∆Cat platelets. Accordingly, the secretome from both transgenic platelets had an overall pro-angiogenic effect as evidenced by an in vitro capillary-tube formation assay with HUVECs (human umbilical vein endothelial cells) and by two in vivo models of heterotopic tumor growth. In addition, transgenic C3G expression in platelets greatly increased mouse melanoma cells metastasis. Moreover, immunofluorescence microscopy showed that the pro-angiogenic factors VEGF and bFGF were partially retained into α-granules in thrombin- and ADP-activated mouse platelets from both, C3G and C3GΔCat transgenic mice. The observed interaction between C3G and Vesicle-associated membrane protein (Vamp)-7 could explain these results. Concomitantly, increased platelet spreading in both transgenic platelets upon thrombin activation supports this novel function of C3G in α-granule exocytosis. Collectively, our data point out to the co-existence of Rap1GEF-dependent and independent mechanisms mediating C3G effects on platelet secretion, which regulates pathological angiogenesis in tumors and other contexts. The results herein support an important role for platelet C3G in angiogenesis and metastasis.

FgBud3, a Rho4-Interacting Guanine Nucleotide Exchange Factor, Is Involved in Polarity Growth, Cell Division and Pathogenicity of Fusarium graminearum.

PubMed

Zhang, Chengkang; Luo, Zenghong; He, Dongdong; Su, Li; Yin, Hui; Wang, Guo; Liu, Hong; Rensing, Christopher; Wang, Zonghua

2018-01-01

Rho GTPases are signaling macromolecules that are associated with developmental progression and pathogenesis of Fusarium graminearum . Generally, enzymatic activities of Rho GTPases are regulated by Rho GTPase guanine nucleotide exchange factors (RhoGEFs). In this study, we identified a putative RhoGEF encoding gene ( FgBUD3 ) in F. graminearum database and proceeded further by using a functional genetic approach to generate FgBUD3 targeted gene deletion mutant. Phenotypic analysis results showed that the deletion of FgBUD3 caused severe reduction in growth of FgBUD3 mutant generated during this study. We also observed that the deletion of FgBUD3 completely abolished sexual reproduction and triggered the production of abnormal asexual spores with nearly no septum in ΔFgbud3 strain. Further results obtained from infection assays conducted during this research revealed that the FgBUD3 defective mutant lost its pathogenicity on wheat and hence, suggests FgBud3 plays an essential role in the pathogenicity of F. graminearum . Additional, results derived from yeast two-hybrid assays revealed that FgBud3 strongly interacted with FgRho4 compared to the interaction with FgRho2, FgRho3, and FgCdc42. Moreover, we found that FgBud3 interacted with both GTP-bound and GDP-bound form of FgRho4. From these results, we subsequently concluded that, the Rho4-interacting GEF protein FgBud3 crucially promotes vegetative growth, asexual and sexual development, cell division and pathogenicity in F. graminearum .

The General Environment Fit Scale: A Factor Analysis and Test of Convergent Construct Validity

PubMed Central

Beasley, Christopher; Jason, Leonard; Miller, Steven

2014-01-01

Person-environment fit (P-E fit) was initially espoused as an important construct in the field of community psychology; however, most of the theoretical and empirical development of the construct has been conducted by industrial/organizational (I/O) psychologists. In the current study, the GEFS—a P-E fit measure was developed from I/O and business management perspectives on fit—was administered to 246 attendees of an annual convention for residents and alumni of Oxford House (OH), a network of over 1400 mutual-help recovery homes. The authors conducted confirmatory factor and convergent construct validity analyses on the GEFS. The results suggested that the theoretical factor structure of the measure adequately fit the data, suggesting that the GEFS is a valid measure of P-E fit. OH resident fit with their recovery home was related to satisfaction, but not expected tenure. Exploratory analyses revealed that the sufficient supply of resident needs by the recovery home and similarity between residents and their housemates predicted satisfaction with the recovery home, but only similarity with housemates predicted how long residents intended to stay in the OHs. PMID:22071911

Comparative Study of Multiplet Structures of Mn4+ in K2SiF6, K2GeF6, and K2TiF6 Based on First-Principles Configuration-Interaction Calculations

NASA Astrophysics Data System (ADS)

Novita, Mega; Ogasawara, Kazuyoshi

2012-02-01

We performed first-principles configuration-interaction calculations of multiplet energies for Mn4+ in K2SiF6, K2GeF6, and K2TiF6 crystals. The results indicate that corrections based on a single-electron calculation are effective for the prediction of 4A2 → 4T2 and 4A2 → 4T1a transition energies, while such corrections are not necessary for the prediction of the 4A2 → 2E transition energy. The cluster size dependence of the multiplet energies is small. However, the 4A2 → 2E transition energy is slightly improved by using larger clusters including K ions. The theoretical multiplet energies are improved further by considering the lattice relaxation effect. As a result, the characteristic multiplet energy shifts depending on the host crystal are well reproduced without using any empirical parameters. Although K2GeF6 and K2TiF6 have lower symmetry than K2SiF6, the results indicate that the variation of the multiplet energy is mainly determined by the Mn-F bond length.

TD-60 links RalA GTPase function to the CPC in mitosis

PubMed Central

Papini, Diana; Langemeyer, Lars; Abad, Maria A.; Kerr, Alastair; Samejima, Itaru; Eyers, Patrick A.; Jeyaprakash, A. Arockia; Higgins, Jonathan M. G.; Barr, Francis A.; Earnshaw, William C.

2015-01-01

TD-60 (also known as RCC2) is a highly conserved protein that structurally resembles the Ran guanine exchange factor (GEF) RCC1, but has not previously been shown to have GEF activity. TD-60 has a typical chromosomal passenger complex (CPC) distribution in mitotic cells, but associates with integrin complexes and is involved in cell motility during interphase. Here we show that TD-60 exhibits GEF activity, in vitro and in cells, for the small GTPase RalA. TD-60 or RalA depletion causes spindle abnormalities in prometaphase associated with abnormal centromeric accumulation of CPC components. TD-60 and RalA apparently work together to contribute to the regulation of kinetochore–microtubule interactions in early mitosis. Importantly, several mitotic phenotypes caused by TD-60 depletion are reverted by the expression of a GTP-locked mutant, RalA (Q72L). The demonstration that a small GTPase participates in the regulation of the CPC reveals a level of mitotic regulation not suspected in previous studies. PMID:26158537

Deconstructing Ras Signaling in the Thymus

PubMed Central

Kortum, Robert L.; Sommers, Connie L.; Pinski, John M.; Alexander, Clayton P.; Merrill, Robert K.; Li, Wenmei; Love, Paul E.

2012-01-01

Thymocytes must transit at least two distinct developmental checkpoints, governed by signals that emanate from either the pre-T cell receptor (pre-TCR) or the TCR to the small G protein Ras before emerging as functional T lymphocytes. Recent studies have shown a role for the Ras guanine exchange factor (RasGEF) Sos1 at the pre-TCR checkpoint. At the second checkpoint, the quality of signaling through the TCR is interrogated to ensure the production of an appropriate T cell repertoire. Although RasGRP1 is the only confirmed RasGEF required at the TCR checkpoint, current models suggest that the intensity and character of Ras activation, facilitated by both Sos and RasGRP1, will govern the boundary between survival (positive selection) and death (negative selection) at this stage. Using mouse models, we have assessed the independent and combined roles for the RasGEFs Sos1, Sos2, and RasGRP1 during thymocyte development. Although Sos1 was the dominant RasGEF at the pre-TCR checkpoint, combined Sos1/RasGRP1 deletion was required to effectively block development at this stage. Conversely, while RasGRP1 deletion efficiently blocked positive selection, combined RasGRP1/Sos1 deletion was required to block negative selection. This functional redundancy in RasGEFs during negative selection may act as a failsafe mechanism ensuring appropriate central tolerance. PMID:22586275

eIF2β is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control

PubMed Central

Jennings, Martin D.; Kershaw, Christopher J.; White, Christopher; Hoyle, Danielle; Richardson, Jonathan P.; Costello, Joseph L.; Donaldson, Ian J.; Zhou, Yu; Pavitt, Graham D.

2016-01-01

In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2β that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2β mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2β mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2•GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2β acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation. PMID:27458202

eIF2β is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control.

PubMed

Jennings, Martin D; Kershaw, Christopher J; White, Christopher; Hoyle, Danielle; Richardson, Jonathan P; Costello, Joseph L; Donaldson, Ian J; Zhou, Yu; Pavitt, Graham D

2016-11-16

In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2β that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2β mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2β mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2•GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2β acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Transforming growth factor‐β enhances Rho‐kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1

PubMed Central

Shaifta, Yasin; MacKay, Charles E.; Irechukwu, Nneka; O'Brien, Katie A.; Wright, David B.; Ward, Jeremy P. T.

2017-01-01

Key points Transforming growth‐factor‐β (TGF‐β) and RhoA/Rho‐kinase are independently implicated in the airway hyper‐responsiveness associated with asthma, but how these proteins interact is not fully understood.We examined the effects of pre‐treatment with TGF‐β on expression and activity of RhoA, Rho‐kinase and ARHGEF1, an activator of RhoA, as well as on bradykinin‐induced contraction, in airway smooth muscle.TGF‐β enhanced bradykinin‐induced RhoA translocation, Rho‐kinase‐dependent phosphorylation and contraction, but partially suppressed bradykinin‐induced RhoA activity (RhoA‐GTP content).TGF‐β enhanced the expression of ARHGEF1, while a small interfering RNA against ARHGEF1 and a RhoGEF inhibitor prevented the effects of TGF‐β on RhoA and Rho‐kinase activity and contraction, respectively.ARHGEF1 expression was also enhanced in airway smooth muscle from asthmatic patients and ovalbumin‐sensitized mice.ARHGEF1 is a key TGF‐β target gene, an important regulator of Rho‐kinase activity and therefore a potential therapeutic target for the treatment of asthmatic airway hyper‐responsiveness. Abstract Transforming growth factor‐β (TGF‐β), RhoA/Rho‐kinase and Src‐family kinases (SrcFK) have independently been implicated in airway hyper‐responsiveness, but how they interact to regulate airway smooth muscle contractility is not fully understood. We found that TGF‐β pre‐treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho‐kinase (Y27632), but not the SrcFK inhibitor PP2, prevented this enhancement. In cultured human airway smooth muscle cells (hASMCs), TGF‐β pre‐treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC20, MYPT‐1 and the actin‐severing protein cofilin, but not of RhoA, ROCK2 or c‐Src. In hASMCs, acute treatment with BK triggered subcellular translocation

Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity

PubMed Central

Miller, Megan B.; Yan, Yan; Machida, Kazuya; Kiraly, Drew D.; Levy, Aaron D.; Wu, Yi I.; Lam, TuKiet T.; Abbott, Thomas; Koleske, Anthony J.; Eipper, Betty A.; Mains, Richard E.

2017-01-01

Kalirin7 (Kal7), a postsynaptic Rho GDP/GTP exchange factor (RhoGEF), plays a crucial role in long term potentiation and in the effects of cocaine on behavior and spine morphology. The KALRN gene has been linked to schizophrenia and other disorders of synaptic function. Mass spectrometry was used to quantify phosphorylation at 26 sites in Kal7 from individual adult rat nucleus accumbens and prefrontal cortex before and after exposure to acute or chronic cocaine. Region- and isoform-specific phosphorylation was observed along with region-specific effects of cocaine on Kal7 phosphorylation. Evaluation of the functional significance of multi-site phosphorylation in a complex protein like Kalirin is difficult. With the identification of five tyrosine phosphorylation (pY) sites, a panel of 71 SH2 domains was screened, identifying subsets that interacted with multiple pY sites in Kal7. In addition to this type of reversible interaction, endoproteolytic cleavage by calpain plays an essential role in long-term potentiation. Calpain cleaved Kal7 at two sites, separating the N-terminal domain, which affects spine length, and the PDZ binding motif from the GEF domain. Mutations preventing phosphorylation did not affect calpain sensitivity or GEF activity; phosphomimetic mutations at specific sites altered protein stability, increased calpain sensitivity and reduced GEF activity. PMID:28418645

Activator-inhibitor coupling between Rho signaling and actin assembly make the cell cortex an excitable medium

PubMed Central

Bement, William M.; Leda, Marcin; Moe, Alison M.; Kita, Angela M.; Larson, Matthew E.; Golding, Adriana E.; Pfeuti, Courtney; Su, Kuan-Chung; Miller, Ann L.; Goryachev, Andrew B.; von Dassow, George

2016-01-01

Animal cell cytokinesis results from patterned activation of the small GTPase Rho, which directs assembly of actomyosin in the equatorial cortex. Cytokinesis is restricted to a portion of the cell cycle following anaphase onset in which the cortex is responsive to signals from the spindle. We show that shortly after anaphase onset oocytes and embryonic cells of frogs and echinoderms exhibit cortical waves of Rho activity and F-actin polymerization. The waves are modulated by cyclin-dependent kinase 1 (Cdk1) activity and require the Rho GEF (guanine nucleotide exchange factor), Ect2. Surprisingly, during wave propagation, while Rho activity elicits F-actin assembly, F-actin subsequently inactivates Rho. Experimental and modeling results show that waves represent excitable dynamics of a reaction diffusion system with Rho as the activator and F-actin the inhibitor. We propose that cortical excitability explains fundamental features of cytokinesis including its cell cycle regulation. PMID:26479320

Developmental lineage priming in Dictyostelium by heterogeneous Ras activation.

PubMed

Chattwood, Alex; Nagayama, Koki; Bolourani, Parvin; Harkin, Lauren; Kamjoo, Marzieh; Weeks, Gerald; Thompson, Christopher R L

2013-11-26

In cell culture, genetically identical cells often exhibit heterogeneous behavior, with only 'lineage primed' cells responding to differentiation inducing signals. It has recently been proposed that such heterogeneity exists during normal embryonic development to allow position independent patterning based on 'salt and pepper' differentiation and sorting out. However, the molecular basis of lineage priming and how it leads to reproducible cell type proportioning are poorly understood. To address this, we employed a novel forward genetic approach in the model organism Dictyostelium discoideum. These studies reveal that the Ras-GTPase regulator gefE is required for normal lineage priming and salt and pepper differentiation. This is because Ras-GTPase activity sets the intrinsic response threshold to lineage specific differentiation signals. Importantly, we show that although gefE expression is uniform, transcription of its target, rasD, is both heterogeneous and dynamic, thus providing a novel mechanism for heterogeneity generation and position-independent differentiation. DOI: http://dx.doi.org/10.7554/eLife.01067.001.

ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores

PubMed Central

Isokane, Mayumi; Walter, Thomas; Mahen, Robert; Nijmeijer, Bianca; Hériché, Jean-Karim; Miura, Kota; Maffini, Stefano; Ivanov, Miroslav Penchev; Kitajima, Tomoya S.; Peters, Jan-Michael

2016-01-01

To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17’s RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment. PMID:26953350

ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores.

PubMed

Isokane, Mayumi; Walter, Thomas; Mahen, Robert; Nijmeijer, Bianca; Hériché, Jean-Karim; Miura, Kota; Maffini, Stefano; Ivanov, Miroslav Penchev; Kitajima, Tomoya S; Peters, Jan-Michael; Ellenberg, Jan

2016-03-14

To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17's RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment. © 2016 Isokane et al.

The Juxtamembrane and carboxy-terminal domains of Arabidopsis PRK2 are critical for ROP-induced growth in pollen tubes

USDA-ARS?s Scientific Manuscript database

Polarized growth of pollen tubes is a critical step for successful reproduction in angiosperms and is controlled by ROP GTPases. Spatiotemporal activation of ROP (Rho GTPases of plants) necessitates a complex and sophisticated regulatory system, in which guanine nucleotide exchange factors (RopGEFs)...

Cadherin-11 regulates protrusive activity in Xenopus cranial neural crest cells upstream of Trio and the small GTPases

PubMed Central

Kashef, Jubin; Köhler, Almut; Kuriyama, Sei; Alfandari, Dominique; Mayor, Roberto; Wedlich, Doris

2009-01-01

Xenopus Cadherin-11 (Xcad-11) is expressed when cranial neural crest cells (CNC) acquire motility. However, its function in stimulating cell migration is poorly understood. Here, we demonstrate that Xcad-11 initiates filopodia and lamellipodia formation, which is essential for CNC to populate pharyngeal pouches. We identified the cytoplasmic tail of Xcad-11 as both necessary and sufficient for proper CNC migration as long as it was linked to the plasma membrane. Our results showing that guanine nucleotide exchange factor (GEF)-Trio binds to Xcad-11 and can functionally substitute for it like constitutively active forms of RhoA, Rac, and cdc42 unravel a novel cadherin function. PMID:19528317

Cadherin-11 regulates protrusive activity in Xenopus cranial neural crest cells upstream of Trio and the small GTPases.

PubMed

Kashef, Jubin; Köhler, Almut; Kuriyama, Sei; Alfandari, Dominique; Mayor, Roberto; Wedlich, Doris

2009-06-15

Xenopus Cadherin-11 (Xcad-11) is expressed when cranial neural crest cells (CNC) acquire motility. However, its function in stimulating cell migration is poorly understood. Here, we demonstrate that Xcad-11 initiates filopodia and lamellipodia formation, which is essential for CNC to populate pharyngeal pouches. We identified the cytoplasmic tail of Xcad-11 as both necessary and sufficient for proper CNC migration as long as it was linked to the plasma membrane. Our results showing that guanine nucleotide exchange factor (GEF)-Trio binds to Xcad-11 and can functionally substitute for it like constitutively active forms of RhoA, Rac, and cdc42 unravel a novel cadherin function.

The Guanine Nucleotide Exchange Factor Tiam1 Affects Neuronal Morphology; Opposing Roles for the Small GTPases Rac and Rho

PubMed Central

van Leeuwen, Frank N.; Kain, Hendrie E.T.; van der Kammen, Rob A.; Michiels, Frits; Kranenburg, Onno W.; Collard, John G.

1997-01-01

The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the α6β1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid– induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process. PMID:9348295

A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction.

PubMed

Kopra, Kari; Ligabue, Alessio; Wang, Qi; Syrjänpää, Markku; Blaževitš, Olga; Veltel, Stefan; van Adrichem, Arjan J; Hänninen, Pekka; Abankwa, Daniel; Härmä, Harri

2014-07-01

A quenching resonance energy transfer (QRET) assay for small GTPase nucleotide exchange kinetic monitoring is demonstrated using nanomolar protein concentrations. Small GTPases are central signaling proteins in all eukaryotic cells acting as a "molecular switches" that are active in the GTP-state and inactive in the GDP-state. GTP-loading is highly regulated by guanine nucleotide exchange factors (GEFs). In several diseases, most prominently cancer, this process in misregulated. The kinetics of the nucleotide exchange reaction reports on the enzymatic activity of the GEF reaction system and is, therefore, of special interest. We determined the nucleotide exchange kinetics using europium-labeled GTP (Eu-GTP) in the QRET assay for small GTPases. After GEF catalyzed GTP-loading of a GTPase, a high time-resolved luminescence signal was found to be associated with GTPase bound Eu-GTP, whereas the non-bound Eu-GTP fraction was quenched by soluble quencher. The association kinetics of the Eu-GTP was measured after GEF addition, whereas the dissociation kinetics could be determined after addition of unlabeled GTP. The resulting association and dissociation rates were in agreement with previously published values for H-Ras(Wt), H-Ras(Q61G), and K-Ras(Wt), respectively. The broader applicability of the QRET assay for small GTPases was demonstrated by determining the kinetics of the Ect2 catalyzed RhoA(Wt) GTP-loading. The QRET assay allows the use of nanomolar protein concentrations, as more than 3-fold signal-to-background ratio was achieved with 50 nM GTPase and GEF proteins. Thus, small GTPase exchange kinetics can be efficiently determined in a HTS compatible 384-well plate format.

Heparanase enhances the generation of activated factor X in the presence of tissue factor and activated factor VII.

PubMed

Nadir, Yona; Brenner, Benjamin; Fux, Liat; Shafat, Itay; Attias, Judith; Vlodavsky, Israel

2010-11-01

Heparanase is an endo-β-D-glucuronidase dominantly involved in tumor metastasis and angiogenesis. Recently, we demonstrated that heparanase is involved in the regulation of the hemostatic system. Our hypothesis was that heparanase is directly involved in activation of the coagulation cascade. Activated factor X and thrombin were studied using chromogenic assays, immunoblotting and thromboelastography. Heparanase levels were measured by enzyme-linked immunosorbent assay. A potential direct interaction between tissue factor and heparanase was studied by co-immunoprecipitation and far-western assays. Interestingly, addition of heparanase to tissue factor and activated factor VII resulted in a 3- to 4-fold increase in activation of the coagulation cascade as shown by increased activated factor X and thrombin production. Culture medium of human embryonic kidney 293 cells over-expressing heparanase and its derivatives increased activated factor X levels in a non-enzymatic manner. When heparanase was added to pooled normal plasma, a 7- to 8-fold increase in activated factor X level was observed. Subsequently, we searched for clinical data supporting this newly identified role of heparanase. Plasma samples from 35 patients with acute leukemia at presentation and 20 healthy donors were studied for heparanase and activated factor X levels. A strong positive correlation was found between plasma heparanase and activated factor X levels (r=0.735, P=0.001). Unfractionated heparin and an inhibitor of activated factor X abolished the effect of heparanase, while tissue factor pathway inhibitor and tissue factor pathway inhibitor-2 only attenuated the procoagulant effect. Using co-immunoprecipitation and far-western analyses it was shown that heparanase interacts directly with tissue factor. Overall, our results support the notion that heparanase is a potential modulator of blood hemostasis, and suggest a novel mechanism by which heparanase increases the generation of activated

Epidermal wound repair is regulated by the planar cell polarity signaling pathway.

PubMed

Caddy, Jacinta; Wilanowski, Tomasz; Darido, Charbel; Dworkin, Sebastian; Ting, Stephen B; Zhao, Quan; Rank, Gerhard; Auden, Alana; Srivastava, Seema; Papenfuss, Tony A; Murdoch, Jennifer N; Humbert, Patrick O; Parekh, Vishwas; Boulos, Nidal; Weber, Thomas; Zuo, Jian; Cunningham, John M; Jane, Stephen M

2010-07-20

The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. Here, we show that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, Celsr1, PTK7, and Scrb1, and the transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects, and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3(-)(/-) mice, we identified RhoGEF19, a homolog of a RhoA activator involved in PCP signaling in Xenopus, as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerization, cellular polarity, and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling and broadly implicate this pathway in epidermal repair. (c) 2010 Elsevier Inc. All rights reserved.

Epidermal wound repair is regulated by the planar cell polarity signaling pathway

PubMed Central

Caddy, Jacinta; Wilanowski, Tomasz; Darido, Charbel; Dworkin, Sebastian; Ting, Stephen B.; Zhao, Quan; Rank, Gerhard; Auden, Alana; Srivastava, Seema; Papenfuss, Tony A.; Murdoch, Jennifer N.; Humbert, Patrick O.; Boulos, Nidal; Weber, Thomas; Zuo, Jian; Cunningham, John M.; Jane, Stephen M.

2010-01-01

SUMMARY The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. Here, we show that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, Celsr1, PTK7, and Scrb1, and the transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3−/− mice, we identified RhoGEF19, a homologue of a RhoA activator involved in PCP signaling in Xenopus, as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerisation, cellular polarity and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling, and broadly implicate this pathway in epidermal repair. PMID:20643356

Gα12 structural determinants of Hsp90 interaction are necessary for serum response element-mediated transcriptional activation.

PubMed

Montgomery, Ellyn R; Temple, Brenda R S; Peters, Kimberly A; Tolbert, Caitlin E; Booker, Brandon K; Martin, Joseph W; Hamilton, Tyler P; Tagliatela, Alicia C; Smolski, William C; Rogers, Stephen L; Jones, Alan M; Meigs, Thomas E

2014-04-01

The G12/13 class of heterotrimeric G proteins, comprising the α-subunits Gα12 and Gα13, regulates multiple aspects of cellular behavior, including proliferation and cytoskeletal rearrangements. Although guanine nucleotide exchange factors for the monomeric G protein Rho (RhoGEFs) are well characterized as effectors of this G protein class, a variety of other downstream targets has been reported. To identify Gα12 determinants that mediate specific protein interactions, we used a structural and evolutionary comparison between the G12/13, Gs, Gi, and Gq classes to identify "class-distinctive" residues in Gα12 and Gα13. Mutation of these residues in Gα12 to their deduced ancestral forms revealed a subset necessary for activation of serum response element (SRE)-mediated transcription, a G12/13-stimulated pathway implicated in cell proliferative signaling. Unexpectedly, this subset of Gα12 mutants showed impaired binding to heat-shock protein 90 (Hsp90) while retaining binding to RhoGEFs. Corresponding mutants of Gα13 exhibited robust SRE activation, suggesting a Gα12-specific mechanism, and inhibition of Hsp90 by geldanamycin or small interfering RNA-mediated lowering of Hsp90 levels resulted in greater downregulation of Gα12 than Gα13 signaling in SRE activation experiments. Furthermore, the Drosophila G12/13 homolog Concertina was unable to signal to SRE in mammalian cells, and Gα12:Concertina chimeras revealed Gα12-specific determinants of SRE activation within the switch regions and a C-terminal region. These findings identify Gα12 determinants of SRE activation, implicate Gα12:Hsp90 interaction in this signaling mechanism, and illuminate structural features that arose during evolution of Gα12 and Gα13 to allow bifurcated mechanisms of signaling to a common cell proliferative pathway.

The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells.

PubMed

Lucato, Christina M; Halls, Michelle L; Ooms, Lisa M; Liu, Heng-Jia; Mitchell, Christina A; Whisstock, James C; Ellisdon, Andrew M

2015-08-21

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP3 and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP3/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP3 and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes.

PubMed

Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

2016-01-15

Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes*

PubMed Central

Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

2016-01-01

Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes. PMID:26620560

Metabolomics reveals the formation of aldehydes and iminium in gefitinib metabolism

USDA-ARS?s Scientific Manuscript database

Gefitinib (GEF), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is widely used for the treatment of cancers, particularly non-small cell lung cancer. However, its clinical use is limited by multiple adverse effects associated with GEF, such as liver and lung injuries, sever...

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation

PubMed Central

Zou, Siying; Teixeira, Alexandra M.; Yin, Mingzhu; Xiang, Yaozu; Xavier-Ferruccio, Juliana; Zhang, Ping-xia; Hwa, John; Min, Wang; Krause, Diane S.

2018-01-01

Summary Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal hemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout, shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bβ3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using 2 different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice. PMID:27345948

Leukaemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation.

PubMed

Zou, Siying; Teixeira, Alexandra M; Yin, Mingzhu; Xiang, Yaozu; Xavier-Ferrucio, Juliana; Zhang, Ping-Xia; Hwa, John; Min, Wang; Krause, Diane S

2016-08-30

Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal haemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout (KO), shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bβ3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using two different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice.

GDP-bound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5·Vps9 system.

PubMed

Uejima, Tamami; Ihara, Kentaro; Goh, Tatsuaki; Ito, Emi; Sunada, Mariko; Ueda, Takashi; Nakano, Akihiko; Wakatsuki, Soichi

2010-11-19

Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.

Multiplex image-based autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression regulator

PubMed Central

Jung, Jennifer; Nayak, Arnab; Schaeffer, Véronique; Starzetz, Tatjana; Kirsch, Achim K; Müller, Stefan; Dikic, Ivan; Mittelbronn, Michel; Behrends, Christian

2017-01-01

Autophagy is an intracellular recycling and degradation pathway that depends on membrane trafficking. Rab GTPases are central for autophagy but their regulation especially through the activity of Rab GEFs remains largely elusive. We employed a RNAi screen simultaneously monitoring different populations of autophagosomes and identified 34 out of 186 Rab GTPase, GAP and GEF family members as potential autophagy regulators, amongst them SMCR8. SMCR8 uses overlapping binding regions to associate with C9ORF72 or with a C9ORF72-ULK1 kinase complex holo-assembly, which function in maturation and formation of autophagosomes, respectively. While focusing on the role of SMCR8 during autophagy initiation, we found that kinase activity and gene expression of ULK1 are increased upon SMCR8 depletion. The latter phenotype involved association of SMCR8 with the ULK1 gene locus. Global mRNA expression analysis revealed that SMCR8 regulates transcription of several other autophagy genes including WIPI2. Collectively, we established SMCR8 as multifaceted negative autophagy regulator. DOI: http://dx.doi.org/10.7554/eLife.23063.001 PMID:28195531

Interplay between Solo and keratin filaments is crucial for mechanical force–induced stress fiber reinforcement

PubMed Central

Fujiwara, Sachiko; Ohashi, Kazumasa; Mashiko, Toshiya; Kondo, Hiroshi; Mizuno, Kensaku

2016-01-01

Mechanical force–induced cytoskeletal reorganization is essential for cell and tissue remodeling and homeostasis; however, the underlying cellular mechanisms remain elusive. Solo (ARHGEF40) is a RhoA-targeting guanine nucleotide exchange factor (GEF) involved in cyclical stretch–induced human endothelial cell reorientation and convergent extension cell movement in zebrafish gastrula. In this study, we show that Solo binds to keratin-8/keratin-18 (K8/K18) intermediate filaments through multiple sites. Solo overexpression promotes the formation of thick actin stress fibers and keratin bundles, whereas knockdown of Solo, expression of a GEF-inactive mutant of Solo, or inhibition of ROCK suppresses stress fiber formation and leads to disorganized keratin networks, indicating that the Solo-RhoA-ROCK pathway serves to precisely organize keratin networks, as well as to promote stress fibers. Of importance, knockdown of Solo or K18 or overexpression of GEF-inactive or deletion mutants of Solo suppresses tensile force–induced stress fiber reinforcement. Furthermore, knockdown of Solo or K18 suppresses tensile force-induced RhoA activation. These results strongly suggest that the interplay between Solo and K8/K18 filaments plays a crucial role in tensile force–induced RhoA activation and consequent actin cytoskeletal reinforcement. PMID:26823019

The activation of plasminogen by Hageman factor (Factor XII) and Hageman factor fragments.

PubMed Central

Goldsmith, G H; Saito, H; Ratnoff, O S

1978-01-01

Activation of plasminogen through surface-mediated reactions is well recognized. In the presence of kaolin, purified Hageman factor (Factor XII) changed plasminogen to plasmin, as assayed upon a synthetic amide substrate and by fibrinolysis. Kinetic studies suggested an enzymatic action of Hageman factor upon its substrate, plasminogen. Hageman factor fragments, at a protein concentration equivalent to whole Hageman factor, activated plasminogen to a lesser extent. These protein preparations were not contaminated with other agents implicated in surface-mediated fibrinolysis. Diisopropyl fluorophosphate treatment of plasminogen did not inhibit its activation by Hageman factor. These studies indicate that Hageman factor has a hitherto unsuspected function, the direct activation of plasminogen. PMID:659637

The Chromobacterium violaceum type III effector CopE, a guanine nucleotide exchange factor for Rac1 and Cdc42, is involved in bacterial invasion of epithelial cells and pathogenesis.

PubMed

Miki, Tsuyoshi; Akiba, Kinari; Iguchi, Mirei; Danbara, Hirofumi; Okada, Nobuhiko

2011-06-01

The type III secretion system (T3SS) encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi-1/-1a) is critical for Chromobacterium violaceum pathogenesis. T3SS-dependent virulence is commonly characterized by type III effector virulence function, but the full repertoire of the effector proteins of Cpi-1/-1a T3SS is unknown. In this study, we showed that expression of Cpi-1/-1a T3SS is controlled by the master regulator CilA. We used transcriptional profiling with DNA microarrays to define CilA regulon and identified genes encoding T3SS effectors whose translocation into host cells was dependent on Cpi-1/-1a T3SS. From these effectors, we found that CopE (CV0296) has similarities to a guanine nucleotide exchange factor (GEF) for Rho GTPases in its C-terminal portion. The N-terminal portions (1-81 amino acids) of CopE and a CivB as a putative chaperone were required for its translocation. CopE specifically activates Rac1 and Cdc42 followed by the induction of actin cytoskeletal rearrangement. Interestingly, C. violaceum invades human epithelial HeLa cells in a Cpi-1/-1a-encoded T3SS- and CopE-dependent manner. Finally, C. violaceum strains lacking copE and expressing a CopE-G168V deficient in GEF activity were attenuated for virulence in mice, suggesting that CopE contributes to the virulence of this pathogen. © 2011 Blackwell Publishing Ltd.

Fission Activities of the Nuclear Reactions Group in Uppsala

NASA Astrophysics Data System (ADS)

Al-Adili, A.; Alhassan, E.; Gustavsson, C.; Helgesson, P.; Jansson, K.; Koning, A.; Lantz, M.; Mattera, A.; Prokofiev, A. V.; Rakopoulos, V.; Sjöstrand, H.; Solders, A.; Tarrío, D.; Österlund, M.; Pomp, S.

This paper highlights some of the main activities related to fission of the nuclear reactions group at Uppsala University. The group is involved for instance in fission yield experiments at the IGISOL facility, cross-section measurements at the NFS facility, as well as fission dynamics studies at the IRMM JRC-EC. Moreover, work is ongoing on the Total Monte Carlo (TMC) methodology and on including the GEF fission code into the TALYS nuclear reaction code. Selected results from these projects are discussed.

Role of Longwave Cloud-Radiation Feedback in the Simulation of the Madden-Julian Oscillation

NASA Technical Reports Server (NTRS)

Kim, Daehyun; Ahn, Min-Seop; Kang, In-Sik; Del Genio, Anthony D.

2015-01-01

The role of the cloud-radiation interaction in the simulation of the Madden-Julian oscillation (MJO) is investigated. A special focus is on the enhancement of column-integrated diabatic heating due to the greenhouse effects of clouds and moisture in the region of anomalous convection. The degree of this enhancement, the greenhouse enhancement factor (GEF), is measured at different precipitation anomaly regimes as the negative ratio of anomalous outgoing longwave radiation to anomalous precipitation. Observations show that the GEF varies significantly with precipitation anomaly and with the MJO cycle. The greenhouse enhancement is greater in weak precipitation anomaly regimes and its effectiveness decreases monotonically with increasing precipitation anomaly. The GEF also amplifies locally when convection is strengthened in association with the MJO, especially in the weak precipitation anomaly regime (less than 5 mm day(exp -1)). A robust statistical relationship is found among CMIP5 climate model simulations between the GEF and the MJO simulation fidelity. Models that simulate a stronger MJO also simulate a greater GEF, especially in the weak precipitation anomaly regime (less than 5 mm day(exp -1)). Models with a greater GEF in the strong precipitation anomaly regime (greater than 30 mm day(-1)) represent a slightly slower MJO propagation speed. Many models that lack the MJO underestimate the GEF in general and in particular in the weak precipitation anomaly regime. The results herein highlight that the cloud-radiation interaction is a crucial process for climate models to correctly represent the MJO.

The Molecular Motor KIF21B Mediates Synaptic Plasticity and Fear Extinction by Terminating Rac1 Activation.

PubMed

Morikawa, Momo; Tanaka, Yosuke; Cho, Hyun-Soo; Yoshihara, Masaharu; Hirokawa, Nobutaka

2018-06-26

Fear extinction is a component of cognitive flexibility that is relevant for important psychiatric diseases, but its molecular mechanism is still largely elusive. We established mice lacking the kinesin-4 motor KIF21B as a model for fear extinction defects. Postsynaptic NMDAR-dependent long-term depression (LTD) is specifically impaired in knockouts. NMDAR-mediated LTD-causing stimuli induce dynamic association of KIF21B with the Rac1GEF subunit engulfment and cell motility protein 1 (ELMO1), leading to ELMO1 translocation out of dendritic spines and its sequestration in endosomes. This process may essentially terminate transient activation of Rac1, shrink spines, facilitate AMPAR endocytosis, and reduce postsynaptic strength, thereby forming a mechanistic link to LTD expression. Antagonizing ELMO1/Dock Rac1GEF activity by the administration of 4-[3'-(2″-chlorophenyl)-2'-propen-1'-ylidene]-1-phenyl-3,5-pyrazolidinedione (CPYPP) significantly reverses the knockout phenotype. Therefore, we propose that KIF21B-mediated Rac1 inactivation is a key molecular event in NMDAR-dependent LTD expression underlying cognitive flexibility in fear extinction. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Rab14 and Its Exchange Factor FAM116 Link Endocytic Recycling and Adherens Junction Stability in Migrating Cells

PubMed Central

Linford, Andrea; Yoshimura, Shin-ichiro; Bastos, Ricardo Nunes; Langemeyer, Lars; Gerondopoulos, Andreas; Rigden, Daniel J.; Barr, Francis A.

2012-01-01

Summary Rab GTPases define the vesicle trafficking pathways underpinning cell polarization and migration. Here, we find that Rab4, Rab11, and Rab14 and the candidate Rab GDP-GTP exchange factors (GEFs) FAM116A and AVL9 are required for cell migration. Rab14 and its GEF FAM116A localize to and act on an intermediate compartment of the transferrin-recycling pathway prior to Rab11 and after Rab5 and Rab4. This Rab14 intermediate recycling compartment has specific functions in migrating cells discrete from early and recycling endosomes. Rab14-depleted cells show increased N-cadherin levels at junctional complexes and cannot resolve cell-cell junctions. This is due to decreased shedding of cell-surface N-cadherin by the ADAM family protease ADAM10/Kuzbanian. In FAM116A- and Rab14-depleted cells, ADAM10 accumulates in a transferrin-positive endocytic compartment, and the cell-surface level of ADAM10 is correspondingly reduced. FAM116 and Rab14 therefore define an endocytic recycling pathway needed for ADAM protease trafficking and regulation of cell-cell junctions. PMID:22595670

Wsc1 and Mid2 Are Cell Surface Sensors for Cell Wall Integrity Signaling That Act through Rom2, a Guanine Nucleotide Exchange Factor for Rho1

PubMed Central

Philip, Bevin; Levin, David E.

2001-01-01

Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae. They have been proposed to function as mechanosensors of cell wall stress induced by wall remodeling during vegetative growth and pheromone-induced morphogenesis. These proteins are required for activation of the cell wall integrity signaling pathway that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade. We show here by two-hybrid experiments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact with Rom2, a guanine nucleotide exchange factor (GEF) for Rho1. At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain. This domain is distinct from the Rho1-interacting domain, suggesting that the GEF can interact simultaneously with a sensor and with Rho1. We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the ability to catalyze GTP loading of Rho1 in vitro, providing evidence that the function of the sensor-Rom2 interaction is to stimulate nucleotide exchange toward this G-protein. In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive cell lysis defect with a wsc1 null allele. Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins. We demonstrate that Mid2 is not mannosylated in a pmt2 mutant and that this modification is important for signaling by Mid2. PMID:11113201

Myosin IIA/IIB restrict adhesive and protrusive signaling to generate front-back polarity in migrating cells.

PubMed

Vicente-Manzanares, Miguel; Newell-Litwa, Karen; Bachir, Alexia I; Whitmore, Leanna A; Horwitz, Alan Rick

2011-04-18

Migratory front-back polarity emerges from the cooperative effect of myosin IIA (MIIA) and IIB (MIIB) on adhesive signaling. We demonstrate here that, during polarization, MIIA and MIIB coordinately promote localized actomyosin bundling, which generates large, stable adhesions that do not signal to Rac and thereby form the cell rear. MIIA formed dynamic actomyosin proto-bundles that mark the cell rear during spreading; it also bound to actin filament bundles associated with initial adhesion maturation in protrusions. Subsequent incorporation of MIIB stabilized the adhesions and actomyosin filaments with which it associated and formed a stable, extended rear. These adhesions did not turn over and no longer signal to Rac. Microtubules fine-tuned the polarity by positioning the front opposite the MIIA/MIIB-specified rear. Decreased Rac signaling in the vicinity of the MIIA/MIIB-stabilized proto-bundles and adhesions was accompanied by the loss of Rac guanine nucleotide exchange factor (GEFs), like βPIX and DOCK180, and by inhibited phosphorylation of key residues on adhesion proteins that recruit and activate Rac GEFs. These observations lead to a model for front-back polarity through local GEF depletion.

The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

PubMed Central

Van Eps, Ned; Thomas, Celestine J.; Hubbell, Wayne L.; Sprang, Stephen R.

2015-01-01

Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (Gα), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β2 adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP- and GTP-bound states of Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF. PMID:25605908

G protein signaling in the parasite Entamoeba histolytica

PubMed Central

Bosch, Dustin E; Siderovski, David P

2013-01-01

The parasite Entamoeba histolytica causes amebic colitis and systemic amebiasis. Among the known amebic factors contributing to pathogenesis are signaling pathways involving heterotrimeric and Ras superfamily G proteins. Here, we review the current knowledge of the roles of heterotrimeric G protein subunits, Ras, Rho and Rab GTPase families in E. histolytica pathogenesis, as well as of their downstream signaling effectors and nucleotide cycle regulators. Heterotrimeric G protein signaling likely modulates amebic motility and attachment to and killing of host cells, in part through activation of an RGS-RhoGEF (regulator of G protein signaling–Rho guanine nucleotide exchange factor) effector. Rho family GTPases, as well as RhoGEFs and Rho effectors (formins and p21-activated kinases) regulate the dynamic actin cytoskeleton of E. histolytica and associated pathogenesis-related cellular processes, such as migration, invasion, phagocytosis and evasion of the host immune response by surface receptor capping. A remarkably large family of 91 Rab GTPases has multiple roles in a complex amebic vesicular trafficking system required for phagocytosis and pinocytosis and secretion of known virulence factors, such as amebapores and cysteine proteases. Although much remains to be discovered, recent studies of G protein signaling in E. histolytica have enhanced our understanding of parasitic pathogenesis and have also highlighted possible targets for pharmacological manipulation. PMID:23519208

Optimal experimental design in an epidermal growth factor receptor signalling and down-regulation model.

PubMed

Casey, F P; Baird, D; Feng, Q; Gutenkunst, R N; Waterfall, J J; Myers, C R; Brown, K S; Cerione, R A; Sethna, J P

2007-05-01

We apply the methods of optimal experimental design to a differential equation model for epidermal growth factor receptor signalling, trafficking and down-regulation. The model incorporates the role of a recently discovered protein complex made up of the E3 ubiquitin ligase, Cbl, the guanine exchange factor (GEF), Cool-1 (beta -Pix) and the Rho family G protein Cdc42. The complex has been suggested to be important in disrupting receptor down-regulation. We demonstrate that the model interactions can accurately reproduce the experimental observations, that they can be used to make predictions with accompanying uncertainties, and that we can apply ideas of optimal experimental design to suggest new experiments that reduce the uncertainty on unmeasurable components of the system.

A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network

PubMed Central

Cheng, Yougan; Othmer, Hans

2016-01-01

Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that Gα2βγ cycling modulated by Ric8, a nonreceptor guanine exchange factor for Gα2 in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both Gα2 and Gβγ are essential for direction sensing, in that membrane-localized Gα2*, the activated GTP-bearing form of Gα2, leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient ‘memory’ to eliminate the ‘back-of-the wave’ problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since the signal pathways we study are highly conserved between Dicty

Synergistic antitumor effect between gefitinib and fractionated irradiation in anaplastic oligodendrogliomas cannot be predicted by the Egfr signaling activity.

PubMed

Pinel, Sophie; Mriouah, Jihane; Vandamme, Marc; Chateau, Alicia; Plénat, François; Guérin, Eric; Taillandier, Luc; Bernier-Chastagner, Valérie; Merlin, Jean-Louis; Chastagner, Pascal

2013-01-01

In high-grade gliomas, the identification of patients that could benefit from EGFR inhibitors remains a challenge, hindering the use of these agents. Using xenografts models, we evaluated the antitumor effect of the combined treatment "gefitinib + radiotherapy" and aimed to identify the profile of responsive tumors. Expression of phosphorylated proteins involved in the EGFR-dependent signaling pathways was analyzed in 10 glioma models. We focused on three models of anaplastic oligodendrogliomas (TCG2, TCG3 and TCG4) harboring high levels of phospho-EGFR, phospho-AKT and phospho-MEK1. They were treated with gefitinib (GEF 75 mg/kg/day x 5 days/week, for 2 weeks) and/or fractionated radiotherapy (RT: 5x2Gy/week for 2 weeks). Our results showed that GEF and/or RT induced significant tumor growth delays. However, only the TCG3 xenografts were highly responsive to the combination GEF+RT, with ∼50% of tumor cure. Phosphoproteins analysis five days after treatment onset demonstrated in TCG3 xenografts, but not in TCG2 model, that the EGFR-dependent pathways were inhibited after GEF treatment. Moreover, TCG3-bearing mice receiving GEF monotherapy exhibited a transient beneficial therapeutic response, rapidly followed by tumor regrowth, along with a major vascular remodeling. Taken together, our data evoked an "EGFR-addictive" behavior for TCG3 tumors. This study confirms that combination of gefitinib with fractionated irradiation could be a potent therapeutic strategy for anaplastic oligodendrogliomas harboring EGFR abnormalities but this treatment seems mainly beneficial for "EGFR-addictive" tumors. Unfortunately, neither the usual molecular markers (EGFR amplification, PTEN loss) nor the basal overexpression of phosphoproteins were useful to distinguish this responsive tumor. Evaluating the impact of TKIs on the EGFR-dependent pathways during the treatment might be more relevant, and requires further validation.

Epac activation sensitizes rat sensory neurons through activation of Ras.

PubMed

Shariati, Behzad; Thompson, Eric L; Nicol, Grant D; Vasko, Michael R

2016-01-01

Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2'-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization. Copyright © 2015 Elsevier Inc. All rights reserved.

Epac activation sensitizes rat sensory neurons via activation of Ras

PubMed Central

Shariati, Behzad; Thompson, Eric L.; Nicol, Grant D.; Vasko, Michael R.

2015-01-01

Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2′-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization. PMID:26596174

Thrombin Promotes Sustained Signaling and Inflammatory Gene Expression through the CDC25 and Ras-associating Domains of Phospholipase Cϵ*

PubMed Central

Dusaban, Stephanie S.; Kunkel, Maya T.; Smrcka, Alan V.; Brown, Joan Heller

2015-01-01

Phospholipase C-epsilon (PLCϵ) plays a critical role in G-protein-coupled receptor-mediated inflammation. In addition to its ability to generate the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, PLCϵ, unlike the other phospholipase C family members, is activated in a sustained manner. We hypothesized that the ability of PLCϵ to function as a guanine nucleotide exchange factor (GEF) for Rap1 supports sustained downstream signaling via feedback of Rap1 to the enzyme Ras-associating (RA2) domain. Using gene deletion and adenoviral rescue, we demonstrate that both the GEF (CDC25 homology domain) and RA2 domains of PLCϵ are required for long term protein kinase D (PKD) activation and subsequent induction of inflammatory genes. PLCϵ localization is largely intracellular and its compartmentalization could contribute to its sustained activation. Here we show that localization of PLCϵ to the Golgi is required for activation of PKD in this compartment as well as for subsequent induction of inflammatory genes. These data provide a molecular mechanism by which PLCϵ mediates sustained signaling and by which astrocytes mediate pathophysiological inflammatory responses. PMID:26350460

Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS

DOE PAGES

Huang, William Y. C.; Yan, Qingrong; Lin, Wan-Chen; ...

2016-07-01

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kineticsmore » and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. In conclusion, the generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.« less

Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS

PubMed Central

Huang, William Y. C.; Yan, Qingrong; Lin, Wan-Chen; Chung, Jean K.; Hansen, Scott D.; Christensen, Sune M.; Tu, Hsiung-Lin; Kuriyan, John; Groves, Jay T.

2016-01-01

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes. PMID:27370798

Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS.

PubMed

Huang, William Y C; Yan, Qingrong; Lin, Wan-Chen; Chung, Jean K; Hansen, Scott D; Christensen, Sune M; Tu, Hsiung-Lin; Kuriyan, John; Groves, Jay T

2016-07-19

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.

RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development

PubMed Central

Martin-Vilchez, Samuel; Whitmore, Leanna; Asmussen, Hannelore; Zareno, Jessica; Horwitz, Rick; Newell-Litwa, Karen

2017-01-01

Small RhoGTPases regulate changes in post-synaptic spine morphology and density that support learning and memory. They are also major targets of synaptic disorders, including Autism. Here we sought to determine whether upstream RhoGTPase regulators, including GEFs, GAPs, and GDIs, sculpt specific stages of synaptic development. The majority of examined molecules uniquely regulate either early spine precursor formation or later maturation. Specifically, an activator of actin polymerization, the Rac1 GEF β-PIX, drives spine precursor formation, whereas both FRABIN, a Cdc42 GEF, and OLIGOPHRENIN-1, a RhoA GAP, regulate spine precursor elongation. However, in later development, a novel Rac1 GAP, ARHGAP23, and RhoGDIs inactivate actomyosin dynamics to stabilize mature synapses. Our observations demonstrate that specific combinations of RhoGTPase regulatory proteins temporally balance RhoGTPase activity during post-synaptic spine development. PMID:28114311

Factor V activation and inactivation by venom proteases.

PubMed

Rosing, J; Govers-Riemslag, J W; Yukelson, L; Tans, G

2001-01-01

Blood coagulation factor V is a single-chain glycoprotein with M(r) = 330,000 which plays an important role in the procoagulant and anticoagulant pathways. Thrombin activates factor V into factor Va, a two-chain molecule which is composed of a heavy (M(r) = 105,000) and a light chain (M(r) = 71,000/74,000). Factor Va accelerates factor Xa-catalysed prothrombin activation more than 1,000-fold and under physiological conditions the cofactor activity of factor Va in prothrombin activation is down-regulated by activated protein C. Factor V can also be activated by a wide variety of snake venoms (e.g. from Vipera species, Naja naja oxiana, Bothrops atrox) and by proteases present in the bristles of a South American caterpillar (Lonomia achelous). Some venoms, notably of Vipera lebetina turanica and Lonomia achelous, contain proteases that are able to inactivate factor V or factor Va. Venom factor V activators are excellent tools in studying the structure-function relationship of factor V(a) and they are also used in diagnostic tests for quantification of plasma factor V levels and for the screening of defects in the protein C pathway. In this review, the structural and functional properties of animal venom factor V activators and inactivators is described. Copyright 2002 S. Karger AG, Basel

Index of /data/nccf/com/gens/prod

Science.gov Websites

-2018 17:38 - cmce.20180527/ 27-May-2018 05:39 - fens.20180526/ 26-May-2018 17:50 - fens.20180527/ 27 -May-2018 06:20 - gefs.20180520/ 20-May-2018 22:41 - gefs.20180521/ 21-May-2018 22:43 - gefs.20180522 / 22-May-2018 22:43 - gefs.20180523/ 23-May-2018 22:42 - gefs.20180524/ 24-May-2018 22:43 - gefs

BLOC-3 Mutated in Hermansky-Pudlak Syndrome Is a Rab32/38 Guanine Nucleotide Exchange Factor

PubMed Central

Gerondopoulos, Andreas; Langemeyer, Lars; Liang, Jin-Rui; Linford, Andrea; Barr, Francis A.

2012-01-01

Summary Hermansky-Pudlak syndrome (HPS) is a human disease characterized by partial loss of pigmentation and impaired blood clotting [1–3]. These symptoms are caused by defects in the biogenesis of melanosomes and platelet dense granules, often referred to as lysosome-related organelles [2]. Genes mutated in HPS encode subunits of the biogenesis of lysosome-related organelles complexes (BLOCs). BLOC-1 and BLOC-2, together with the AP-3 clathrin adaptor complex, act at early endosomes to sort components required for melanin formation and melanosome biogenesis away from the degradative lysosomal pathway toward early stage melanosomes [4–6]. However the molecular functions of the Hps1-Hps4 complex BLOC-3 remain mysterious [7–9]. Like other trafficking pathways, melanosome biogenesis and transport of enzymes involved in pigmentation involves specific Rab GTPases, in this instance Rab32 and Rab38 [10–12]. We now demonstrate that BLOC-3 is a Rab32 and Rab38 guanine nucleotide exchange factor (GEF). Silencing of the BLOC-3 subunits Hps1 and Hps4 results in the mislocalization of Rab32 and Rab38 and reduction in pigmentation. In addition, we show that BLOC-3 can promote specific membrane recruitment of Rab32/38. BLOC-3 therefore defines a novel Rab GEF family with a specific function in the biogenesis of lysosome-related organelles. PMID:23084991

Targeted Sos1 deletion reveals its critical role in early T-cell development

PubMed Central

Kortum, Robert L.; Sommers, Connie L.; Alexander, Clayton P.; Pinski, John M.; Li, Wenmei; Grinberg, Alex; Lee, Jan; Love, Paul E.; Samelson, Lawrence E.

2011-01-01

Activation of the small G protein Ras is required for thymocyte differentiation. In thymocytes, Ras is activated by the Ras guanine exchange factors (RasGEFs) Sos1, Sos2, and RasGRP1. We report the development of a floxed allele of sos1 to assess the role of Sos1 during thymocyte development. Sos1 was required for pre–T-cell receptor (pre-TCR)– but not TCR-stimulated developmental signals. Sos1 deletion led to a partial block at the DN-to-DP transition. Sos1-deficient thymocytes showed reduced pre-TCR–stimulated proliferation, differentiation, and ERK phosphorylation. In contrast, TCR-stimulated positive selection, and negative selection under strong stimulatory conditions, remained intact in Sos1-deficient mice. Comparison of RasGEF expression at different developmental stages showed that relative to Sos2 and RasGRP1, Sos1 is most abundant in DN thymocytes, but least abundant in DP thymocytes. These data reveal that Sos1 is uniquely positioned to affect signal transduction early in thymocyte development. PMID:21746917

Structural basis for the recognition of Asef by adenomatous polyposis coli

PubMed Central

Zhang, Zhenyi; Chen, Leyi; Gao, Lei; Lin, Kui; Zhu, Liang; Lu, Yang; Shi, Xiaoshan; Gao, Yuan; Zhou, Jing; Xu, Ping; Zhang, Jian; Wu, Geng

2012-01-01

Adenomatous polyposis coli (APC) regulates cell-cell adhesion and cell migration through activating the APC-stimulated guanine nucleotide-exchange factor (GEF; Asef), which is usually autoinhibited through the binding between its Src homology 3 (SH3) and Dbl homology (DH) domains. The APC-activated Asef stimulates the small GTPase Cdc42, which leads to decreased cell-cell adherence and enhanced cell migration. In colorectal cancers, truncated APC constitutively activates Asef and promotes cancer cell migration and angiogenesis. Here, we report crystal structures of the human APC/Asef complex. We find that the armadillo repeat domain of APC uses a highly conserved surface groove to recognize the APC-binding region (ABR) of Asef, conformation of which changes dramatically upon binding to APC. Key residues on APC and Asef for the complex formation were mutated and their importance was demonstrated by binding and activity assays. Structural superimposition of the APC/Asef complex with autoinhibited Asef suggests that the binding between APC and Asef might create a steric clash between Asef-DH domain and APC, which possibly leads to a conformational change in Asef that stimulates its GEF activity. Our structures thus elucidate the molecular mechanism of Asef recognition by APC, as well as provide a potential target for pharmaceutical intervention against cancers. PMID:21788986

The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2*

PubMed Central

Hosokawa, Hiroyuki; Dip, Phat Vinh; Merkulova, Maria; Bakulina, Anastasia; Zhuang, Zhenjie; Khatri, Ashok; Jian, Xiaoying; Keating, Shawn M.; Bueler, Stephanie A.; Rubinstein, John L.; Randazzo, Paul A.; Ausiello, Dennis A.; Grüber, Gerhard; Marshansky, Vladimir

2013-01-01

Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H+-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1–17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1–17) and its amino acids Phe5, Met10, and Gln14 involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1–17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1–a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor. PMID:23288846

Global environment facility: Independent evaluation of the pilot phase; Fondo para el medio ambiente mundial: evaluacion independiente de la etapa experimental

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1994-06-01

This study responds to a request by participants in the Global Environment Facility (GEF) for an independent evaluation of the pilot phase. It profiles the GEF, discusses its policy framework, and reviews project development procedures and the strategies and projects in each of the GEF`s four focal areas. The study concludes that fundamental changes must occur and recommends specific reforms, such as articulating more clearly the GEF`s mandate, objectives, and strategies; addressing deficiencies in meeting its global focus; improving capacities and procedures within implementing agencies for managing the portfolio; and increasing non-government organization (NGO), country and community-level participation.

Global environment facility: Independent evaluation of the pilot phase; Fonds pour l`environnement mondial: evaluation independante de la phase pilote

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1994-12-31

This study responds to a request by participants in the Global Environment Facility (GEF) for an independent evaluation of the pilot phase. It profiles the GEF, discusses its policy framework, and reviews project development procedures and the strategies and projects in each of the GEF`s four focal areas. The study concludes that fundamental changes must occur and recommends specific reforms, such as articulating more clearly the GEF`s mandate, objectives, and strategies; addressing deficiencies in meeting its global focus; improving capacities and procedures within implementing agencies for managing the portfolio; and increasing non-government organization (NGO), country and community-level participation.

BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein regulates neurite development via PI3K-AKT and ERK signaling pathways.

PubMed

Zhou, C; Li, C; Li, D; Wang, Y; Shao, W; You, Y; Peng, J; Zhang, X; Lu, L; Shen, X

2013-12-19

The elongation of neuron is highly dependent on membrane trafficking. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein 1 (BIG1) functions in the membrane trafficking between the Golgi apparatus and the plasma membrane. BFA, an uncompetitive inhibitor of BIG1 can inhibit neurite outgrowth and polarity development. In this study, we aimed to define the possible role of BIG1 in neurite development and to further investigate the potential mechanism. By immunostaining, we found that BIG1 was extensively colocalized with synaptophysin, a marker for synaptic vesicles in soma and partly in neurites. The amount of both protein and mRNA of BIG1 were up-regulated during rat brain development. BIG1 depletion significantly decreased the neurite length and inhibited the phosphorylation of phosphatidylinositide 3-kinase (PI3K) and protein kinase B (AKT). Inhibition of BIG1 guanine nucleotide-exchange factor (GEF) activity by BFA or overexpression of the dominant-negative BIG1 reduced PI3K and AKT phosphorylation, indicating regulatory effects of BIG1 on PI3K-AKT signaling pathway is dependent on its GEF activity. BIG1 siRNA or BFA treatment also significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of wild-type BIG1 significantly increased ERK phosphorylation, but the dominant-negative BIG1 had no effect on ERK phosphorylation, indicating the involvement of BIG1 in ERK signaling regulation may not be dependent on its GEF activity. Our result identified a novel function of BIG1 in neurite development. The newly recognized function integrates the function of BIG1 in membrane trafficking with the activation of PI3K-AKT and ERK signaling pathways which are critical in neurite development. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Role of hepsin in factor VII activation in zebrafish.

PubMed

Khandekar, Gauri; Jagadeeswaran, Pudur

2014-01-01

Factor VII, the initiator of the extrinsic coagulation cascade, circulates in human plasma mainly in its zymogen form, factor VII and in small amounts in its activated form, factor VIIa. However, the mechanism of initial generation of factor VIIa is not known despite intensive research using currently available model systems. Earlier findings suggested serine proteases factor VII activating protease and hepsin play a role in activating factor VII, however, it has remained controversial. In this paper we estimated the levels of factor VIIa and factor VII for the first time in zebrafish adult population and also reevaluated the role of the above two serine proteases in activating factor VII in vivo using zebrafish as a model system. Knockdown of factor VII activating protease and hepsin was performed followed by assaying for their effect on factor VIIa concentration and extrinsic coagulation as measured by the kinetic prothrombin time. Factor VII activating protease knockdown showed no change in kinetic prothrombin time and no effect on factor VIIa levels while hepsin knockdown increased the kinetic prothrombin time and significantly reduced the factor VIIa plasma levels. Our results thus indicate that hepsin plays a physiologically important role in factor VII activation and hemostasis in zebrafish. © 2013.

cAMP signalling in the vasculature: the role of Epac (exchange protein directly activated by cAMP).

PubMed

Roberts, Owain Llŷr; Dart, Caroline

2014-02-01

The second messenger cAMP plays a central role in mediating vascular smooth muscle relaxation in response to vasoactive transmitters and in strengthening endothelial cell-cell junctions that regulate the movement of solutes, cells and macromolecules between the blood and the surrounding tissue. The vasculature expresses three cAMP effector proteins: PKA (protein kinase A), CNG (cyclic-nucleotide-gated) ion channels, and the most recently discovered Epacs (exchange proteins directly activated by cAMP). Epacs are a family of GEFs (guanine-nucleotide-exchange factors) for the small Ras-related GTPases Rap1 and Rap2, and are being increasingly implicated as important mediators of cAMP signalling, both in their own right and in parallel with the prototypical cAMP target PKA. In the present paper, we review what is currently known about the role of Epac within blood vessels, particularly with regard to the regulation of vascular tone, endothelial barrier function and inflammation.

Interplay between Solo and keratin filaments is crucial for mechanical force-induced stress fiber reinforcement.

PubMed

Fujiwara, Sachiko; Ohashi, Kazumasa; Mashiko, Toshiya; Kondo, Hiroshi; Mizuno, Kensaku

2016-03-15

Mechanical force-induced cytoskeletal reorganization is essential for cell and tissue remodeling and homeostasis; however, the underlying cellular mechanisms remain elusive. Solo (ARHGEF40) is a RhoA-targeting guanine nucleotide exchange factor (GEF) involved in cyclical stretch-induced human endothelial cell reorientation and convergent extension cell movement in zebrafish gastrula. In this study, we show that Solo binds to keratin-8/keratin-18 (K8/K18) intermediate filaments through multiple sites. Solo overexpression promotes the formation of thick actin stress fibers and keratin bundles, whereas knockdown of Solo, expression of a GEF-inactive mutant of Solo, or inhibition of ROCK suppresses stress fiber formation and leads to disorganized keratin networks, indicating that the Solo-RhoA-ROCK pathway serves to precisely organize keratin networks, as well as to promote stress fibers. Of importance, knockdown of Solo or K18 or overexpression of GEF-inactive or deletion mutants of Solo suppresses tensile force-induced stress fiber reinforcement. Furthermore, knockdown of Solo or K18 suppresses tensile force-induced RhoA activation. These results strongly suggest that the interplay between Solo and K8/K18 filaments plays a crucial role in tensile force-induced RhoA activation and consequent actin cytoskeletal reinforcement. © 2016 Fujiwara et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Evaluations of Extended-Range tropical Cyclone Forecasts in the Western North Pacific by using the Ensemble Reforecasts: Preliminary Results

NASA Astrophysics Data System (ADS)

Tsai, Hsiao-Chung; Chen, Pang-Cheng; Elsberry, Russell L.

2017-04-01

The objective of this study is to evaluate the predictability of the extended-range forecasts of tropical cyclone (TC) in the western North Pacific using reforecasts from National Centers for Environmental Prediction (NCEP) Global Ensemble Forecast System (GEFS) during 1996-2015, and from the Climate Forecast System (CFS) during 1999-2010. Tsai and Elsberry have demonstrated that an opportunity exists to support hydrological operations by using the extended-range TC formation and track forecasts in the western North Pacific from the ECMWF 32-day ensemble. To demonstrate this potential for the decision-making processes regarding water resource management and hydrological operation in Taiwan reservoir watershed areas, special attention is given to the skill of the NCEP GEFS and CFS models in predicting the TCs affecting the Taiwan area. The first objective of this study is to analyze the skill of NCEP GEFS and CFS TC forecasts and quantify the forecast uncertainties via verifications of categorical binary forecasts and probabilistic forecasts. The second objective is to investigate the relationships among the large-scale environmental factors [e.g., El Niño Southern Oscillation (ENSO), Madden-Julian Oscillation (MJO), etc.] and the model forecast errors by using the reforecasts. Preliminary results are indicating that the skill of the TC activity forecasts based on the raw forecasts can be further improved if the model biases are minimized by utilizing these reforecasts.

Gain-of-function SOS1 mutations cause a distinctive form of noonansyndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tartaglia, Marco; Pennacchio, Len A.; Zhao, Chen

2006-09-01

Noonan syndrome (NS) is a developmental disordercharacterized by short stature, facial dysmorphia, congenital heartdefects and skeletal anomalies1. Increased RAS-mitogenactivated proteinkinase (MAPK) signaling due to PTPN11 and KRAS mutations cause 50 percentof NS2-6. Here, we report that 22 of 129 NS patients without PTPN11 orKRAS mutation (17 percent) have missense mutations in SOS1, which encodesa RAS-specific guanine nucleotide exchange factor (GEF). SOS1 mutationscluster at residues implicated in the maintenance of SOS1 in itsautoinhibited form and ectopic expression of two NS-associated mutantsinduced enhanced RAS activation. The phenotype associated with SOS1defects is distinctive, although within NS spectrum, with a highprevalence of ectodermal abnormalitiesmore » but generally normal developmentand linear growth. Our findings implicate for the first timegain-of-function mutations in a RAS GEF in inherited disease and define anew mechanism by which upregulation of the RAS pathway can profoundlychange human development.« less

Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate

PubMed Central

Lee, Young Kwang; Low-Nam, Shalini T.; Chung, Jean K.; Hansen, Scott D.; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T.

2017-01-01

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification. PMID:28452363

Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

PubMed

Lee, Young Kwang; Low-Nam, Shalini T; Chung, Jean K; Hansen, Scott D; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T

2017-04-28

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

Gα12 facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3-kinase-mediated activation in a RhoA-dependent manner.

PubMed

Yoo, Edwin J; Cao, Gaoyuan; Koziol-White, Cynthia J; Ojiaku, Christie A; Sunder, Krishna; Jude, Joseph A; Michael, James V; Lam, Hong; Pushkarsky, Ivan; Damoiseaux, Robert; Di Carlo, Dino; Ahn, Kwangmi; An, Steven S; Penn, Raymond B; Panettieri, Reynold A

2017-12-01

PI3K-dependent activation of Rho kinase (ROCK) is necessary for agonist-induced human airway smooth muscle cell (HASMC) contraction, and inhibition of PI3K promotes bronchodilation of human small airways. The mechanisms driving agonist-mediated PI3K/ROCK axis activation, however, remain unclear. Given that G 12 family proteins activate ROCK pathways in other cell types, their role in M 3 muscarinic acetylcholine receptor-stimulated PI3K/ROCK activation and contraction was examined. Gα 12 coupling was evaluated using co-immunoprecipitation and serum response element (SRE)-luciferase reporter assays. siRNA and pharmacological approaches, as well as overexpression of a regulator of G-protein signaling (RGS) proteins were applied in HASMCs. Phosphorylation levels of Akt, myosin phosphatase targeting subunit-1 (MYPT1), and myosin light chain-20 (MLC) were measured. Contraction and shortening were evaluated using magnetic twisting cytometry (MTC) and micro-pattern deformation, respectively. Human precision-cut lung slices (hPCLS) were utilized to evaluate bronchoconstriction. Knockdown of M 3 receptors or Gα 12 attenuated activation of Akt, MYPT1, and MLC phosphorylation. Gα 12 coimmunoprecipitated with M 3 receptors, and p115RhoGEF-RGS overexpression inhibited carbachol-mediated induction of SRE-luciferase reporter. p115RhoGEF-RGS overexpression inhibited carbachol-induced activation of Akt, HASMC contraction, and shortening. Moreover, inhibition of RhoA blunted activation of PI3K. Lastly, RhoA inhibitors induced dilation of hPCLS. Gα 12 plays a crucial role in HASMC contraction via RhoA-dependent activation of the PI3K/ROCK axis. Inhibition of RhoA activation induces bronchodilation in hPCLS, and targeting Gα 12 signaling may elucidate novel therapeutic targets in asthma. These findings provide alternative approaches to the clinical management of airway obstruction in asthma. © 2017 The British Pharmacological Society.

Two distinct mTORC2-dependent pathways converge on Rac1 to drive breast cancer metastasis.

PubMed

Morrison Joly, Meghan; Williams, Michelle M; Hicks, Donna J; Jones, Bayley; Sanchez, Violeta; Young, Christian D; Sarbassov, Dos D; Muller, William J; Brantley-Sieders, Dana; Cook, Rebecca S

2017-06-30

The importance of the mTOR complex 2 (mTORC2) signaling complex in tumor progression is becoming increasingly recognized. HER2-amplified breast cancers use Rictor/mTORC2 signaling to drive tumor formation, tumor cell survival and resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapy. Cell motility, a key step in the metastatic process, can be activated by mTORC2 in luminal and triple negative breast cancer cell lines, but its role in promoting metastases from HER2-amplified breast cancers is not yet clear. Because Rictor is an obligate cofactor of mTORC2, we genetically engineered Rictor ablation or overexpression in mouse and human HER2-amplified breast cancer models for modulation of mTORC2 activity. Signaling through mTORC2-dependent pathways was also manipulated using pharmacological inhibitors of mTOR, Akt, and Rac. Signaling was assessed by western analysis and biochemical pull-down assays specific for Rac-GTP and for active Rac guanine nucleotide exchange factors (GEFs). Metastases were assessed from spontaneous tumors and from intravenously delivered tumor cells. Motility and invasion of cells was assessed using Matrigel-coated transwell assays. We found that Rictor ablation potently impaired, while Rictor overexpression increased, metastasis in spontaneous and intravenously seeded models of HER2-overexpressing breast cancers. Additionally, migration and invasion of HER2-amplified human breast cancer cells was diminished in the absence of Rictor, or upon pharmacological mTOR kinase inhibition. Active Rac1 was required for Rictor-dependent invasion and motility, which rescued invasion/motility in Rictor depleted cells. Rictor/mTORC2-dependent dampening of the endogenous Rac1 inhibitor RhoGDI2, a factor that correlated directly with increased overall survival in HER2-amplified breast cancer patients, promoted Rac1 activity and tumor cell invasion/migration. The mTORC2 substrate Akt did not affect RhoGDI2 dampening, but partially

Vav family exchange factors: an integrated regulatory and functional view

PubMed Central

Bustelo, Xosé R

2014-01-01

The Vav family is a group of tyrosine phosphorylation-regulated signal transduction molecules hierarchically located downstream of protein tyrosine kinases. The main function of these proteins is to work as guanosine nucleotide exchange factors (GEFs) for members of the Rho GTPase family. In addition, they can exhibit a variety of catalysis-independent roles in specific signaling contexts. Vav proteins play essential signaling roles for both the development and/or effector functions of a large variety of cell lineages, including those belonging to the immune, nervous, and cardiovascular systems. They also contribute to pathological states such as cancer, immune-related dysfunctions, and atherosclerosis. Here, I will provide an integrated view about the evolution, regulation, and effector properties of these signaling molecules. In addition, I will discuss the pros and cons for their potential consideration as therapeutic targets. PMID:25483299

Evaluating the influence of geo-environmental factors on gully erosion in a semi-arid region of Iran: An integrated framework.

PubMed

Rahmati, Omid; Tahmasebipour, Naser; Haghizadeh, Ali; Pourghasemi, Hamid Reza; Feizizadeh, Bakhtiar

2017-02-01

Despite the importance of soil erosion in sustainable development goals in arid and semi-arid areas, the study of the geo-environmental conditions and factors influencing gully erosion occurrence is rarely undertaken. As effort to this challenge, the main objective of this study is to apply an integrated approach of Geographic Object-Based Image Analysis (GEOBIA) together with high-spatial resolution imagery (SPOT-5) for detecting gully erosion features at the Kashkan-Poldokhtar watershed, Iran. We also aimed to apply a Conditional Probability (CP) model for establishing the spatial relationship between gullies and the Geo-Environmental Factors (GEFs). The gully erosion inventory map prepared using GEOBIA and field surveying was randomly partitioned into two subsets: (1) part 1 that contains 70% was used in the training phase of the CP model; (2) part 2 is a validation dataset (30%) for validation of the model and to confirm its accuracy. Prediction performances of the GEOBIA and CP model were checked by overall accuracy and Receiver Operating Characteristics (ROC) curve methods, respectively. In addition, the influence of all GEFs on gully erosion was evaluated by performing a sensitivity analysis model. The validation findings illustrated that overall accuracy for GEOBIA approach and the area under the ROC curve for the CP model were 92.4% and 89.9%, respectively. Also, based on sensitivity analysis, soil texture, drainage density, and lithology represent significantly effects on the gully erosion occurrence. This study has shown that the integrated framework can be successfully used for modeling gully erosion occurrence in a data-poor environment. Copyright © 2016 Elsevier B.V. All rights reserved.

The Secondary Structure of Human Hageman Factor (Factor XII) and its Alteration by Activating Agents

PubMed Central

McMillin, Carl R.; Saito, Hidehiko; Ratnoff, Oscar D.; Walton, Alan G.

1974-01-01

Hageman factor (factor XII) is activated by exposure to surfaces such as glass or by solutions of certain compounds, notably ellagic acid. Changes in the structure of Hageman factor accompanying activation have been examined in this study by circular dichroism spectroscopy. The spectrum of unactivated Hageman factor in aqueous solutions suggests that its conformation is mainly aperiodic. Various perturbants altered the conformation of Hageman factor in differing ways, demonstrating the sensitivity of Hageman factor to its environment. After activation of Hageman factor with solutions of ellagic acid, a negative trough appeared in the region of the circular dichroism spectrum commonly assigned to tyrosine residues, along with other minor changes in the peptide spectral region. Some of these changes are similar to changes that occurred upon partial neutralization of the basic residues at alkali pH. Activation of Hageman factor by adsorption to quartz surfaces (in an aqueous environment) also produced changes similar to those in the ellagic acid-activated Hageman factor, including the negative ellipticity in the tyrosine region. These observations suggest that the activation process may be related to a change in status of some of the basic amino acid residues, coupled with a specific change in the environment of some tyrosine residues. The importance of these changes during the activation process remains to be determined. The sensitivity of Hageman factor to its environment is consistent with the view that the initiation of clotting by exposure of plasma to appropriate agents is brought about by alterations in the conformation of Hageman factor that occur in the apparent absence of Fletcher factor or other recognized clotting factors. Images PMID:4373492

Arf6 guanine-nucleotide exchange factor, cytohesin-2, interacts with actinin-1 to regulate neurite extension.

PubMed

Torii, Tomohiro; Miyamoto, Yuki; Nakamura, Kazuaki; Maeda, Masahiro; Yamauchi, Junji; Tanoue, Akito

2012-09-01

Proper regulation of morphological changes in neuronal cells is essential for their differentiation. Complex signaling mechanisms mediate a variety of morphological changes such as formation of neurites. It is well established that a number of small GTPases control neurite behavior before the connection with the target tissue. However, their regulatory mechanisms remain to be fully understood. Here, we show that the Arf6 guanine-nucleotide exchange factor (GEF), cytohesin-2 (CYTH2), interacts with the cytoskeletal protein actinin-1 (ACTN1) and regulates neurite extension in N1E-115 cells used as the model. Knockdown of ACTN1, as well as that of CYTH2, in cells inhibits cellular Arf6 activity and neurite extension. The C-terminal polybasic region of CYTH2 participates in interacting directly with the EFh2 domain of ACTN1. Expression of CYTH2 mutant deficient of the EFh2 domain in cells also inhibits Arf6 activation and neurite extension. Furthermore, FRET analysis detects that the respective interactive region peptides, tagged with cell-permeable short peptides, greatly decrease Arf6 activation at growth cones in a time-dependent manner. Collectively, the signaling through CYTH2 and ACTN1 properly regulates neurite extension in N1E-115 cells, demonstrating the unexpected interaction of CYTH2 and ACTN1 in the regulation of cellular Arf6 activity involved in neurite extension. Copyright © 2012 Elsevier Inc. All rights reserved.

Pak and Rac GTPases promote oncogenic KIT–induced neoplasms

PubMed Central

Martin, Holly; Mali, Raghuveer Singh; Ma, Peilin; Chatterjee, Anindya; Ramdas, Baskar; Sims, Emily; Munugalavadla, Veerendra; Ghosh, Joydeep; Mattingly, Ray R.; Visconte, Valeria; Tiu, Ramon V.; Vlaar, Cornelis P.; Dharmawardhane, Suranganie; Kapur, Reuben

2013-01-01

An acquired somatic mutation at codon 816 in the KIT receptor tyrosine kinase is associated with poor prognosis in patients with systemic mastocytosis and acute myeloid leukemia (AML). Treatment of leukemic cells bearing this mutation with an allosteric inhibitor of p21–activated kinase (Pak) or its genetic inactivation results in growth repression due to enhanced apoptosis. Inhibition of the upstream effector Rac abrogates the oncogene-induced growth and activity of Pak. Although both Rac1 and Rac2 are constitutively activated via the guanine nucleotide exchange factor (GEF) Vav1, loss of Rac1 or Rac2 alone moderately corrected the growth of KIT-bearing leukemic cells, whereas the combined loss resulted in 75% growth repression. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of myeloproliferative neoplasms (MPNs) and corrected the associated pathology in mice. To assess the role of Rac GEFs in oncogene-induced transformation, we used an inhibitor of Rac, EHop-016, which specifically targets Vav1 and found that EHop-016 was a potent inhibitor of human and murine leukemic cell growth. These studies identify Pak and Rac GTPases, including Vav1, as potential therapeutic targets in MPN and AML involving an oncogenic form of KIT. PMID:24091327

Activation of Rab GTPase Sec4 by its GEF Sec2 is required for prospore membrane formation during sporulation in yeast Saccharomyces cerevisiae.

PubMed

Suda, Yasuyuki; Tachikawa, Hiroyuki; Inoue, Ichiro; Kurita, Tomokazu; Saito, Chieko; Kurokawa, Kazuo; Nakano, Akihiko; Irie, Kenji

2018-02-01

Sec2 activates Sec4 Rab GTPase as a guanine nucleotide exchange factor for the recruitment of downstream effectors to facilitate tethering and fusion of post-Golgi vesicles at the plasma membrane. During the meiosis and sporulation of budding yeast, post-Golgi vesicles are transported to and fused at the spindle pole body (SPB) to form a de novo membrane, called the prospore membrane. Previous studies have revealed the role of the SPB outer surface called the meiotic outer plaque (MOP) in docking and fusion of post-Golgi vesicles. However, the upstream molecular machinery for post-Golgi vesicular fusion that facilitates prospore membrane formation remains enigmatic. Here, we demonstrate that the GTP exchange factor for Sec4, Sec2, participates in the formation of the prospore membrane. A conditional mutant in which the SEC2 expression is shut off during sporulation showed sporulation defects. Inactivation of Sec2 caused Sec4 targeting defects along the prospore membranes, thereby causing insufficient targeting of downstream effectors and cargo proteins to the prospore membrane. These results suggest that the activation of Sec4 by Sec2 is required for the efficient supply of post-Golgi vesicles to the prospore membrane and thus for prospore membrane formation/extension and subsequent deposition of spore wall materials. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Losac, the First Hemolin that Exhibits Procogulant Activity through Selective Factor X Proteolytic Activation*

PubMed Central

Alvarez-Flores, Miryam Paola; Furlin, Daniel; Ramos, Oscar H. P.; Balan, Andrea; Konno, Katsuhiro; Chudzinski-Tavassi, Ana Marisa

2011-01-01

Envenoming by the contact of human skin with Lonomia obliqua caterpillars promotes a hemorrhagic syndrome characterized by a consumptive coagulopathy. Losac (Lonomia obliqua Stuart factor activator) is a component of the bristle of L. obliqua that is probably partially responsible for the observed syndrome because it activates factor X and is recognized by an effective antilonomic serum. Here we unveil the proteolytic activity of Losac and demonstrate the feasibility of its recombinant production. On the other hand, Losac has no homology to known proteases, but it can be inhibited by PMSF, a serine protease inhibitor. Instead, it shows closer homology to members of the hemolin family of proteins, a group of cell adhesion molecules. The recombinant protein (rLosac) shortened the coagulation time of normal and deficient plasmas, whereas it was ineffective in factor X-deficient plasma unless reconstituted with this protein. rLosac was able to activate factor X in a dose- and time-dependent manner but not γ-carboxyglutamic acid domainless factor X. Moreover, phospholipids and calcium ions increased rLosac activity. Also, rLosac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation. Linear double reciprocal plots indicate that rLosac follows a Michaelis-Menten kinetics. Cleavage of factor X by rLosac resulted in fragments that are compatible with those generated by RVV-X (a well known factor X activator). Together, our results validate Losac as the first protein from the hemolin family exhibiting procoagulant activity through selective proteolysis on coagulation factor X. PMID:21177860

Mechanism of the Exchange Reaction in HRAS from Multiscale Modeling

PubMed Central

Kapoor, Abhijeet; Travesset, Alex

2014-01-01

HRAS regulates cell growth promoting signaling processes by cycling between active (GTP-bound) and inactive (GDP-bound) states. Understanding the transition mechanism is central for the design of small molecules to inhibit the formation of RAS-driven tumors. Using a multiscale approach involving coarse-grained (CG) simulations, all-atom classical molecular dynamics (CMD; total of 3.02 µs), and steered molecular dynamics (SMD) in combination with Principal Component Analysis (PCA), we identified the structural features that determine the nucleotide (GDP) exchange reaction. We show that weakening the coupling between the SwitchI (residues 25–40) and SwitchII (residues 59–75) accelerates the opening of SwitchI; however, an open conformation of SwitchI is unstable in the absence of guanine nucleotide exchange factors (GEFs) and rises up towards the bound nucleotide to close the nucleotide pocket. Both I21 and Y32, play a crucial role in SwitchI transition. We show that an open SwitchI conformation is not necessary for GDP destabilization but is required for GDP/Mg escape from the HRAS. Further, we present the first simulation study showing displacement of GDP/Mg away from the nucleotide pocket. Both SwitchI and SwitchII, delays the escape of displaced GDP/Mg in the absence of GEF. Based on these results, a model for the mechanism of GEF in accelerating the exchange process is hypothesized. PMID:25272152

Solo/Trio8, a membrane-associated short isoform of Trio, modulates endosome dynamics and neurite elongation.

PubMed

Sun, Ying-Jie; Nishikawa, Kaori; Yuda, Hideki; Wang, Yu-Lai; Osaka, Hitoshi; Fukazawa, Nobuna; Naito, Akira; Kudo, Yoshihisa; Wada, Keiji; Aoki, Shunsuke

2006-09-01

With DNA microarrays, we identified a gene, termed Solo, that is downregulated in the cerebellum of Purkinje cell degeneration mutant mice. Solo is a mouse homologue of rat Trio8-one of multiple Trio isoforms recently identified in rat brain. Solo/Trio8 contains N-terminal sec14-like and spectrin-like repeat domains followed by a single guanine nucleotide exchange factor 1 (GEF1) domain, but it lacks the C-terminal GEF2, immunoglobulin-like, and kinase domains that are typical of Trio. Solo/Trio8 is predominantly expressed in Purkinje neurons of the mouse brain, and expression begins following birth and increases during Purkinje neuron maturation. We identified a novel C-terminal membrane-anchoring domain in Solo/Trio8 that is required for enhanced green fluorescent protein-Solo/Trio8 localization to early endosomes (positive for both early-endosome antigen 1 [EEA1] and Rab5) in COS-7 cells and primary cultured neurons. Solo/Trio8 overexpression in COS-7 cells augmented the EEA1-positive early-endosome pool, and this effect was abolished via mutation and inactivation of the GEF domain or deletion of the C-terminal membrane-anchoring domain. Moreover, primary cultured neurons transfected with Solo/Trio8 showed increased neurite elongation that was dependent on these domains. These results suggest that Solo/Trio8 acts as an early-endosome-specific upstream activator of Rho family GTPases for neurite elongation of developing Purkinje neurons.

Coupling of the Matched Gravity and Electromagnetic Fields of the Sun with Jupiter and its Moons Together in Nearest Portion of Jupiter's Orbit to the Sun as the Main Cause of the Peak of Approximately 11 Yearly Solar Cycles and Hazards from Solar Storms

NASA Astrophysics Data System (ADS)

Gholibeigian, Kazem; Gholibeigian, Hassan

2016-04-01

On March 13, 1989 the entire province of Quebec Blackout by solar storm during solar cycle 22. The solar storm of 1859, also known as the Carrington event, was a powerful geomagnetic solar storm during solar cycle 10. The solar storm of 2012 during solar cycle 24 was of similar magnitude, but it passed Earth's orbit without striking the plane. All of these solar storms occurred in the peak of 11 yearly solar cycles. In this way, the White House in its project which is focusing on hazards from solar system, in a new strategy and action plan to increase protection from damaging solar emissions, should focus on coupling of the matched Gravity and Electromagnetic Fields)GEFs) of the Sun with Jupiter and its moons together. On the other hand, in solar system, the Jupiter's gravity has largest effect to the Sun's core and its dislocation, because the gravity force between the Jupiter and the Sun is 11.834 times, In addition overlapping of the solar cycles with the Jupiter's orbit period is 11.856 years. These observable factors lead us to the effect of the Jupiter and Sun gravity fields coupling as the main cause of the approximately 11 years duration for solar cycles. Its peak in each cycle is when the Jupiter is in nearest portion to the Sun in its orbit. In this way, the other planets in their coupling with Sun help to the variations and strengthening solar cycles. [Gholibeigian, 7/24/2015http://adsabs.harvard.edu/abs/2014EGU]. In other words, the both matched GEFs are generating by the large scale forced convection system inside the stars and planets [Gholibeigian et. al, AGU Fall Meeting 2015]. These two fields are couple and strengthening each other. The Jupiter with its 67 moons generate the largest coupled and matched GEFs in its core and consequently strongest effect on the Sun's core. Generation and coupling of the Jupiter's GEFs with its moons like Europa, Io and Ganymede make this planet of thousands of times brighter and many times bigger than Earth as the

PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

PubMed

Dunn, Henry A; Ferguson, Stephen S G

2015-10-01

G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

Epidermal growth factor- and hepatocyte growth factor-receptor activity in serum-free cultures of human hepatocytes.

PubMed

Runge, D M; Runge, D; Dorko, K; Pisarov, L A; Leckel, K; Kostrubsky, V E; Thomas, D; Strom, S C; Michalopoulos, G K

1999-02-01

Serum-free primary cultures of hepatocytes are a useful tool to study factors triggering hepatocyte proliferation and regeneration. We have developed a chemically defined serum-free system that allows human hepatocyte proliferation in the presence of epidermal growth factor and hepatocyte growth factor. DNA synthesis and accumulation were determined by [3H]thymidine incorporation and fluorometry, respectively. Western blot analyses and co-immunoprecipitations were used to investigate the association of proteins involved in epidermal growth factor and hepatocyte growth factor activation and signaling: epidermal growth factor receptor, hepatocyte growth factor receptor (MET), urokinase-type plasminogen activator and its receptor, and a member of the signal transducer and activator of transcription family, STAT-3. Primary human hepatocytes proliferated under serum-free conditions in a chemically defined medium for up to 12 days. Epidermal growth factor-receptor and MET were present and functional, decreasing over time. MET, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor co-precipitated to varying degrees during the culture period. STAT-3 co-precipitated with epidermal growth factor-receptor and MET to varying degrees. Proliferation of human hepatocytes can improve by modification of a chemically defined medium originally used for rat hepatocyte cultures. In these long-term cultures of human hepatocytes, hepatocyte growth factor and epidermal growth factor can stimulate growth and differentiation by interacting with their receptors and initiating downstream signaling. This involves complex formation of the receptors with other plasma membrane components for MET (urokinase-type plasminogen activator in context of its receptor) and activation of STAT-3 for both receptors.

Asymmetric Mbc, active Rac1 and F-actin foci in the fusion-competent myoblasts during myoblast fusion in Drosophila

PubMed Central

Haralalka, Shruti; Shelton, Claude; Cartwright, Heather N.; Katzfey, Erin; Janzen, Evan; Abmayr, Susan M.

2011-01-01

Myoblast fusion is an intricate process that is initiated by cell recognition and adhesion, and culminates in cell membrane breakdown and formation of multinucleate syncytia. In the Drosophila embryo, this process occurs asymmetrically between founder cells that pattern the musculature and fusion-competent myoblasts (FCMs) that account for the bulk of the myoblasts. The present studies clarify and amplify current models of myoblast fusion in several important ways. We demonstrate that the non-conventional guanine nucleotide exchange factor (GEF) Mbc plays a fundamental role in the FCMs, where it functions to activate Rac1, but is not required in the founder cells for fusion. Mbc, active Rac1 and F-actin foci are highly enriched in the FCMs, where they localize to the Sns:Kirre junction. Furthermore, Mbc is crucial for the integrity of the F-actin foci and the FCM cytoskeleton, presumably via its activation of Rac1 in these cells. Finally, the local asymmetric distribution of these proteins at adhesion sites is reminiscent of invasive podosomes and, consistent with this model, they are enriched at sites of membrane deformation, where the FCM protrudes into the founder cell/myotube. These data are consistent with models promoting actin polymerization as the driving force for myoblast fusion. PMID:21389053

Recombinant activated factor VII in cardiac surgery: single-center experience.

PubMed

Singh, Sarvesh Pal; Chauhan, Sandeep; Choudhury, Minati; Malik, Vishwas; Choudhary, Shiv Kumar

2014-02-01

The widespread off-label use of recombinant activated factor VII for the control of refractory postoperative hemorrhage continues despite a warning from the Food and Drug Administration. Although effective in reducing the need for transfusion of blood and blood products, safety concerns still prevail. To compare the dosing and efficacy of recombinant activated factor VII between pediatric and adult patients, and in the operating room and intensive care unit. The records of 69 patients (33 children and 36 adults) who underwent cardiovascular surgery and received recombinant activated factor VII were reviewed retrospectively. The dose of recombinant activated factor VII, mediastinal drainage, use of blood and blood products, incidence of thrombosis, and 28-day mortality were studied. the efficacy of recombinant activated factor VII was comparable in adults and children, despite the lower dose in adults. Prophylactic use of recombinant activated factor VII decreased the incidence of mediastinal exploration and the duration of intensive care unit stay. A 4.3% incidence of thrombotic complications was observed in this study. The efficacious dose of recombinant activated factor VII is much less in adults compared to children. Prophylactic use of recombinant activated factor VII decreases the dose required, the incidence of mediastinal exploration, and intensive care unit stay, with no survival benefit.

Regulation of neurite morphogenesis by interaction between R7 regulator of G protein signaling complexes and G protein subunit Gα13.

PubMed

Scherer, Stephanie L; Cain, Matthew D; Kanai, Stanley M; Kaltenbronn, Kevin M; Blumer, Kendall J

2017-06-16

The R7 regulator of G protein signaling family (R7-RGS) critically regulates nervous system development and function. Mice lacking all R7-RGS subtypes exhibit diverse neurological phenotypes, and humans bearing mutations in the retinal R7-RGS isoform RGS9-1 have vision deficits. Although each R7-RGS subtype forms heterotrimeric complexes with Gβ 5 and R7-RGS-binding protein (R7BP) that regulate G protein-coupled receptor signaling by accelerating deactivation of G i/o α-subunits, several neurological phenotypes of R7-RGS knock-out mice are not readily explained by dysregulated G i/o signaling. Accordingly, we used tandem affinity purification and LC-MS/MS to search for novel proteins that interact with R7-RGS heterotrimers in the mouse brain. Among several proteins detected, we focused on Gα 13 because it had not been linked to R7-RGS complexes before. Split-luciferase complementation assays indicated that Gα 13 in its active or inactive state interacts with R7-RGS heterotrimers containing any R7-RGS isoform. LARG (leukemia-associated Rho guanine nucleotide exchange factor (GEF)), PDZ-RhoGEF, and p115RhoGEF augmented interaction between activated Gα 13 and R7-RGS heterotrimers, indicating that these effector RhoGEFs can engage Gα 13 ·R7-RGS complexes. Because Gα 13 /R7-RGS interaction required R7BP, we analyzed phenotypes of neuronal cell lines expressing RGS7 and Gβ 5 with or without R7BP. We found that neurite retraction evoked by Gα 12/13 -dependent lysophosphatidic acid receptors was augmented in R7BP-expressing cells. R7BP expression blunted neurite formation evoked by serum starvation by signaling mechanisms involving Gα 12/13 but not Gα i/o These findings provide the first evidence that R7-RGS heterotrimers interact with Gα 13 to augment signaling pathways that regulate neurite morphogenesis. This mechanism expands the diversity of functions whereby R7-RGS complexes regulate critical aspects of nervous system development and function. © 2017 by

Segmentierung von Aortenaneurysmen in CTA-Bildern mit dem statistischen Verfahren der Active Appearance Models

NASA Astrophysics Data System (ADS)

Greiner, Katharina; Egger, Jan; Großkopf, Stefan; Kaftan, Jens N.; Dörner, Ralf; Freisleben, Bernd

In diesem Beitrag werden Active Appearance Models (AAMs) zur Segmentierung der äußeren Kontur von Aortenaneurysmen eingesetzt. Diese Aufgabe ist wegen des geringen Kontrastes zum umliegenden Gewebe und des Aufbaus der teils thrombotisierten oder kalzifizierten Gefäßwände im Bereich eines Aneurysmas so komplex, dass sie aufgrund der Vielgestalt der Kontur in CT-Angiographie-Bildern die Verwendung eines statistischen Modells für Form und eingeschlossene Textur rechtfertigt. Für die Evaluation des Verfahrens wurden verschiedene statistische Modelle aus Schichten von neun CTA-Datensätzen trainiert und die Segmentierung anhand von Leave-One-Out-Tests überprüft.

Salivary Platelet Activating Factor Levels in Periodontal Disease

DTIC Science & Technology

1991-05-01

Factor Levels in Periodontal Disease 6. AUTHOR(S) Martha L. Garito, Major 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATO;N...ABSTRACT 98 0801 SALIVARY PLATELET ACTIVATING FACTOR LEVELS IN PERIODONTAL DISEASE A THESIS Presented to the Faculty of The University of Texas Graduate...B.S., D.M.D. San Antonio, Texas May 1991 SALIVARY PLATELET ACTIVATING FACTOR LEVELS IN PERIODONTAL DISEASE Martha Laura Gar’to APPROVED: - Supervising

Pigment epithelium-derived factor blocks tumor extravasation by suppressing amoeboid morphology and mesenchymal proteolysis.

PubMed

Ladhani, Omar; Sánchez-Martinez, Cristina; Orgaz, Jose L; Jimenez, Benilde; Volpert, Olga V

2011-07-01

Metastatic melanoma cells are highly adaptable to their in vivo microenvironment and can switch between protease-dependent mesenchymal and protease-independent amoeboid invasion to facilitate metastasis. Such adaptability can be visualized in vitro, when cells are cultured in conditions that recapitulate three-dimensional microenvironments. Using thick collagen layers in cell culture and in vivo extravasation assays, we found that pigment epithelium-derived factor (PEDF) suppressed lung extravasation of aggressive melanoma by coordinated regulation of cell shape and proteolysis. In cells grown on a thick collagen bed, PEDF overexpression and exogenous PEDF blocked the rapidly invasive, rounded morphology, and promoted an elongated, mesenchymal-like phenotype associated with reduced invasion. These changes in cell shape depended on decreased RhoA and increased Rac1 activation and were mediated by the up-regulation of Rac1-GEF, DOCK3 and down-regulation of Rac1-GAP, ARHGAP22. Surprisingly, we found that PEDF overexpression also blocked the trafficking of membrane-tethered, MT1-MMP to the cell surface through RhoA inhibition and Rac1 activation. In vivo, knockdown of Rac1 and DOCK3 or overexpression of MT1-MMP was sufficient to reverse the inhibitory effect of PEDF on extravasation. Using functional studies, we demonstrated that PEDF suppressed the rounded morphology and MT1-MMP surface localization through its antiangiongenic, 34-mer epitope and the recently identified PEDF receptor candidate, PNPLA2. Our findings unveil the coordinated regulation of cell shape and proteolysis and identify an unknown mechanism for PEDF's antimetastatic activity.

Recurrent De Novo Mutations Disturbing the GTP/GDP Binding Pocket of RAB11B Cause Intellectual Disability and a Distinctive Brain Phenotype.

PubMed

Lamers, Ideke J C; Reijnders, Margot R F; Venselaar, Hanka; Kraus, Alison; Jansen, Sandra; de Vries, Bert B A; Houge, Gunnar; Gradek, Gyri Aasland; Seo, Jieun; Choi, Murim; Chae, Jong-Hee; van der Burgt, Ineke; Pfundt, Rolph; Letteboer, Stef J F; van Beersum, Sylvia E C; Dusseljee, Simone; Brunner, Han G; Doherty, Dan; Kleefstra, Tjitske; Roepman, Ronald

2017-11-02

The Rab GTPase family comprises ∼70 GTP-binding proteins, functioning in vesicle formation, transport and fusion. They are activated by a conformational change induced by GTP-binding, allowing interactions with downstream effectors. Here, we report five individuals with two recurrent de novo missense mutations in RAB11B; c.64G>A; p.Val22Met in three individuals and c.202G>A; p.Ala68Thr in two individuals. An overlapping neurodevelopmental phenotype, including severe intellectual disability with absent speech, epilepsy, and hypotonia was observed in all affected individuals. Additionally, visual problems, musculoskeletal abnormalities, and microcephaly were present in the majority of cases. Re-evaluation of brain MRI images of four individuals showed a shared distinct brain phenotype, consisting of abnormal white matter (severely decreased volume and abnormal signal), thin corpus callosum, cerebellar vermis hypoplasia, optic nerve hypoplasia and mild ventriculomegaly. To compare the effects of both variants with known inactive GDP- and active GTP-bound RAB11B mutants, we modeled the variants on the three-dimensional protein structure and performed subcellular localization studies. We predicted that both variants alter the GTP/GDP binding pocket and show that they both have localization patterns similar to inactive RAB11B. Evaluation of their influence on the affinity of RAB11B to a series of binary interactors, both effectors and guanine nucleotide exchange factors (GEFs), showed induction of RAB11B binding to the GEF SH3BP5, again similar to inactive RAB11B. In conclusion, we report two recurrent dominant mutations in RAB11B leading to a neurodevelopmental syndrome, likely caused by altered GDP/GTP binding that inactivate the protein and induce GEF binding and protein mislocalization. Copyright © 2017 American Society of Human Genetics. All rights reserved.

[Exon-dependent Subgroup-analysis of the Non-interventional REASON-Study: PFS and OS in EGFR-mutated NSCLC Patients Treated with Gefitinib or Chemotherapy].

PubMed

Schuette, W; Dietel, M; Thomas, M; Eberhardt, W; Griesinger, F; Zirrgiebel, U; Radke, S; Schirmacher, P

2016-08-01

To analyze the influence of the localization of mutations in the epidermal growth factor receptor (EGFR) gene on progression-free (PFS) and overall survival (OS) in patients (pts) with locally advanced or metastatic non-small cell lung cancer (NSCLC) treated with gefitinib (gef) or chemotherapy (CT) under real world conditions within the REASON study. Subgroups of pts with mutations in exon 19 (n = 141), 18/20 (n = 43), and 21 (n = 104) were analyzed for PFS and OS according to gef or CT treatment and compared using the log-rank test. Pts with mutations in exon 19 and 18/20 treated with gef as first line therapy showed increased PFS and OS compared to CT. This increase was statistically significant in pts with exon 19 mutation (11.3 vs. 6.5 months), but was not found in pts with exon 21 mutation (9.1 vs. 9.3 months). Also, OS was significantly increased in patients with mutation in exon 19 treated with gef ever over all treatment lines compared to CT (21.8 vs. 10.6 months), whereas this was not found in pts with mutation in exon 21 (14.1 vs. 13.9 months). Localization and nature of EGFR mutations influences gefitinib treatment outcomes under routine conditions and should therefore be analyzed in detail. © Georg Thieme Verlag KG Stuttgart · New York.

[Virulence determinant of Chromobacterium violaceum].

PubMed

Miki, Tsuyoshi

2014-01-01

Chromobacterium violaceum is a Gram-negative bacterium that infects humans and animals with fatal sepsis. The infection with C. violaceum is rare in case of those who are healthy, but once established, C. violaceum causes sever disease accompanied by abscess formation in the lungs, liver and spleen. Furthermore, C. violaceum is resistant to a broad range of antibiotics, which in some cases renders the antimicrobial therapy for this infection difficult. Thus, the infection with C. violaceum displays high mortality rates unless initial proper antimicrobial therapy. In contrast, the infection mechanism had completely remained unknown. To this end, we have tried to identify virulence factors-associated with C. violaceum infection. Two distinct type III secretion systems (TTSSs) were thought to be one of the most important virulence factors, which are encoded by Chromobacterium pathogenicity island 1/1a and 2 (Cpi-1/-1a and -2) respectively. Our results have shown that Cpi-1/-1a-encoded TTSS, but not Cpi-2, is indispensable for the virulence in a mouse infection model. C. violaceum caused fulminant hepatitis in a Cpi-1/-1a-encoded TTSS-dependent manner. We next have identified 16 novel effectors secreted from Cpi-1/-1a-encoded TTS machinery. From these effectors, we found that CopE (Chromobacterium outer protein E) has similarities to a guanine nucleotide exchange factor (GEF) for Rho GTPases. CopE acts as GEF for Rac1 and Cdc42, leading to induction of actin cytoskeletal rearrangement. Interestingly, C. violaceum invades cultured human epithelial cells in a CopE-dependent manner. Finally, an inactivation of CopE by disruption of copE gene or amino acid point mutation leading to loss of GEF activity attenuates significantly the mouse virulence of C. violaceum. These results suggest that Cpi-1/-1a-encoded TTSS is a major virulence determinant for C. violaceum infection, and that CopE contributes to the virulence in part of this pathogen.

Rho proteins of plants--functional cycle and regulation of cytoskeletal dynamics.

PubMed

Mucha, Elena; Fricke, Inka; Schaefer, Antje; Wittinghofer, Alfred; Berken, Antje

2011-11-01

Rho-related ROP proteins are molecular switches that essentially regulate a wide variety of processes. Of central interest is their influence on the plant cytoskeleton by which they affect vital processes like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors to ultimately provoke a biological response. However, the mode of action of the engaged regulators and effectors as well as their upstream and downstream interaction partners have long been largely unknown. As opposed to analogous systems in animals and fungi, plants use specific GTPase activating proteins (RopGAPs) with a unique domain composition and novel guanine nucleotide exchange factors (RopGEFs) with a probable link to cell surface receptors. Moreover, plants comprise novel effector molecules and adapters connecting ROPs to mostly unknown downstream targets on the route to the cytoskeleton. This review aims to summarize recent knowledge on the molecular mechanisms and reaction cascades involved in ROP dependent cytoskeletal rearrangements, addressing the structure and function of the unusual RopGAPs, RopGEFs and effectors, and the upstream and downstream pathways linking ROPs to cell receptor-like kinases, actin filaments, and microtubules. Copyright © 2010 Elsevier GmbH. All rights reserved.

Structure activity relationship of phenolic diterpenes from Salvia officinalis as activators of the nuclear factor E2-related factor 2 pathway

PubMed Central

Fischedick, Justin T; Standiford, Miranda; Johnson, Delinda A.; Johnson, Jeffrey A.

2013-01-01

Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to activate cytoprotective genes which may be useful in the treatment of neurodegenerative disease. In order to better understand the structure activity relationship of phenolic diterpenes from Salvia officinalis L., we isolated carnosic acid, carnosol, epirosmanol, rosmanol, 12-methoxy-carnosic acid, sageone, and carnosaldehyde using polyamide column, centrifugal partition chromatography, and semi-preparative high performance liquid chromatography. Isolated compounds were screened in-vitro for their ability to active the Nrf2 and general cellular toxicity using mouse primary cortical cultures. All compounds except 12-methoxy-carnosic acid were able to activate the antioxidant response element. Furthermore both carnosol and carnoasldehyde were able to induce Nrf2-dependent gene expression as well as protect mouse primary cortical neuronal cultures from H2O2 induced cell death. PMID:23507152

PI3K regulates endocytosis after insulin secretion by mediating signaling crosstalk between Arf6 and Rab27a.

PubMed

Yamaoka, Mami; Ando, Tomomi; Terabayashi, Takeshi; Okamoto, Mitsuhiro; Takei, Masahiro; Nishioka, Tomoki; Kaibuchi, Kozo; Matsunaga, Kohichi; Ishizaki, Ray; Izumi, Tetsuro; Niki, Ichiro; Ishizaki, Toshimasa; Kimura, Toshihide

2016-02-01

In secretory cells, endocytosis is coupled to exocytosis to enable proper secretion. Although endocytosis is crucial to maintain cellular homeostasis before and after secretion, knowledge about secretagogue-induced endocytosis in secretory cells is still limited. Here, we searched for proteins that interacted with the Rab27a GTPase-activating protein (GAP) EPI64 (also known as TBC1D10A) and identified the Arf6 guanine-nucleotide-exchange factor (GEF) ARNO (also known as CYTH2) in pancreatic β-cells. We found that the insulin secretagogue glucose promotes phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generation through phosphoinositide 3-kinase (PI3K), thereby recruiting ARNO to the intracellular side of the plasma membrane. Peripheral ARNO promotes clathrin assembly through its GEF activity for Arf6 and regulates the early stage of endocytosis. We also found that peripheral ARNO recruits EPI64 to the same area and that the interaction requires glucose-induced endocytosis in pancreatic β-cells. Given that GTP- and GDP-bound Rab27a regulate exocytosis and the late stage of endocytosis, our results indicate that the glucose-induced activation of PI3K plays a pivotal role in exocytosis-endocytosis coupling, and that ARNO and EPI64 regulate endocytosis at distinct stages. © 2016. Published by The Company of Biologists Ltd.

LPS-inducible factor(s) from activated macrophages mediates cytolysis of Naegleria fowleri amoebae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cleary, S.F.; Marciano-Cabral, F.

1986-03-01

Soluble cytolytic factors of macrophage origin have previously been described with respect to their tumoricidal activity. The purpose of this study was to investigate the mechanism and possible factor(s) responsible for cytolysis of the amoeba Naegleria fowleri by activated peritoneal macrophages from B6C3F1 mice. Macrophages or conditioned medium (CM) from macrophage cultures were incubated with /sup 3/H-Uridine labeled amoebae. Percent specific release of label served as an index of cytolysis. Bacille Calmette-Guerin (BCG) and Corynebacterium parvum macrophages demonstrated significant cytolysis of amoebae at 24 h with an effector to target ratio of 10:1. Treatment of macrophages with inhibitors of RNAmore » or protein synthesis blocked amoebicidal activity. Interposition of a 1 ..mu..m pore membrane between macrophages and amoebae inhibited killing. Inhibition in the presence of the membrane was overcome by stimulating the macrophages with LPS. CM from SPS-stimulated, but not unstimulated, cultures of activated macrophages was cytotoxic for amoebae. The activity was heat sensitive and was recovered from ammonium sulfate precipitation of the CM. Results indicate that amoebicidal activity is mediated by a protein(s) of macrophage origin induced by target cell contact or stimulation with LPS.« less

Short-term Drought Prediction in India.

NASA Astrophysics Data System (ADS)

Shah, R.; Mishra, V.

2014-12-01

Medium range soil moisture drought forecast helps in decision making in the field of agriculture and water resources management. Part of skills in medium range drought forecast comes from precipitation. Proper evaluation and correction of precipitation forecast may improve drought predictions. Here, we evaluate skills of ensemble mean precipitation forecast from Global Ensemble Forecast System (GEFS) for medium range drought predictions over India. Climatological mean (CLIM) of historic data (OBS) are used as reference forecast to evaluate GEFS precipitation forecast. Analysis was conducted based on forecast initiated on 1st and 15th dates of each month for lead up to 7-days. Correlation and RMSE were used to estimate skill scores of accumulated GEFS precipitation forecast from lead 1 to 7-days. Volumetric indices based on the 2X2 contingency table were used to check missed and falsely predicted historic volume of daily precipitation from GEFS in different regions and at different thresholds. GEFS showed improvement in correlation of 0.44 over CLIM during the monsoon season and 0.55 during the winter season. Lower RMSE was showed by GEFS than CLIM. Ratio of RMSE in GEFS and CLIM comes out as 0.82 and 0.4 (perfect skill is at zero) during the monsoon and winter season, respectively. We finally used corrected GEFS forecast to derive the Variable Infiltration Capacity (VIC) model, which was used to develop short-term forecast of hydrologic and agricultural (soil moisture) droughts in India.

COACTIVATOR ACTIVATOR (CoAA) PREVENTS THE TRANSCRIPTIONAL ACTIVITY OF RUNT DOMAIN TRANSCRIPTION FACTORS

PubMed Central

Li, Xiaodong; Hoeppner, Luke H.; Jensen, Eric D.; Gopalakrishnan, Rajaram; Westendorf, Jennifer J.

2013-01-01

Runx proteins are essential for a number of developmental processes and are aberrantly expressed in many human cancers. Runx factors bind DNA and co-factors to activate or repress genes crucial for bone formation, hematopoiesis, and neuronal development. Co-activator activator (CoAA) is a nuclear protein that regulates gene expression, RNA splicing and is overexpressed in many human tumors. In this study, we identified CoAA as a Runx2 binding protein. CoAA repressed Runx factor-dependent activation of reporter genes in a histone deacetylase-independent manner. CoAA also blocked Runx2-mediated repression of the Axin2 promoter, a novel Runx target gene. The carboxy-terminus of CoAA is essential for binding the Runt domains of Runx1 and Runx2. In electophoretic mobility shift assays, CoAA inhibited Runx2 interactions with DNA. These data indicate that CoAA is an inhibitor of Runx factors and can negate Runx factor regulation of gene expression. CoAA is expressed at high levels in human fetal osteoblasts and osteosarcoma cell lines. Suppression of CoAA expression by RNA interference reduced osteosarcoma cell viability in vitro, suggesting that it contributes to the proliferation and/or survival of osteoblast lineage cells. PMID:19585539

Complementary Phosphorylation Sites in the Adaptor Protein SLP-76 Promote Synergistic Activation of Natural Killer Cells

PubMed Central

Kim, Hun Sik; Long, Eric O.

2013-01-01

The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require the induction of synergistic signals from co-activation receptors, such as CD314 (NKG2D) and CD244 (2B4), which bind to ligands expressed on target cells. Synergy is required to overcome inhibition of the guanine nucleotide exchange factor (GEF) Vav1, a central regulator of NK cell activation, by the E3 ubiquitin ligase c-Cbl. However, the molecular basis for this synergy is unknown. Here, we showed that the adaptor protein Src homology 2 (SH2) domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) was required for this synergy, and that distinct tyrosine residues in SLP-76 were phosphorylated by each receptor of a synergistic pair. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76, each of which enables binding of SLP-76 to Vav1, was unique to receptors that stimulate ligand-dependent target cell killing, because antibody-dependent stimulation by Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that complementation of separate phospho-tyrosine targets in SLP-76 forms the basis of synergistic NK cell activation. PMID:22786724

Kelch-like ECH-associated Protein 1-dependent Nuclear Factor-E2-related Factor 2 Activation in Relation to Antioxidation Induced by Sevoflurane Preconditioning.

PubMed

Cai, Min; Tong, Li; Dong, Beibei; Hou, Wugang; Shi, Likai; Dong, Hailong

2017-03-01

The authors have reported that antioxidative effects play a crucial role in the volatile anesthetic-induced neuroprotection. Accumulated evidence shows that endogenous antioxidation could be up-regulated by nuclear factor-E2-related factor 2 through multiple pathways. However, whether nuclear factor-E2-related factor 2 activation is modulated by sevoflurane preconditioning and, if so, what is the signaling cascade underlying upstream of this activation are still unknown. Sevoflurane preconditioning in mice was performed with sevoflurane (2.5%) 1 h per day for five consecutive days. Focal cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion. Expression of nuclear factor-E2-related factor 2, kelch-like ECH-associated protein 1, manganese superoxide dismutase, thioredoxin-1, and nicotinamide adenine dinucleotide phosphate quinolone oxidoreductase-1 was detected (n = 6). The antioxidant activities and oxidative product expression were also examined. To determine the role of kelch-like ECH-associated protein 1 inhibition-dependent nuclear factor-E2-related factor 2 activation in sevoflurane preconditioning-induced neuroprotection, the kelch-like ECH-associated protein 1-nuclear factor-E2-related factor 2 signal was modulated by nuclear factor-E2-related factor 2 knockout, kelch-like ECH-associated protein 1 overexpression lentivirus, and kelch-like ECH-associated protein 1 deficiency small interfering RNA (n = 8). The infarct volume, neurologic scores, and cellular apoptosis were assessed. Sevoflurane preconditioning elicited neuroprotection and increased nuclear factor-E2-related factor 2 nuclear translocation, which in turn up-regulated endogenous antioxidation and reduced oxidative injury. Sevoflurane preconditioning reduced kelch-like ECH-associated protein 1 expression. Nuclear factor-E2-related factor 2 ablation abolished neuroprotection and reversed sevoflurane preconditioning by mediating the up-regulation of antioxidants. Kelch

Pigment Epithelium-Derived Factor Blocks Tumor Extravasation by Suppressing Amoeboid Morphology and Mesenchymal Proteolysis12

PubMed Central

Ladhani, Omar; Sánchez-Martinez, Cristina; Orgaz, Jose L; Jimenez, Benilde; Volpert, Olga V

2011-01-01

Metastatic melanoma cells are highly adaptable to their in vivo microenvironment and can switch between protease-dependent mesenchymal and protease-independent amoeboid invasion to facilitate metastasis. Such adaptability can be visualized in vitro, when cells are cultured in conditions that recapitulate three-dimensional microenvironments. Using thick collagen layers in cell culture and in vivo extravasation assays, we found that pigment epithelium-derived factor (PEDF) suppressed lung extravasation of aggressive melanoma by coordinated regulation of cell shape and proteolysis. In cells grown on a thick collagen bed, PEDF overexpression and exogenous PEDF blocked the rapidly invasive, rounded morphology, and promoted an elongated, mesenchymal-like phenotype associated with reduced invasion. These changes in cell shape depended on decreased RhoA and increased Rac1 activation and were mediated by the up-regulation of Rac1-GEF, DOCK3 and down-regulation of Rac1-GAP, ARHGAP22. Surprisingly, we found that PEDF overexpression also blocked the trafficking of membrane-tethered, MT1-MMP to the cell surface through RhoA inhibition and Rac1 activation. In vivo, knockdown of Rac1 and DOCK3 or overexpression of MT1-MMP was sufficient to reverse the inhibitory effect of PEDF on extravasation. Using functional studies, we demonstrated that PEDF suppressed the rounded morphology and MT1-MMP surface localization through its antiangiongenic, 34-mer epitope and the recently identified PEDF receptor candidate, PNPLA2. Our findings unveil the coordinated regulation of cell shape and proteolysis and identify an unknown mechanism for PEDF's antimetastatic activity. PMID:21750657

Free factor XIII activation peptide (fAP-FXIII) is a regulator of factor XIII activity via factor XIII-B.

PubMed

Dodt, Johannes; Pasternack, Ralf; Seitz, Rainer; Volkers, Peter

2016-02-01

In a factor XIIIa (FXIIIa) generation assay with recombinant FXIII-A2 (rFXIII-A2 ) free FXIII activation peptide (fAP-FXII) prolonged the time to peak (TTP) but did not affect the area under the curve (AUC) or concentration at peak (CP). Addition of recombinant factorXIII-B2 (rFXIII-B2 ) restored the characteristics of the FXIIIa generation parameters (AUC, TTP and CP) to those observed for plasma FXIII (FXIII-A2 B2 ). FXIII-A2 B2 reconstituted from rFXIII-A2 and rFXIII-B2 showed a similar effect on AUC, TTP and CP in the presence of fAP-FXII as observed for plasma FXIII-A2 B2 , indicating a role for FXIII-B in this observation. An effect of fAP-FXIII on thrombin, the proteolytic activator of FXIII, was excluded by thrombin generation assays and clotting experiments. In a purified system, fAP-FXIII did not interfere with the FXIIIa activity development of thrombin-cleaved rFXIII-A2 (rFXIII-A2 ') also excluding direct inhibition of FXIIIa. However, FXIIIa activity development of FXIII-A2 'B2 was inhibited in a concentration-dependent manner by fAP-FXIII, indicating that an interaction between AP-FXIII and FXIII-B2 contributes to the overall stability of FXIII-A2 'B2 . In addition to its well-known role, FXIII-B also contributes to FXIII-A2 B2 stability or dissociation depending on fAP-FXIII and calcium concentrations. © 2015 John Wiley & Sons Ltd.

Cross-species antiviral activity of goose interferon lambda against duck plague virus is related to its positive self-regulatory feedback loop.

PubMed

Chen, Shun; Zhang, Wei; Zhou, Qin; Wang, Anqi; Sun, Lipei; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Chen, Xiaoyue; Cheng, Anchun

2017-06-01

Duck plague virus (DPV) is a virus of the Herpesviridae family that leads to acute disease with a high mortality rate in ducks. Control of the disease contributes to the development of poultry breeding. Type III IFN family (IFN-λs) is a novel member of the IFN family, and goose IFN-λ (goIFN-λ) is a newly identified gene whose antiviral function has only been investigated to a limited extent. Here, the cross-species antiviral activity of goIFN-λ against DPV in duck embryo fibroblasts (DEFs) was studied. We found that pre-treatment with goIFN-λ greatly increased the expression of IFN-λ in both heterologous DEFs and homologous goose embryo fibroblasts (GEFs), while differentially inducing IFNα- and IFN-stimulated genes. Additionally, a positive self-regulatory feedback loop of goIFN-λ was blocked by a mouse anti-goIFN-λ polyclonal antibody, which was confirmed in both homologous GEFs and goose peripheral blood mononuclear cells (PBMCs). The suppression of the BAC-DPV-EGFP by goIFN-λ in DEFs was confirmed by fluorescence microscopy, flow cytometry (FCM) analysis, viral copies and titre detection, which can be rescued by mouse anti-goIFN-λ polyclonal antibody incubation. Finally, reporter gene assays indicated that the cross-species antiviral activity of goIFN-λ against BAC-DPV-EGFP is related to its positive self-regulatory feedback loop and subsequent ISG induction. Our data shed light on the fundamental mechanisms of goIFN-λ antiviral function in vitro and extend the considerable range of therapeutic applications in multiple-poultry disease.

Factor VIII-bypassing activity of bovine tissue factor using the canine hemophilic model.

PubMed Central

O'Brien, D P; Giles, A R; Tate, K M; Vehar, G A

1988-01-01

The bleeding disorder of hemophilia A currently treated by replacement therapy of the missing coagulation factor, factor VIII, is frequently complicated by the development of neutralizing antibodies. The therapeutic potential of attenuated forms of the lipid-associated glycoprotein tissue factor, a known initiator of coagulation, was investigated as a factor VIII-by-passing activity. The protein moiety of tissue factor (Apo-TF) was partially purified and exhibited minimal procoagulant activity before relipidation in vitro. In pilot studies, Apo-TF injection into rabbits previously anticoagulated with an antibody to factor VIII was found to have a procoagulant effect. The efficacy of the material was further demonstrated when injection of Apo-TF in hemophilic dogs resulted in a normalization of the cuticle bleeding time. Little or no change in the blood parameters associated with disseminated intravascular coagulation was observed at lower doses, although mild to moderate effects were seen at higher doses. These data suggest a novel role for Apo-TF preparations as a potential therapeutic agent for hemophiliacs with antibodies to factor VIII once the potential thrombogenicity of such materials is evaluated. Images PMID:3134399

Kinetics of Interaction between ADP-ribosylation Factor-1 (Arf1) and the Sec7 Domain of Arno Guanine Nucleotide Exchange Factor, Modulation by Allosteric Factors, and the Uncompetitive Inhibitor Brefeldin A

PubMed Central

Rouhana, Jad; Padilla, André; Estaran, Sébastien; Bakari, Sana; Delbecq, Stephan; Boublik, Yvan; Chopineau, Joel; Pugnière, Martine; Chavanieu, Alain

2013-01-01

The GDP/GTP nucleotide exchange of Arf1 is catalyzed by nucleotide exchange factors (GEF), such as Arno, which act through their catalytic Sec7 domain. This exchange is a complex mechanism that undergoes conformational changes and intermediate complex species involving several allosteric partners such as nucleotides, Mg2+, and Sec7 domains. Using a surface plasmon resonance approach, we characterized the kinetic binding parameters for various intermediate complexes. We first confirmed that both GDP and GTP counteract equivalently to the free-nucleotide binary Arf1-Arno complex stability and revealed that Mg2+ potentiates by a factor of 2 the allosteric effect of GDP. Then we explored the uncompetitive inhibitory mechanism of brefeldin A (BFA) that conducts to an abortive pentameric Arf1-Mg2+-GDP-BFA-Sec7 complex. With BFA, the association rate of the abortive complex is drastically reduced by a factor of 42, and by contrast, the 15-fold decrease of the dissociation rate concurs to stabilize the pentameric complex. These specific kinetic signatures have allowed distinguishing the level and nature as well as the fate in real time of formed complexes according to experimental conditions. Thus, we showed that in the presence of GDP, the BFA-resistant Sec7 domain of Arno can also associate to form a pentameric complex, which suggests that the uncompetitive inhibition by BFA and the nucleotide allosteric effect combine to stabilize such abortive complex. PMID:23255605

Differential roles of tissue factor and phosphatidylserine in activation of coagulation.

PubMed

Spronk, Henri M H; ten Cate, Hugo; van der Meijden, Paola E J

2014-05-01

It has been suggested that the main physiological trigger of coagulation, tissue factor, possesses limited procoagulant activity and occurs in an inactive or so-called encrypted state. For the conversion of encrypted into decrypted tissue factor with sufficient procoagulant activity, four distinct models have been proposed: 1; dimer formation, 2; lipid rafts, 3; disulfide bonds, and 4; phosphatidylserine exposure. Pro and cons can be given for each of these mechanisms of tissue factor encryption/decryption, however, it seems most likely that two or more mechanisms act together in activating the procoagulant activity. The exposure of phosphatidylserine in the outer layer of cell membranes supports coagulation through enhanced formation of the tenase (factors IXa, VIIIa and X) and prothrombinase (factors Xa, Va and prothrombin) complexes. The proposed role for phosphatidylserine in decryption of tissue factor could contribute to the correct orientation of the tissue factor - factor VII complex. Overall, the contribution of both tissue factor and phosphatidylserine to coagulation seems distinct with tissue factor being the physiological activator and phosphatidylserine the driving force of propagation of coagulation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Potent and Selective Peptide-based Inhibition of the G Protein Gαq*

PubMed Central

Charpentier, Thomas H.; Waldo, Gary L.; Lowery-Gionta, Emily G.; Krajewski, Krzysztof; Strahl, Brian D.; Kash, Thomas L.; Harden, T. Kendall; Sondek, John

2016-01-01

In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gαq binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gαq within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gαq in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gαq. A representative peptide was specific for active Gαq because it did not bind inactive Gαq or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ1γ2. In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gαq; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gαq in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gαq-dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gαq in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gαq in cells. PMID:27742837

Absence of in vitro Procoagulant Activity in Immunoglobulin Preparations due to Activated Coagulation Factors

PubMed Central

Oviedo, Adriana E.; Bernardi, María E.; Guglielmone, Hugo A.; Vitali, María S.

2015-01-01

Summary Background Immunoglobulin (IG) products, including intravenous (IVIG) or subcutaneous (SCIG) immunoglobulins are considered safe and effective for medical therapy; however, a sudden and unexpected increase in thromboembolic events (TE) after administration of certain batches of IVIG products has been attributed to the presence of activated coagulation factors, mainly factor XIa. Our aims were to examine the presence of enduring procoagulant activity during the manufacturing process of IGs, with special focus on monitoring factor XIa, and to evaluate the presence of in vitro procoagulant activity attributed to coagulation factors in different lots of IVIG and SCIG. Methods Samples of different steps of IG purification, 19 lots of IVIG and 9 of SCIG were analyzed and compared with 1 commercial preparation of IVIG and 2 of SCIG, respectively. Factors II, VII, IX, XI and XIa and non-activated partial thromboplastin time (NAPTT) were assayed. Results The levels of factors II, VII, IX, X and XI were non-quantifiable once fraction II had been re-dissolved and in all analyzed lots of IVIG and SCIG. The level of factor XIa at that point was under the detection limits of the assay, and NAPTT yielded values greater than the control during the purification process. In SCIG, we detected higher concentrations of factor XIa in the commercial products, which reached values up to 5 times higher than the average amounts found in the 9 batches produced by UNC-Hemoderivados. Factor XIa in commercial IVIG reached levels slightly higher than those of the 19 batches produced by UNC-Hemoderivados. Conclusion IVIG and SCIG manufactured by UNC-Hemoderivados showed a lack of thrombogenic potential, as demonstrated not only by the laboratory data obtained in this study but also by the absence of any reports of TE registered by the post marketing pharmacovigilance department. PMID:26733772

Far-side geometrical enhancement in surface-enhanced Raman scattering with Ag plasmonic films

NASA Astrophysics Data System (ADS)

Perera, M. Nilusha M. N.; Gibbs, W. E. Keith; Juodkazis, Saulius; Stoddart, Paul R.

2018-01-01

Surface-enhanced Raman scattering (SERS) is a surface sensitive technique where the large increase in scattering has primarily been attributed to electromagnetic and chemical enhancements. While smaller geometrical enhancements due to thin film interference and cavity resonances have also been reported, an additional enhancement in the SERS signal, referred to as the `far-side geometrical enhancement', occurs when the SERS substrate is excited through an underlying transparent dielectric substrate. Here the far-side geometrically-enhanced SERS signal has been explored experimentally in more detail. Thermally evaporated Ag plasmonic films functionalised with thiophenol were used to study the dependence of the geometrically-enhanced SERS signal on the excitation wavelength, supporting substrate material and excitation angle of incidence. The results were interpreted using a `geometrical enhancement factor' (GEF), defined as the ratio of far-side to near-side SERS signal intensity. The experimental results confirmed that the highest GEFs of 3.2-3.5× are seen closer to the localized surface plasmon resonance peak of the Ag metallic nanostructures. Interestingly, the GEF for Ag plasmonic films deposited on glass and sapphire were the same within the measurement errors, whereas increasing angle of incidence showed a decrease in the GEF. Given this improved understanding of the far-side geometrical SERS enhancement, the potential for further signal amplification and optimisation for practical sensing applications can now be considered, especially for SERS detection modes at the farend of optical fibre probes and through process windows.

Platelet Activating Factor: A Growth Factor for Breast Cancer

DTIC Science & Technology

2006-09-01

synthase (ADS) increases ether lipid content, growth and PAF synthesis in MCF-7 cells. 4. Eicosapentaenoic acid (EPA) inhibits the synthesis of PAF...Schmitt, J. D., Bullock, B. C. Wykle, R. L. Reacylation of platelet activating factor with eicosapentaenoic acid in fish-oil-enriched monkey...breast cancer. Recent studies have shown that the ratio of two families of essential fatty acids is important in regulating many cellular processes

Increased volume of distribution for recombinant activated factor VII and longer plasma-derived factor VII half-life may explain their long lasting prophylactic effect.

PubMed

Mathijssen, Natascha C J; Masereeuw, Rosalinde; Holme, Pal Andre; van Kraaij, Marian G J; Laros-van Gorkom, Britta A P; Peyvandi, Flora; van Heerde, Waander L

2013-08-01

Prophylaxis with plasma-derived or recombinant activated factor VII is beneficial in severe factor VII deficiency. To understand why prophylactic treatment with both products is efficacious, we conducted a pharmacokinetic study. Ten factor VII deficient patients were treated with either recombinant activated (20 μg/kg) or plasma-derived (25 IU/kg) factor VII in a cross-over design. Pharmacokinetic parameters were analyzed through activated factor VII activity, factor VII clotting activity, and factor VII antigen levels on depicted time points. Factor VII activity half-lifes, determined by non-compartmental and one-compartmental analysis (results in brackets), were shorter for recombinant activated (1.4h; 0.7h) than for plasma-derived factor VII (6.8h; 3.2h); both recombinant activated (5.1h; 2.1h and plasma-derived factor VII (5.8h; 3.2h) resulted in longer half-lives of factor VII antigen. Activated factor VII half-lives (based on activated factor VII activity levels) were significantly higher compared to factor VII clotting activity (1.6h; 0.9h). Volumes of distribution were significantly higher for activated factor VII (236 ml/kg; 175 ml/kg, measured by activated factor VII) as compared to plasma-derived factor VII (206 ml/kg; 64 ml/kg, measured by factor FVII activity), suggesting a plasma- and extracellular fluid distribution for recombinant activated factor VII. Recombinant activated factor VII showed significantly shorter half-lifes than plasma-derived factor VII. Volumes of distribution were significantly higher for treatment with recombinant activated factor VII. The longer half-life for plasma-derived factor VII, compared to recombinant activated factor VII, and the increased volume of distribution for recombinant activated factor VII, compared to plasma-derived factor VII may further elucidate the beneficial effect of prophylactic treatment of both products. Copyright © 2013 Elsevier Ltd. All rights reserved.

Factor XI and Contact Activation as Targets for Antithrombotic Therapy

PubMed Central

Gailani, David; Bane, Charles E.; Gruber, Andras

2015-01-01

Summary The most commonly used anticoagulants produce therapeutic antithrombotic effects either by inhibiting thrombin or factor Xa, or by lowering the plasma levels of the precursors of these key enzymes, prothrombin and factor X. These drugs do not distinguish between thrombin generation contributing to thrombosis from thrombin generation required for hemostasis. Thus, anticoagulants increase bleeding risk, and many patients who would benefit from therapy go untreated because of comorbidities that place them at unacceptable risk for hemorrhage. Studies in animals demonstrate that components of the plasma contact activation system contribute to experimentally-induced thrombosis, despite playing little or no role in hemostasis. Attention has focused on factor XII, the zymogen of a protease (factor XIIa) that initiates contact activation when blood is exposed to foreign surfaces; and factor XI, the zymogen of the protease factor XIa, which links contact activation to the thrombin generation mechanism. In the case of factor XI, epidemiologic data indicate this protein contributes to stroke and venous thromboembolism, and perhaps myocardial infarction, in humans. A phase 2 trial showing that reduction of factor XI may be more effective than low-molecular-weight heparin at preventing venous thrombosis during knee replacement surgery provides proof of concept for the premise that an antithrombotic effect can be uncoupled from an anticoagulant effect in humans by targeting components of contact activation. Here we review data on the role of factor XI and factor XII in thrombosis, and results of pre-clinical and human trials for therapies targeting these proteins. PMID:25976012

A critical role for phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac exchanger 1 in endothelial junction disruption and vascular hyperpermeability

PubMed Central

Naikawadi, Ram P.; Cheng, Ni; Vogel, Stephen M.; Qian, Feng; Wu, Dianqing; Malik, Asrar B.; Ye, Richard D.

2013-01-01

Rationale The small GTPase Rac is critical to vascular endothelial functions, yet its regulation in endothelial cells remains unclear. Understanding the upstream pathway may delineate Rac activation mechanisms and its role in maintaining vascular endothelial barrier integrity. Objective By investigating P-Rex1, one of the Rac-specific guanine nucleotide exchange factors (GEFs) previously known for G protein-coupled receptor (GPCR) signaling, we sought to determine whether Rac-GEF is a nodal for signal integration and potential target for drug intervention. Methods and Results Using gene deletion and siRNA silencing approach, we investigated the role of P-Rex1 in lung microvascular endothelial cells (HLMVECs). TNF-α exposure led to disruption of endothelial junctions, and silencing P-Rex1 protected junction integrity. TNF-α stimulated Rac activation and ROS production in a P-Rex1-dependent manner. Removal of P-Rex1 significantly reduced ICAM-1 expression, PMN transendothelial migration and leukocyte sequestration in TNF-α challenged mouse lungs. The P-Rex1 knockout mice were also refractory to lung vascular hyper-permeability and edema in a LPS-induced sepsis model. Conclusions These results demonstrate for the first time that P-Rex1 expressed in endothelial cells is activated downstream of TNF-α, which is not a GPCR agonist. Our data identify P-Rex1 as a critical mediator of vascular barrier disruption. Targeting P-Rex1 may effectively protect against TNF-α and LPS-induced endothelial junction disruption and vascular hyper-permeability. PMID:22965143

Differential procoagulant activity of microparticles derived from monocytes, granulocytes, platelets and endothelial cells: impact of active tissue factor.

PubMed

Shustova, Olga N; Antonova, Olga A; Golubeva, Nina V; Khaspekova, Svetlana G; Yakushkin, Vladimir V; Aksuk, Svetlana A; Alchinova, Irina B; Karganov, Mikhail Y; Mazurov, Alexey V

2017-07-01

: Microparticles released by activated/apoptotic cells exhibit coagulation activity as they express phosphatidylserine and some of them - tissue factor. We compared procoagulant properties of microparticles from monocytes, granulocytes, platelets and endothelial cells and assessed the impact of tissue factor in observed differences. Microparticles were sedimented (20 000g, 30 min) from the supernatants of activated monocytes, monocytic THP-1 cells, granulocytes, platelets and endothelial cells. Coagulation activity of microparticles was examined using plasma recalcification assay. The size of microparticles was evaluated by dynamic light scattering. Tissue factor activity was measured by its ability to activate factor X. All microparticles significantly accelerated plasma coagulation with the shortest lag times for microparticles derived from monocytes, intermediate - for microparticles from THP-1 cells and endothelial cells, and the longest - for microparticles from granulocytes and platelets. Average diameters of microparticles ranged within 400-600 nm. The largest microparticles were produced by endothelial cells and granulocytes, smaller - by monocytes, and the smallest - by THP-1 cells and platelets. The highest tissue factor activity was detected in microparticles from monocytes, lower activity - in microparticles from endothelial cells and THP-1 cells, and no activity - in microparticles from platelets and granulocytes. Anti-tissue factor antibodies extended coagulation lag times for microparticles from monocytes, endothelial cells and THP-1 cells and equalized them with those for microparticles from platelets and granulocytes. Higher coagulation activity of microparticles from monocytes, THP-1 cells and endothelial cells in comparison with microparticles from platelets and granulocytes is determined mainly by the presence of active tissue factor.

Factors Modulating Estrogen Receptor Activity

DTIC Science & Technology

1997-07-01

public release; distribution unlimited The views, opinions and/or findings contained in this report are those of the author( s ) and should not be...TITLE AND SUBTITLE Activity Factors Modulating Estrogen Receptor 6. AUTHOR( S ) Michael J. Garabedian, Ph.D. 7. PERFORMING ORGANIZATION NAME( S ) AND...ADDRESS(ES) New York University Medical Center New York, New York 10016 9. SPONSORING/MONITORING AGENCY NAME( S ) AND ADDRESS(ES) Commander U.S

Regulating the Regulator: Post-Translational Modification of Ras

PubMed Central

Ahearn, Ian M.; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R.

2013-01-01

Ras proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on Ras is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which regulate the activation state of Ras without covalently modifying it. In contrast, post-translational modifications (PTMs) of Ras proteins direct them to various cellular membranes and, in some cases, modulate GTP–GDP exchange. Important Ras PTMs include the constitutive and irreversible remodelling of its C-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications including phosphorylation, peptidyl-proly isomerisation, mono- and di-ubiquitination, nitrosylation, ADP ribosylation and glucosylation. PMID:22189424

Macrophages Exhibit a Large Repertoire of Activation States via Multiple Mechanisms of Macrophage-activating Factors.

PubMed

Sumiya, Y U; Inoue, Takahiro; Ishikawa, Mami; Inui, Toshio; Kuchiike, Daisuke; Kubo, Kentaro; Uto, Yoshihiro; Nishikata, Takahito

2016-07-01

Macrophages are important components of human defense systems and consequently key to antitumor immunity. Human-serum macrophage activation factor (serum MAF) can activate macrophages, making it a promising reagent for anticancer therapy. We established four different macrophage subtypes through introduction of different culture conditions to THP-1- and U937-derived macrophages. We assessed phagocytic activity to understand subtype responses to typical macrophage activation factors (MAFs) and the activation mechanisms of serum MAF. All four macrophage subtypes differed in their response to all MAFs. Moreover, serum MAF had two different activation mechanisms: N-acetylgalactosamine (GalNAc)-dependent and GalNAc-independent. Macrophage activation states and mechanisms are heterogeneous. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

T-Cell Receptor- and CD28-induced Vav1 activity is required for the accumulation of primed T cells into antigenic tissue

PubMed Central

David, Rachel; Ma, Liang; Ivetic, Aleksandar; Takesono, Aya; Ridley, Anne J.; Chai, Jian-Guo; Tybulewicz, Victor; Marelli-Berg, Federica M.

2016-01-01

Localization of primed T cells to antigenic tissue is essential for the development of effective immunity. Together with tissue-selective homing molecules, T-cell receptor (TCR)- and CD28-mediated signals have been shown to promote transendothelial migration of specific T cells into non-lymphoid antigen-rich tissue tissue. However, the cellular and molecular requirements for T-cell accumulation to target tissue following their recruitment are largely undefined. The guanine nucleotide exchange factor (GEF) Vav1 has an integral role in coupling TCR and CD28 to signalling pathways that regulate T cell activation and migration. Here, we have investigated the contribution of TCR- and CD28-induced Vav1 activity to the trafficking and localization of primed HY-specific CD4+ T cells to antigenic sites. Severe migratory defects displayed by Vav1-/- T cells in vitro were fully compensated by a combination of shear flow and chemokines, leading to normal recruitment of Vav1-/- T cells in vivo. In contrast, Vav1-/- T-cell retention into antigen-rich tissue was severely impaired, reflecting their inability to engage in sustained TCR- and CD28-mediated interactions with tissue-resident antigen-presenting cells (APCs). This novel function of APC-induced, TCR- and CD28-mediated Vav1 activity in the regulation of effector T-cell immunity highlights its potential as a therapeutic target in T-cell-mediated tissue damage. PMID:19060239

Leukemia inhibitory factor: a novel bone-active cytokine.

PubMed

Reid, L R; Lowe, C; Cornish, J; Skinner, S J; Hilton, D J; Willson, T A; Gearing, D P; Martin, T J

1990-03-01

A number of cytokines have been found to be potent regulators of bone resorption and to share the properties originally attributed to osteoclast-activating factor. One such activity, differentiation-inducing factor (DIF, D-factor) from mouse spleen cells, shares a number of biological and biochemical properties with the recently characterized and cloned leukemia inhibitory factor (LIF). We have assessed the effects of recombinant LIF on bone resorption and other parameters in neonatal mouse calvaria. Both recombinant murine and human (h) LIFs stimulated 45Ca release from prelabeled calvaria in a dose-dependent manner. The increase in bone resorption was associated with an increase in the number of osteoclasts per mm2 bone. The osteolytic effect of hLIF were blocked by 10(-7) M indomethacin. hLIF also stimulated incorporation of [3H] thymidine into calvaria, but the dose-response relationship was distinct from that for bone resorption, and this effect was not blocked by indomethacin. Similarly, hLIF increased [3H]phenylalanine incorporation into calvaria, and this was also not inhibited by indomethacin. It is concluded that LIF stimulates bone resorption by a mechanism involving prostaglandin production, but that a distinct mechanism is responsible for its stimulation of DNA and protein synthesis. The primary structure of LIF differs from that of other fully characterized, bone-active cytokines, and it, thus, represents a novel factor which may be involved in the normal regulation of bone cell function.

Factors related to meeting physical activity guidelines in active college students: A social cognitive perspective.

PubMed

Farren, G L; Zhang, T; Martin, S B; Thomas, K T

2017-01-01

To examine the relations of sex, exercise self-efficacy, outcome expectations, and social support with meeting physical activity guidelines (PAGs). Three hundred ninety-six college students participated in this study in the summer 2013. Students completed online questionnaires that assessed physical activity behaviors and psychosocial factors (ie, self-efficacy, outcome expectancies, and social support). Students' physical activity profile was categorized as meeting no PAGs, meeting aerobic PAGs only, meeting muscle-strengthening PAGs only, or meeting both PAGs. A multinomial logistic regression revealed that students' sex and psychosocial factors significantly affected the odds of meeting any and all PAGs. Sex significantly moderated the relationship between outcome expectancy and meeting aerobic PAGs and between outcome expectancy meeting muscle-strengthening PAGs. Results indicate that interventions designed to increase psychosocial factors may increase the likelihood of students meeting any and all PAGs. Social support may be especially beneficial for increasing muscle-strengthening activity.

Platelet-derived growth factor inhibits platelet activation in heparinized whole blood.

PubMed

Selheim, F; Holmsen, H; Vassbotn, F S

1999-08-15

We previously have demonstrated that human platelets have functionally active platelet-derived growth factor alpha-receptors. Studies with gel-filtered platelets showed that an autocrine inhibition pathway is transduced through this tyrosine kinase receptor during platelet activation. The physiological significance of this inhibitory effect of platelet-derived growth factor on gel-filtered platelets activation is, however, not known. In the present study, we investigated whether platelet-derived growth factor inhibits platelet activation under more physiological conditions in heparinized whole blood, which represents a more physiological condition than gel-filtered platelets. Using flow cytometric assays, we demonstrate here that platelet-derived growth factor inhibits thrombin-, thrombin receptor agonist peptide SFLLRN-, and collagen-induced platelet aggregation and shedding of platelet-derived microparticles from the platelet plasma membrane during platelet aggregation in stirred heparinized whole blood. The inhibitory effect of platelet-derived growth factor was dose dependent. However, under nonaggregating conditions (no stirring), we could not demonstrate any significant effect of platelet-derived growth factor on thrombin- and thrombin receptor agonist peptide-induced platelet surface expression of P-selectin. Our results demonstrate that platelet-derived growth factor appears to be a true antithrombotic agent only under aggregating conditions in heparinized whole blood.

Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein*

PubMed Central

García-Márquez, Adrián; Gijsbers, Abril; de la Mora, Eugenio; Sánchez-Puig, Nuria

2015-01-01

Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a guanine nucleotide exchange factor (GEF) for EFL1 promoting its activation by the release of GDP. Finally, fluorescence anisotropy measurements showed that the S143L mutation present in the Shwachman-Diamond syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1. PMID:25991726

Role of platelet activating factor in pathogenesis of acute pancreatitis in rats.

PubMed Central

Konturek, S J; Dembinski, A; Konturek, P J; Warzecha, Z; Jaworek, J; Gustaw, P; Tomaszewska, R; Stachura, J

1992-01-01

The importance of platelet activating factor in acute pancreatitis was examined by determining the tissue content of endogenous platelet activating factor and the protective effects of TCV-309, a highly selective platelet activating factor blocker, against caerulein induced pancreatitis in rats. Infusion of caerulein (10 micrograms/kg/h) for five hours resulted in about 70% increase in pancreatic weight, 22% rise in protein content, 50% reduction in tissue blood flow, nine fold increase in tissue level of platelet activating factor and 165% rise in plasma amylase as well as histological evidence of acute pancreatitis. Such infusion of caerulein in chronic pancreatic fistula rats caused a marked increase in protein output from basal secretion of 10 mg/30 minutes to 40 mg/30 minutes in the first hour of infusion followed by a decline in protein output to 15-20 mg/30 minutes in the following hours of the experiment. Exogenous platelet activating factor (50 micrograms/kg) injected ip produced similar alterations in weight, protein content, blood flow, and histology of the pancreas but the increment in serum amylase was significantly smaller and pancreatic secretion was reduced below the basal level. TCV-309 (50 micrograms/kg) given ip before caerulein or platelet activating factor administration significantly reduced the biochemical and morphological alterations caused by caerulein and abolished those induced by exogenous platelet activating factor. These results indicate that platelet activating factor plays an important role in the pathogenesis of acute pancreatitis probably by reducing the blood flow and increasing vascular permeability in the pancreas. PMID:1385272

The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts.

PubMed

Huang, Su; Eleniste, Pierre P; Wayakanon, Kornchanok; Mandela, Prashant; Eipper, Betty A; Mains, Richard E; Allen, Matthew R; Bruzzaniti, Angela

2014-03-01

Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation in the activity of the bone cells can lead to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. Kalirin is a novel GTP-exchange factor protein that has been shown to play a role in cytoskeletal remodeling and dendritic spine formation in neurons. We examined Kalirin expression in skeletal tissue and found that it was expressed in osteoclasts and osteoblasts. Furthermore, micro-CT analyses of the distal femur of global Kalirin knockout (Kal-KO) mice revealed significantly reduced trabecular and cortical bone parameters in Kal-KO mice, compared to WT mice, with significantly reduced bone mass in 8, 14 and 36week-old female Kal-KO mice. Male mice also exhibited a decrease in bone parameters but not to the level seen in female mice. Histomorphometric analyses also revealed decreased bone formation rate in 14week-old female Kal-KO mice, as well as decreased osteoblast number/bone surface and increased osteoclast surface/bone surface. Consistent with our in vivo findings, the bone resorbing activity and differentiation of Kal-KO osteoclasts was increased in vitro. Although alkaline phosphatase activity by Kal-KO osteoblasts was increased in vitro, Kal-KO osteoblasts showed decreased mineralizing activity, as well as decreased secretion of OPG, which was inversely correlated with ERK activity. Taken together, our findings suggest that deletion of Kalirin directly affects osteoclast and osteoblast activity, leading to decreased OPG secretion by osteoblasts which is likely to alter the RANKL/OPG ratio and promote osteoclastogenesis. Therefore, Kalirin may play a role in paracrine and/or endocrine signaling events that control skeletal bone remodeling and the maintenance of bone mass. Copyright © 2013 Elsevier Inc. All rights reserved.

The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts

PubMed Central

Huang, Su; Eleniste, Pierre P.; Wayakanon, Kornchanok; Mandela, Prashant; Eipper, Betty A.; Mains, Richard E.; Allen, Matthew R.; Bruzzaniti, Angela

2014-01-01

Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation in the activity of the bone cells can lead to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. Kalirin is a novel GTP-exchange factor protein that has been shown to play a role in cytoskeletal remodeling and dendritic spine formation in neurons. We examined Kalirin expression in skeletal tissue and found that it was expressed in osteoclasts and osteoblasts. Furthermore, micro-CT analyses of the distal femur of global Kalirin knockout (Kal-KO) mice revealed significantly reduced trabecular and cortical bone parameters in Kal-KO mice, compared to WT mice, with significantly reduced bone mass in 8, 14 and 36 week-old female Kal-KO mice. Male mice also exhibited a decrease in bone parameters but not to the level seen in female mice. Histomorphometric analyses also revealed decreased bone formation rate in 14 week-old female Kal-KO mice, as well as decreased osteoblast number/bone surface and increased osteoclast surface/bone surface. Consistent with our in vivo findings, the bone resorbing activity and differentiation of Kal-KO osteoclasts was increased in vitro. Although alkaline phosphatase activity by Kal-KO osteoblasts was increased in vitro, Kal-KO osteoblasts showed decreased mineralizing activity, as well as decreased secretion of OPG, which was inversely correlated with ERK activity. Taken together, our findings suggest that deletion of Kalirin directly affects osteoclast and osteoblast activity, leading to decreased OPG secretion by osteoblasts which is likely to alter the RANKL/OPG ratio and promote osteoclastogenesis. Therefore, Kalirin may play a role in paracrine and/or endocrine signaling events that control skeletal bone remodeling and the maintenance of bone mass. PMID:24380811

Impact of nonsynonymous mutations of factor X on the functions of factor X and anticoagulant activity of edoxaban.

PubMed

Noguchi, Kengo; Morishima, Yoshiyuki; Takahashi, Shinichi; Ishihara, Hiroaki; Shibano, Toshiro; Murata, Mitsuru

2015-03-01

Edoxaban is an oral direct factor Xa (FXa) inhibitor and its efficacy as an oral anticoagulant is less subject to drug-food and drug-drug interaction than existing vitamin K antagonists. Although this profile of edoxaban suggests it is well suited for clinical use, it is not clear whether genetic variations of factor X influence the activity of edoxaban. Our aim was to investigate a possible impact of single-nucleotide polymorphisms (SNPs) in the factor X gene on the functions of factor X and the activity of edoxaban. Two nonsynonymous SNPs within mature factor X, Ala152Thr and Gly192Arg, were selected as possible candidates that might affect the functions of FXa and the activity of edoxaban. We measured catalytic activities of wild type and mutant FXas in a chromogenic assay using S-2222 and coagulation times including prothrombin time (PT) and activated partial thrombin time (aPTT) of plasma-containing recombinant FXs in the presence and absence of edoxaban. Michaelis-Menten kinetic parameters of FXas, Km and Vmax values, PT and aPTT were not influenced by either mutation indicating these mutations do not affect the FXa catalytic and coagulation activities. The Ki values of edoxaban for the FXas and the concentrations of edoxaban required to double PT and aPTT were not different between wild type and mutated FXas indicating that both mutations have little impact on the activity of edoxaban. In conclusion, these data suggest that edoxaban has little interpatient variability stemming from SNPs in the factor X gene.

Potent and Selective Peptide-based Inhibition of the G Protein Gαq.

PubMed

Charpentier, Thomas H; Waldo, Gary L; Lowery-Gionta, Emily G; Krajewski, Krzysztof; Strahl, Brian D; Kash, Thomas L; Harden, T Kendall; Sondek, John

2016-12-02

In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gα q binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gα q within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gα q in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gα q A representative peptide was specific for active Gα q because it did not bind inactive Gα q or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ 1 γ 2 In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gα q ; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gα q in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gα q -dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gα q in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gα q in cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Contextual factors related to implementation of classroom physical activity breaks.

PubMed

Carlson, Jordan A; Engelberg, Jessa K; Cain, Kelli L; Conway, Terry L; Geremia, Carrie; Bonilla, Edith; Kerner, Jon; Sallis, James F

2017-09-01

Brief structured physical activity in the classroom is effective for increasing student physical activity. The present study investigated the association between implementation-related contextual factors and intervention implementation after adoption of a structured classroom physical activity intervention. Six elementary-school districts adopted structured classroom physical activity programs in 2013-2014. Implementation contextual factors and intervention implementation (structured physical activity provided in past week or month, yes/no) were assessed using surveys of 337 classroom teachers from 24 schools. Mixed-effects models accounted for the nested design. Availability of resources (yes/no, ORs = 1.91-2.93) and implementation climate z-scores (ORs = 1.36-1.47) were consistently associated with implementation. Teacher-perceived classroom behavior benefits (OR = 1.29) but not student enjoyment or health benefits, and time (OR = 2.32) and academic (OR = 1.63) barriers but not student cooperation barriers were associated with implementation (all z-scores). Four implementation contextual factor composites had an additive association with implementation (OR = 1.64 for each additional favorable composite). Training and technical assistance alone may not support a large proportion of teachers to implement structured classroom physical activity. In addition to lack of time and interference with academic lessons, school climate related to whether administrators and other teachers were supportive of the intervention were key factors explaining whether teachers implemented the intervention. Evidence-based implementation strategies are needed for effectively communicating the benefits of classroom physical activity on student behavior and improving teacher and administrator climate/attitudes around classroom physical activity.

Stress-activated MAPKs and CRM1 regulate the subcellular localization of Net1A to control cell motility and invasion.

PubMed

Ulu, Arzu; Oh, Wonkyung; Zuo, Yan; Frost, Jeffrey A

2018-02-01

The neuroepithelial cell transforming gene 1A (Net1A, an isoform of Net1) is a RhoA subfamily guanine nucleotide exchange factor (GEF) that localizes to the nucleus in the absence of stimulation, preventing it from activating RhoA. Once relocalized in the cytosol, Net1A stimulates cell motility and extracellular matrix invasion. In the present work, we investigated mechanisms responsible for the cytosolic relocalization of Net1A. We demonstrate that inhibition of MAPK pathways blocks Net1A relocalization, with cells being most sensitive to JNK pathway inhibition. Moreover, activation of the JNK or p38 MAPK family pathway is sufficient to elicit Net1A cytosolic localization. Net1A relocalization stimulated by EGF or JNK activation requires nuclear export mediated by CRM1. JNK1 (also known as MAPK8) phosphorylates Net1A on serine 52, and alanine substitution at this site prevents Net1A relocalization caused by EGF or JNK activation. Glutamic acid substitution at this site is sufficient for Net1A relocalization and results in elevated RhoA signaling to stimulate myosin light chain 2 (MLC2, also known as MYL2) phosphorylation and F-actin accumulation. Net1A S52E expression stimulates cell motility, enables Matrigel invasion and promotes invadopodia formation. These data highlight a novel mechanism for controlling the subcellular localization of Net1A to regulate RhoA activation, cell motility, and invasion. © 2018. Published by The Company of Biologists Ltd.

Opposing Effects of Platelet-Activating Factor and Lyso-Platelet-Activating Factor on Neutrophil and Platelet ActivationS⃞

PubMed Central

Welch, Emily J.; Naikawadi, Ram P.; Li, Zhenyu; Lin, Phoebe; Ishii, Satoshi; Shimizu, Takao; Tiruppathi, Chinnaswamy; Du, Xiaoping; Subbaiah, Papasani V.; Ye, Richard D.

2009-01-01

Platelet-activating factor (PAF) is a potent, bioactive phospholipid that acts on multiple cells and tissues through its G protein-coupled receptor (GPCR). PAF is not stored but is rapidly generated via enzymatic acetylation of the precursor 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPAF). The bioactivity of PAF is effectively and tightly regulated by PAF acetylhydrolases, which convert PAF back to lysoPAF. Previous studies report that lysoPAF is an inactive precursor and metabolite of PAF. However, lysoPAF has not been carefully studied in its own context. Here we report that lysoPAF has an opposing effect of PAF in the activation of neutrophils and platelets. Whereas PAF potentiates neutrophil NADPH oxidase activation, lysoPAF dose-dependently inhibits this function. Inhibition by lysoPAF is not affected by the use of a PAF receptor antagonist or genetic deletion of the PAF receptor gene. The mechanism of lysoPAF-mediated inhibition of neutrophils involves an elevation in the intracellular cAMP level, and pharmacological blockade of adenylyl cyclase completely reverses the inhibitory effect of lysoPAF. In addition, lysoPAF increases intracellular cAMP levels in platelets and inhibits thrombin-induced platelet aggregation, which can be reversed by inhibition of protein kinase A. These findings identify lysoPAF as a bioactive lipid with opposing functions of PAF and suggest a novel and intrinsic regulatory mechanism for balance of the potent activity of PAF. PMID:18931035

Factors influencing women in physical activity programs in Malaysia.

PubMed

Hanlon, C; Khoo, S; Morris, T; Eime, R

2017-12-11

The majority of research on factors associated with women participation in physical activity (PA) has been in developed countries with limited research in developing countries. Few women in Malaysia are active at the recommended levels for health, and activity rates are less than developed countries. Little research has focused specially on physically active Malaysian women and the factors that contribute to them becoming and staying active in PA programs. This lack of knowledge hinders the tailored development and implementation of PA programs to meet their needs. The aim of this study was to identify the factors of participation in PA programs for Malaysian women. The social-ecological model was used to investigate and theme the factors. Focus group discussion was conducted with participants in six PA programs targeted specifically to women. Thirty-seven women were involved in the focus group discussion, with ages ranging from 19 to 82 years. Inductive and deductive content analysis was conducted from verbatim transcripts using NVivo. Inductive content analysis allowed raw data and second-order themes to emerge. Findings revealed social support structures, tailored programs for women, and location were key contributors that encouraged women to participate in these programs. The similarity in contributors between women in non-western and western countries signifies a prime opportunity for bi-lateral relationships to be formed to enable the enhancement of program development relevant to different ethnicities and cultures within or across countries. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Poliovirus replication requires the N-terminus but not the catalytic Sec7 domain of ArfGEF GBF1.

PubMed

Belov, George A; Kovtunovych, Gennadiy; Jackson, Catherine L; Ehrenfeld, Ellie

2010-10-01

Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the ADP-ribosylation factor (Arf) GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to brefeldin A (BFA), an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism. © Published 2010. This article is a US Government work and is in the public domain in the USA.

Activated factor XI and tissue factor in aortic stenosis: Links with thrombin generation

PubMed Central

Luszczak, Joanna; Undas, Anetta; Gissel, Matthew; Olszowska, Maria; Butenas, Saulius

2011-01-01

Introduction In our previous studies we showed that a significant proportion of patients with various cardiovascular diseases have active tissue factor (TF) and factor (F)XIa in their plasma. Objective To evaluate these two proteins in plasma from patients with aortic stenosis (AS) and established their relationship with the severity of the disease. Methods Fifty-four consecutive patients with AS, including 38 (70.4%) severe AS patients, were studied. Plasma FXIa and TF activity were determined in clotting assays by measuring the response to inhibitory monoclonal antibodies. Results TF activity was detectible in plasma from 14 of 54 patients (25.9%), including 13 of 38 with severe AS (34.2%) and 1 of 16 (6.25%) with moderate AS (p=0.052). FXIa activity was found in 12 (22.2%) patients, mostly in individuals with severe AS (11 of 38, 28.9%, p=0.067). All 12 patients with circulating FXIa had active TF in their plasma as well. Severe AS patients with detectable TF had higher maximal (111±20 vs 97±16 mm Hg, p=0.02) and mean (61±12 vs 53±8 mm Hg, p=0.02) transvalvular gradient, compared with those without such activity in plasma. In severe AS patients with detectable active TF, prothrombin fragment 1.2, a thrombin generation marker, was higher than in patients without TF (375±122 vs. 207±64 pM, p<0.001). Conclusions Detectable FXIa and TF activity was observed for the first time in AS patients, primarily in severe ones. This activity correlates with thrombin generation in those patients. PMID:21519234

[Functional analysis of Oct4 promoter in Xuhuai goat].

PubMed

Wei, Guanghui; Li, Dong; Zuo, Qisheng; Zhang, Yani; Zhu, Rui; Zhang, Lei; Liu, Zhiyong; Qiu, Fenglong; Li, Bichun

2014-08-01

The aim of this study was to determine the activity region of Oct4 (octamer-binding transcription factor 4) promoter in Xuhuai goat, and to investigate the effect of TSA (trichostatin A) and VPA(valproicacid) on Oct4 promoter activity. Specific PCR primers of Oct4 promoter including different lengths of fragments were designed by Primer 5.0, then were amplified and cloned into PGL3-Bacic luciferase reporter vector. All the reconstruction vectors were transfected into gEF, P19 and COS7 cells, respectively. After TSA and VPA treatment, the activity of dual-luciferase reporter gene in these three transfected cells was detected. In addition, the CMV promoter of pEGFP-N1 was replaced by the -1516─+30 bp fragment of Oct4 promoter, GFP fluorescence was used to detect the activity of Oct4 promoter. The results indicated that different fragments of Oct4 promoter showed different degrees of activity in gEF, P19 and COS7 cells, and the maximal activity region of Oct4 promoter was -1516─+30 bp, the basal activity region was -238─+30 bp. Positive regulatory domains existed in the region of -1516─-946 bp and -615─-96 bp, while negative regulatory domains existed in the region of -1936─-1516 bp and -946─-615 bp. The optimum induction concentration to enhance the activity of Oct4 promoter was 1 μmol/L of TSA and 4 mmol/L of VPA. The GFP expression can be started by the fragment of -1516─+30 bp. This study provides an experimental basis for revealing the mechanism of expression and regulation of Oct4 in goat.

Effects of Recombinant Activated Factor VII in Traumatic Nonsurgical Intracranial Hemorrhage

DTIC Science & Technology

2006-09-01

with inhibitors to factors VIII and IX, and it is ap- proved in Europe for the treatment of patients with acquired hemophilia, congenital FVII deficiency...GARY P. WRATTEN SURGICAL SYMPOSIUM Effects of Recombinant Activated Factor VII in Traumatic Nonsurgical Intracranial Hemorrhage Christopher E. White...OBJECTIVE: To determine whether treatment with recombi- nant activated factor VII (rFVIIa) will prevent progression of bleeding in nonsurgical

Factors Influencing Intrinsic Motivation towards Physical Activity.

ERIC Educational Resources Information Center

Rutherford, William J.; And Others

1992-01-01

Study examined factors that influenced intrinsic motivation toward physical activity in 60 adult males who completed questionnaires and physical fitness tests. Results showed that performance information given immediately after task performance increased perceived competence and intrinsic motivation levels toward exercise of individuals with…

Recombinant activated factor VII: 30 years of research and innovation.

PubMed

Hedner, Ulla

2015-06-01

Recombinant activated factor VII (rFVIIa) was initially developed to treat bleeding episodes in patients with congenital haemophilia and inhibitors. The story of its development began in the 1970s, when FVIIa was identified as one of the activated coagulation factors that has minimal potential for inducing thromboembolic side-effects. Extensive research over the last 30 years has greatly increased our knowledge of the characteristics of FVII, its activation, and the mechanisms by which rFVIIa restores haemostasis. In haemophilia, the haemostatic effect of rFVIIa is mediated via binding to thrombin-activated platelets at the site of injury, thereby enhancing thrombin generation also in the absence of factor (F) VIII or FIX. The mechanism of action of rFVIIa has also allowed its successful use in other clinical scenarios characterised by impaired thrombin generation, and its licensed uses have now been extended to acquired haemophilia, congenital FVII deficiency and Glanzmann's thrombasthenia. Copyright © 2015 Elsevier Ltd. All rights reserved.

Kruppel-like factor 2 (KLF2) regulates proinflammatory activation of monocytes

PubMed Central

Das, Hiranmoy; Kumar, Ajay; Lin, Zhiyong; Patino, Willmar D.; Hwang, Paul M.; Feinberg, Mark W.; Majumder, Pradip K.; Jain, Mukesh K.

2006-01-01

The mechanisms regulating activation of monocytes remain incompletely understood. Herein we provide evidence that Kruppel-like factor 2 (KLF2) inhibits proinflammatory activation of monocytes. In vitro, KLF2 expression in monocytes is reduced by cytokine activation or differentiation. Consistent with this observation, KLF2 expression in circulating monocytes is reduced in patients with chronic inflammatory conditions such as coronary artery disease. Adenoviral overexpression of KLF2 inhibits the LPS-mediated induction of proinflammatory factors, cytokines, and chemokines and reduces phagocytosis. Conversely, short interfering RNA-mediated reduction in KLF2 increased inflammatory gene expression. Reconstitution of immunodeficient mice with KLF2-overexpressing monocytes significantly reduced carrageenan-induced acute paw edema formation. Mechanistically, KLF2 inhibits the transcriptional activity of both NF-κB and activator protein 1, in part by means of recruitment of transcriptional coactivator p300/CBP-associated factor. These observations identify KLF2 as a novel negative regulator of monocytic activation. PMID:16617118

Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice.

PubMed

Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y

2014-05-22

Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR-Ras-Raf-MEK-ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [(3)H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras-MAPK activity could be important in its anticancer activity.

Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice

PubMed Central

Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y

2014-01-01

Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR–Ras–Raf–MEK–ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [3H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras–MAPK activity could be important in its anticancer activity. PMID:24853419

GNOM regulates root hydrotropism and phototropism independently of PIN-mediated auxin transport.

PubMed

Moriwaki, Teppei; Miyazawa, Yutaka; Fujii, Nobuharu; Takahashi, Hideyuki

2014-02-01

Plant roots exhibit tropisms in response to gravity, unilateral light and moisture gradients. During gravitropism, an auxin gradient is established by PIN auxin transporters, leading to asymmetric growth. GNOM, a guanine nucleotide exchange factor of ARF GTPase (ARF-GEF), regulates PIN localization by regulating subcellular trafficking of PINs. Therefore, GNOM is important for gravitropism. We previously isolated mizu-kussei2 (miz2), which lacks hydrotropic responses; MIZ2 is allelic to GNOM. Since PIN proteins are not required for root hydrotropism in Arabidopsis, the role of GNOM in root hydrotropism should differ from that in gravitropism. To examine this possibility, we conducted genetic analysis of gnom(miz2) and gnom trans-heterozygotes. The mutant gnom(miz2), which lacks hydrotropic responses, was partially recovered by gnom(emb30-1), which lacks GEF activity, but not by gnom(B4049), which lacks heterotypic domain interactions. Furthermore, the phototropic response of gnom trans-heterozygotes differed from that of the pin2 mutant allele eir1-1. Moreover, defects in the polarities of PIN2 and auxin distribution in a severe gnom mutant were recovered by gnom(miz2). Therefore, an unknown GNOM-mediated vesicle trafficking system may mediate root hydrotropism and phototropism independently of PIN trafficking. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Mutation-Specific Mechanisms of Hyperactivation of Noonan Syndrome SOS Molecules Detected with Single-molecule Imaging in Living Cells.

PubMed

Nakamura, Yuki; Umeki, Nobuhisa; Abe, Mitsuhiro; Sako, Yasushi

2017-10-26

Noonan syndrome (NS) is a congenital hereditary disorder associated with developmental and cardiac defects. Some patients with NS carry mutations in SOS, a guanine nucleotide exchange factor (GEF) for the small GTPase RAS. NS mutations have been identified not only in the GEF domain, but also in various domains of SOS, suggesting that multiple mechanisms disrupt SOS function. In this study, we examined three NS mutations in different domains of SOS to clarify the abnormality in its translocation to the plasma membrane, where SOS activates RAS. The association and dissociation kinetics between SOS tagged with a fluorescent protein and the living cell surface were observed in single molecules. All three mutants showed increased affinity for the plasma membrane, inducing excessive RAS signalling. However, the mechanisms by which their affinity was increased were specific to each mutant. Conformational disorder in the resting state, increased probability of a conformational change on the plasma membrane, and an increased association rate constant with the membrane receptor are the suggested mechanisms. These different properties cause the specific phenotypes of the mutants, which should be rescuable with different therapeutic strategies. Therefore, single-molecule kinetic analyses of living cells are useful for the pathological analysis of genetic diseases.

Assembly and activation of neurotrophic factor receptor complexes.

PubMed

Simi, Anastasia; Ibáñez, Carlos F

2010-04-01

Neurotrophic factors play important roles in the development and function of both neuronal and glial elements of the central and peripheral nervous systems. Their functional diversity is in part based on their ability to interact with alternative complexes of receptor molecules. This review focuses on our current understanding of the mechanisms that govern the assembly and activation of neurotrophic factor receptor complexes. The realization that many, if not the majority, of these complexes exist in a preassembled form at the plasma membrane has forced the revision of classical ligand-mediated oligomerization models, and led to the discovery of novel mechanisms of receptor activation and generation of signaling diversity which are likely to be shared by many different classes of receptors.

Tissue factor-dependent coagulation activation by heme: A thromboelastometry study.

PubMed

de Souza, Gleice Regina; Hounkpe, Bidossessi Wilfried; Fiusa, Maiara Marx Luz; Colella, Marina Pereira; Annichino-Bizzacchi, Joyce M; Traina, Fabiola; Costa, Fernando Ferreira; De Paula, Erich Vinicius

2017-01-01

Heme has been characterized as potent trigger of inflammation. In hemostasis, although heme has been shown to both induce and inhibit different compartments of hemostasis, its net effect on the hemostatic balance, and the biological relevance of these effects remain to be determined. Herein we evaluated the effect of heme on hemostasis using a global assay able to generate clinically relevant data in several other complex hemostatic diseases. Citrated whole blood samples from healthy participants were stimulated by heme or vehicle and incubated for 4h at 37°C. Rotational thromboelastometry was immediately performed. The participation of tissue factor in coagulation activation was evaluated using inhibitory antibody. Heme was able of inducing ex vivo coagulation activation in whole blood, affecting predominantly parameters associated with the initial phases of clot formation. This activation effect was at least partially dependent on hematopoietic tissue factor, since the effects of heme were partially abrogated by the inhibition of human tissue factor. In conclusion, using a global hemostasis assay, our study confirmed that heme is able to activate coagulation in whole blood, in a tissue factor-dependent way. These findings could explain the disturbance in hemostatic balance observed in conditions associated with the release of heme such as sickle cell disease.

Tissue factor-dependent vascular endothelial growth factor production by human fibroblasts in response to activated factor VII.

PubMed

Ollivier, V; Bentolila, S; Chabbat, J; Hakim, J; de Prost, D

1998-04-15

The transmembrane protein tissue factor (TF) is the cell surface receptor for coagulation factor VII (FVII) and activated factor VII (FVIIa). Recently, TF has been identified as a regulator of angiogenesis, tumor growth, and metastasis. This study was designed to link the binding of FVII(a) to its receptor, TF, with the subsequent triggering of angiogenesis through vascular endothelial growth factor (VEGF) production by human lung fibroblasts. We report that incubation of fibroblasts, which express constitutive surface TF, with FVII(a) induces VEGF synthesis. FVII(a)-induced VEGF secretion, assessed by a specific enzyme-linked immunosorbent assay, was time- and concentration-dependent. VEGF secretion was maximal after 24 hours of incubation of the cells with 100 nmol/L FVII(a) and represented a threefold induction of the basal VEGF level. Reverse transcriptase-polymerase chain reaction analysis of VEGF detected three mRNA species of 180, 312, and 384 bp corresponding, respectively, to VEGF121, VEGF165, and VEGF189. A 2.5- to 3.5-fold increase was observed for the 180- and 312-bp transcripts at 12 and 24 hours, respectively. FVII(a)-dependent VEGF production was inhibited by a pool of antibodies against TF, pointing to the involvement of this receptor. On specific active-site inhibition with dansyl-glutamyl-glycinyl-arginyl chloromethyl ketone, FVIIa lost 70% of its capacity to elicit VEGF production. Consistent with this, the native form (zymogen) of FVII only had a 1.8-fold stimulating effect. Protein tyrosine kinase and protein kinase C are involved in signal transduction leading to VEGF production, as shown by the inhibitory effects of genistein and GF 109203X. The results of this study indicate that TF is essential for VIIa-induced VEGF production by human fibroblasts and that its role is mainly linked to the proteolytic activity of the TF-VIIa complex.

Factor analysis shows association between family activity environment and children's health behaviour.

PubMed

Hendrie, Gilly A; Coveney, John; Cox, David N

2011-12-01

To characterise the family activity environment in a questionnaire format, assess the questionnaire's reliability and describe its predictive ability by examining the relationships between the family activity environment and children's health behaviours - physical activity, screen time and fruit and vegetable intake. This paper describes the creation of a tool, based on previously validated scales, adapted from the food domain. Data are from 106 children and their parents (Adelaide, South Australia). Factor analysis was used to characterise factors within the family activity environment. Pearson-Product Moment correlations between the family environment and child outcomes, controlling for demographic variation, were examined. Three factors described the family activity environment - parental activity involvement, opportunity for role modelling and parental support for physical activity - and explained 37.6% of the variance. Controlling for demographic factors, the scale was significantly correlated with children's health behaviour - physical activity (r=0.27), screen time (r=-0.24) and fruit and vegetable intake (r=0.34). The family activity environment questionnaire shows high internal consistency and moderate predictive ability. This study has built on previous research by taking a more comprehensive approach to measuring the family activity environment. This research suggests the family activity environment should be considered in family-based health promotion interventions. © 2011 The Authors. ANZJPH © 2011 Public Health Association of Australia.

Comparative studies on the human serum albumin binding of the clinically approved EGFR inhibitors gefitinib, erlotinib, afatinib, osimertinib and the investigational inhibitor KP2187.

PubMed

Dömötör, Orsolya; Pelivan, Karla; Borics, Attila; Keppler, Bernhard K; Kowol, Christian R; Enyedy, Éva A

2018-05-30

Binding interactions between human serum albumin (HSA) and four approved epidermal growth factor receptor (EGFR) inhibitors gefitinib (GEF), erlotinib (ERL), afatinib (AFA), osimertinib (OSI), as well as the experimental drug KP2187, were investigated by means of spectrofluorometric and molecular modelling methods. Steady-state and time resolved spectrofluorometric techniques were carried out, including direct quenching of protein fluorescence and site marker displacement measurements. Proton dissociation processes and solvent dependent fluorescence properties were investigated as well. The EGFR inhibitors were predominantly presented in their single protonated form (HL + ) at physiological pH except ERL, which is charge-neutral. Significant solvent dependent fluorescence properties were found for GEF, ERL and KP2187, namely their emission spectra show strong dependence on the polarity and the hydrogen bonding ability of the solvents. The inhibitors proved to be bound at site I of HSA (in subdomain IIA) in a weak-to-moderate fashion (logK' 3.9-4.9) using spectrofluorometry. OSI (logK' 4.3) and KP2187 can additionally bind in site II (in subdomain IIIA), while GEF, ERL and AFA clearly show no interaction here. Docking methods qualitatively confirmed binding site preferences of compounds GEF and KP2187, and indicated that they probably bind to HSA in their neutral forms. Binding constants calculated on the basis of the various experimental data indicate a weak-to-moderate binding on HSA, only OSI exhibits somewhat higher affinity towards this protein. However, model calculations performed at physiological blood concentrations of HSA resulted in high (ca. 90%) bound fractions for the inhibitors, highlighting the importance of plasma protein binding. Copyright © 2018 Elsevier B.V. All rights reserved.

Social and Psychological Factors Associated With Adolescent Physical Activity

PubMed Central

Garcia, Jeanette M.; Sirard, John R.; Larsen, Ross; Bruening, Meg; Wall, Melanie; Neumark-Sztainer, Dianne

2017-01-01

Objective The purpose of this study was to examine, using structural equation modeling, the associations between nominated friend physical activity (PA), friend social support with individual psychological factors, and adolescent PA. Methods Data were obtained from EAT 2010 (Eating and Activity Among Teens), a large cross-sectional study conducted in 20 middle and high schools. The sample consisted of 1951 adolescents (mean age: 14.25 ± 1.96, 54% female, 68% ethnic minorities). PA, parent and friend socia l support (perceived social support for PA from parents and friends), and psychological measures (PA enjoyment, PA self-efficacy, and PA barriers) were assessed by self-report questionnaires. The SEM analysis consisted of 1 observed variable: friend PA, and 2 latent constructs: psychological factors, perceived social support. Results The model was a good fit, indicating that there were significant direct effects of both friend PA (P < .01) and psychological factors (P < .0001) on adolescent PA. In addition, psychological factors mediated the association between friend PA and adolescent PA. Conclusion The results of this model suggest that psychological factors and friend PA are associated with adolescent PA, and that psychological factors may play an important role. Future studies should further examine the association of both friend PA and psychological variables with adolescent PA. PMID:27172613

Social and Psychological Factors Associated With Adolescent Physical Activity.

PubMed

Garcia, Jeanette M; Sirard, John R; Larsen, Ross; Bruening, Meg; Wall, Melanie; Neumark-Sztainer, Dianne

2016-09-01

The purpose of this study was to examine, using structural equation modeling, the associations between nominated friend physical activity (PA), friend social support with individual PA-related psychological factors, and adolescent PA. Data were obtained from EAT 2010 (Eating and Activity Among Teens), a large cross-sectional study conducted in 20 middle and high schools. The sample consisted of 1951 adolescents (mean age: 14.25 ± 1.96, 54% female, 68% ethnic minorities). PA, parent and friend social support (perceived social support for PA from parents and friends), and psychological measures (PA enjoyment, PA self-efficacy, and PA barriers) were assessed by self-report questionnaires. The SEM analysis consisted of 1 observed variable: friend PA, and 2 latent constructs: psychological factors, perceived social support. The model was a good fit, indicating that there were significant direct effects of both friend PA (P < .01) and psychological factors (P < .0001) on adolescent PA. In addition, psychological factors mediated the association between friend PA and adolescent PA. The results of this model suggest that psychological factors and friend PA are associated with adolescent PA, and that psychological factors may play an important role. Future studies should further examine the association of both friend PA and psychological variables with adolescent PA.

Neutrophil-mediated oxidative burst and host defense are controlled by a Vav-PLCγ2 signaling axis in mice

PubMed Central

Graham, Daniel B.; Robertson, Charles M.; Bautista, Jhoanne; Mascarenhas, Francesca; Diacovo, M. Julia; Montgrain, Vivianne; Lam, Siu Kit; Cremasco, Viviana; Dunne, W. Michael; Faccio, Roberta; Coopersmith, Craig M.; Swat, Wojciech

2007-01-01

Oxidative burst, a critical antimicrobial mechanism of neutrophils, involves the rapid generation and release of reactive oxygen intermediates (ROIs) by the NADPH oxidase complex. Genetic mutations in an NADPH oxidase subunit, gp91 (also referred to as NOX2), are associated with chronic granulomatous disease (CGD), which is characterized by recurrent and life-threatening microbial infections. To combat such infections, ROIs are produced by neutrophils after stimulation by integrin-dependent adhesion to the ECM in conjunction with stimulation from inflammatory mediators, or microbial components containing pathogen-associated molecular patterns. In this report, we provide genetic evidence that both the Vav family of Rho GTPase guanine nucleotide exchange factors (GEFs) and phospholipase C–γ2 (PLC-γ2) are critical mediators of adhesion-dependent ROI production by neutrophils in mice. We also demonstrated that Vav was critically required for neutrophil-dependent host defense against systemic infection by Staphylococcus aureus and Pseudomonas aeruginosa, 2 common pathogens associated with fatal cases of hospital-acquired pneumonia. We identified a molecular pathway in which Vav GEFs linked integrin-mediated signaling with PLC-γ2 activation, release of intracellular Ca2+ cations, and generation of diacylglycerol to control assembly of the NADPH oxidase complex and ROI production by neutrophils. Taken together, our data indicate that integrin-dependent signals generated during neutrophil adhesion contribute to the activation of NADPH oxidase by a variety of distinct effector pathways, all of which require Vav. PMID:17932569

Factors associated with Spanish older people's membership in political organizations: the role of active aging activities.

PubMed

Serrat, Rodrigo; Villar, Feliciano; Celdrán, Montserrat

2015-09-01

This study explores older people's membership in political organizations by using data from the Survey on older people 2010, carried out by Spain's National Institute for older people and social services. The objectives were to describe the extent of this kind of participation among Spaniards aged 65 and over, and to analyze the factors that are associated with it. Results show that only slightly less than 7 % of the sample belonged to a political organization. To analyze the factors related to this membership, a set of models of multivariate analyses were run, including socioeconomic resources and participation in other types of active aging activity (participation in leisure, learning, and productive activities). Educational level, leisure activities, learning activities, and only volunteering in the case of productive activities were found to be associated with membership in political organizations. Results provide partial support for the socioeconomic resources model and suggest that engagement in leisure activities, learning activities, and volunteering might have an enhancing effect on membership in political organizations.

Nutrient Intake, Physical Activity, and CVD Risk Factors in Children

PubMed Central

Day, R. Sue; Fulton, Janet E.; Dai, Shifan; Mihalopoulos, Nicole L.; Barradas, Danielle T.

2009-01-01

Background Associations among dietary intake, physical activity, and cardiovascular disease (CVD) risk factors are inconsistent among male and female youth, possibly from lack of adjustment for pubertal status. The purpose of this report is to describe the associations of CVD risk factors among youth, adjusted for sexual maturation. Methods Data analyzed in 2007 from a sumsample of 556 children aged 8, 11, and 14 years in Project HeartBeat!, 1991–1993, provide cross-sectional patterns of CVD risk factors by age and gender, adjusting for sexual maturation, within dietary fat and physical activity categories. Results Girls consuming moderate- to high-fat diets were significantly less physically active than those consuming low-fat diets. Boys and girls consuming high-fat diets had higher saturated fat and cholesterol intakes than children in low-fat categories. Boys had no significant differences in physical activity, blood pressure, waist circumference, or plasma cholesterol levels across fat categories. Girls’ plasma cholesterol levels showed no significant differences across fat categories. Dietary intake did not differ across moderate-to-vigorous physical activity (MVPA) categories within gender. There were no differences in BMI by fat or MVPA categories for either gender. Girls’ waist circumference differed significantly by fat category, and systolic blood pressure differed significantly across fat and MVPA categories. Boys’ fifth-phase diastolic blood pressure was significantly different across MVPA categories. Conclusions Girls consuming atherogenic diets were significantly less physically active than those with low fat intakes, whereas boys consuming high-fat diets did not show differences in physical activity measures. With the prevalence of overweight rising among youth, the impact of atherogenic diets and sedentary lifestyles on CVD risk factors is of concern to public health professionals. PMID:19524152

Factors Influencing Farmers' Willingness to Participate in the Conversion of Cultivated Land to Wetland Program in Sanjiang National Nature Reserve, China

NASA Astrophysics Data System (ADS)

Zhang, Chunli; Robinson, Daniel; Wang, Jing; Liu, Jibin; Liu, Xiaohui; Tong, Lianjun

2011-01-01

Sanjiang National Nature Reserve (NNR) is a state-owned natural wetland in China that has suffered severe degradation due to cultivation and wetland reclamation by farmers. As a consequence, the conversion of cultivated land to wetlands (CCW) was proposed by the government of Heilongjiang province and the United Nations Development Programme/Global Environment Facility (UNDP/GEF) project team in 2007. We suggest that voluntary participation in the CCW could be an important tool for accomplishing the integrated objectives of wetland conservation and local development. The purpose of this study was to examine the main factors that influence farmers' willingness to participate in the CCW through a field investigation and a questionnaire. Based on the data from our questionnaire, which provided an effective sample of 310 households in 11 villages, the influencing factors of farmers' willingness to participate were analyzed through binary logistic regression analyses. It was concluded that age, education, the amount of cultivated land, geographical location, and the perceived benefits and risks were important factors for participation. Furthermore, suggestions for improving the wetland compensation system and providing alternative livelihoods are proposed to strengthen participation.

Factors Associated with Evaluating Public Relations Activities.

ERIC Educational Resources Information Center

McElreath, Mark P.

More than 150 public relations practitioners responded to a survey designed to identify and clarify factors associated with evaluative research in public relations. Responses indicated that (1) no more than half the practitioners formally evaluate their public relations activities on a regular basis; (2) the majority of evaluation is done…

Factors associated with participation in physical activity among adolescents in Malaysia.

PubMed

Cheah, Yong Kang; Lim, Hock Kuang; Kee, Chee Cheong; Ghazali, Sumarni Mohd

2016-11-01

The rising prevalence of non-communicable diseases (NCDs) has become a serious public health issue. Among the multi-factorial drivers behind NCDs are modifiable health risk factors, most notably, physical inactivity. In response to the nearly global policy priority of encouraging regular participation in physical activity, the objective of the present study is to examine the factors that determine participation in physical activity among Malaysian adolescents. Nationally representative data consisting of a large sample size was used. A censored regression model was developed to estimate the likelihood of participation and time spent on physical activity. There are significant relationships between physical activity and gender, ethnicity, self-rated academic performance, maternal education, household size and time spent on physical education. The present study provides new insights into the factors affecting physical activity participation among adolescents. Specifically, self-rated excellent academic performance, household size and physical education can increase the likelihood of being physically active. Evidence of the present study implies that policy makers should pay special attention to females, Chinese, adolescents with self-rated poor academic performance and adolescents who have low maternal education.

Preparation of factor VII concentrate using CNBr-activated Sepharose 4B immunoaffinity chromatography

PubMed Central

Mousavi Hosseini, Kamran; Nasiri, Saleh

2015-01-01

Background: Factor VII concentrates are used in patients with congenital or acquired factor VII deficiency or treatment of hemophilia patients with inhibitors. In this research, immunoaffinity chromatography was used to purify factor VII from prothrombin complex (Prothrombin- Proconvertin-Stuart Factor-Antihemophilic Factor B or PPSB) which contains coagulation factors II, VII, IX and X. The aim of this study was to improve purity, safety and tolerability as a highly purified factor VII concentrate. Methods: PPSB was prepared using DEAE-Sephadex and was used as the starting material for purification of coagulation factor VII. Prothrombin complex was treated by solvent/detergent at 24°C for 6 h with constant stirring. The mixture of PPSB in the PBS buffer was filtered and then chromatographed using CNBr-activated Sepharose 4B coupled with specific antibody. Factors II, IX, VII, X and VIIa were assayed on the fractions. Fractions of 48-50 were pooled and lyophilized as a factor VII concentrate. Agarose gel electrophoresis was performed and Tween 80 was measured in the factor VII concentrate. Results: Specific activity of factor VII concentrate increased from 0.16 to 55.6 with a purificationfold of 347.5 and the amount of activated factor VII (FVIIa) was found higher than PPSB (4.4-fold). Results of electrophoresis on agarose gel indicated higher purity of Factor VII compared to PPSB; these finding revealed that factor VII migrated as alpha-2 proteins. In order to improve viral safety, solvent-detergent treatment was applied prior to further purification and nearly complete elimination of tween 80 (2 μg/ml). Conclusion: It was concluded that immuonoaffinity chromatography using CNBr-activated Sepharose 4B can be a suitable choice for large-scale production of factor VII concentrate with higher purity, safety and activated factor VII. PMID:26034723

Preparation of factor VII concentrate using CNBr-activated Sepharose 4B immunoaffinity chromatography.

PubMed

Mousavi Hosseini, Kamran; Nasiri, Saleh

2015-01-01

Factor VII concentrates are used in patients with congenital or acquired factor VII deficiency or treatment of hemophilia patients with inhibitors. In this research, immunoaffinity chromatography was used to purify factor VII from prothrombin complex (Prothrombin- Proconvertin-Stuart Factor-Antihemophilic Factor B or PPSB) which contains coagulation factors II, VII, IX and X. The aim of this study was to improve purity, safety and tolerability as a highly purified factor VII concentrate. PPSB was prepared using DEAE-Sephadex and was used as the starting material for purification of coagulation factor VII. Prothrombin complex was treated by solvent/detergent at 24°C for 6 h with constant stirring. The mixture of PPSB in the PBS buffer was filtered and then chromatographed using CNBr-activated Sepharose 4B coupled with specific antibody. Factors II, IX, VII, X and VIIa were assayed on the fractions. Fractions of 48-50 were pooled and lyophilized as a factor VII concentrate. Agarose gel electrophoresis was performed and Tween 80 was measured in the factor VII concentrate. Specific activity of factor VII concentrate increased from 0.16 to 55.6 with a purificationfold of 347.5 and the amount of activated factor VII (FVIIa) was found higher than PPSB (4.4-fold). RESULTS of electrophoresis on agarose gel indicated higher purity of Factor VII compared to PPSB; these finding revealed that factor VII migrated as alpha-2 proteins. In order to improve viral safety, solvent-detergent treatment was applied prior to further purification and nearly complete elimination of tween 80 (2 μg/ml). It was concluded that immuonoaffinity chromatography using CNBr-activated Sepharose 4B can be a suitable choice for large-scale production of factor VII concentrate with higher purity, safety and activated factor VII.

Potentially Modifiable Factors Associated With Physical Activity in Individuals With Multiple Sclerosis.

PubMed

Reider, Nadia; Salter, Amber R; Cutter, Gary R; Tyry, Tuula; Marrie, Ruth Ann

2017-04-01

Physical activity levels among persons with multiple sclerosis (MS) are worryingly low. We aimed to identify the factors associated with physical activity for people with MS, with an emphasis on factors that have not been studied previously (bladder and hand dysfunction) and are potentially modifiable. This study was a secondary analysis of data collected in the spring of 2012 during the North American Research Committee on Multiple Sclerosis (NARCOMS) Registry. NARCOMS participants were surveyed regarding smoking using questions from the Behavioral Risk Factor Surveillance Survey; disability using the Patient Determined Disease Steps; fatigue, cognition, spasticity, sensory, bladder, vision and hand function using self-reported Performance Scales; health literacy using the Medical Term Recognition Test; and physical activity using questions from the Health Information National Trends Survey. We used a forward binary logistic regression to develop a predictive model in which physical activity was the outcome variable. Of 8,755 respondents, 1,707 (19.5%) were classified as active and 7,068 (80.5%) as inactive. In logistic regression, being a current smoker, moderate or severe level of disability, depression, fatigue, hand, or bladder dysfunction and minimal to mild spasticity were associated with lower odds of meeting physical activity guidelines. MS type was not linked to activity level. Several modifiable clinical and lifestyle factors influenced physical activity in MS. Prospective studies are needed to evaluate whether modification of these factors can increase physical activity participation in persons with MS. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

[Factors conditioning taking up sport activity by disabled people].

PubMed

Plinta, Ryszard; Sobiecka, Joanna

2002-01-01

The aim of the study was to answer the following questions: What factors determine that disabled people take up systematic sport activity or should make such a decision? What reasons are able to eliminate the handicapped from process of regular going in for sport? Three groups of men participated. The group of disabled sportsmen included 39 subjects, the group of non-active disabled people 36 subjects and the group of able-bodied students 45 subjects. All the involved people answered the questions of the questionnaire, which concerned factors associated with sport activity of the handicapped and their socio-demographic characteristics, among other things. The opportunity of improvement of the health status is the main advantage of the sport for disabled people (53%). The chance of self-testing and sport competition was pointed out by 47% of subjects. The most common reason for taking up sport activity was sport passion (over 50%). The others were: possibility of creating new contacts with people, improvement of the physical condition, chance of self-testing and passing free time (23%). The lack of free time was the factor eliminating from sport activity in the second group (28%). The other problem was the shortage of appropriate clubs, equipment, infrastructure etc. (almost 25%). Students concluded that sport for disabled people aims to improve the mental condition mainly (42%). Quite similar percentage of the group (38%) didn't see any reason that was able to exclude the handicapped from goingin for sport. (1) Not only does active going in for sport improve the physical condition of the handicapped but strengthen them psychically as well. (2) The lack of sport activity in disabled people is to a high degree a result of low number of sport clubs, organisations, buildings as well as presence of architectural barriers.

Revisiting the Roco G-protein cycle.

PubMed

Terheyden, Susanne; Ho, Franz Y; Gilsbach, Bernd K; Wittinghofer, Alfred; Kortholt, Arjan

2015-01-01

Mutations in leucine-rich-repeat kinase 2 (LRRK2) are the most frequent cause of late-onset Parkinson's disease (PD). LRRK2 belongs to the Roco family of proteins which share a conserved Ras-like G-domain (Roc) and a C-terminal of Roc (COR) domain tandem. The nucleotide state of small G-proteins is strictly controlled by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). Because of contradictory structural and biochemical data, the regulatory mechanism of the LRRK2 Roc G-domain and the RocCOR tandem is still under debate. In the present study, we solved the first nucleotide-bound Roc structure and used LRRK2 and bacterial Roco proteins to characterize the RocCOR function in more detail. Nucleotide binding induces a drastic structural change in the Roc/COR domain interface, a region strongly implicated in patients with an LRRK2 mutation. Our data confirm previous assumptions that the C-terminal subdomain of COR functions as a dimerization device. We show that the dimer formation is independent of nucleotide. The affinity for GDP/GTP is in the micromolar range, the result of which is high dissociation rates in the s-1 range. Thus Roco proteins are unlikely to need GEFs to achieve activation. Monomeric LRRK2 and Roco G-domains have a similar low GTPase activity to small G-proteins. We show that GTPase activity in bacterial Roco is stimulated by the nucleotide-dependent dimerization of the G-domain within the complex. We thus propose that the Roco proteins do not require GAPs to stimulate GTP hydrolysis but stimulate each other by one monomer completing the catalytic machinery of the other.

Socioeconomic Factors Influence Physical Activity and Sport in Quebec Schools

ERIC Educational Resources Information Center

Morin, Pascale; Lebel, Alexandre; Robitaille, Éric; Bisset, Sherri

2016-01-01

Background: School environments providing a wide selection of physical activities and sufficient facilities are both essential and formative to ensure young people adopt active lifestyles. We describe the association between school opportunities for physical activity and socioeconomic factors measured by low-income cutoff index, school size…

Mechanisms of Hepatocyte Growth Factor Activation in Cancer Tissues

PubMed Central

Kawaguchi, Makiko; Kataoka, Hiroaki

2014-01-01

Hepatocyte growth factor/scatter factor (HGF/SF) plays critical roles in cancer progression through its specific receptor, MET. HGF/SF is usually synthesized and secreted as an inactive proform (pro-HGF/SF) by stromal cells, such as fibroblasts. Several serine proteases are reported to convert pro-HGF/SF to mature HGF/SF and among these, HGF activator (HGFA) and matriptase are the most potent activators. Increased activities of both proteases have been observed in various cancers. HGFA is synthesized mainly by the liver and secreted as an inactive pro-form. In cancer tissues, pro-HGFA is likely activated by thrombin and/or human kallikrein 1-related peptidase (KLK)-4 and KLK-5. Matriptase is a type II transmembrane serine protease that is expressed by most epithelial cells and is also synthesized as an inactive zymogen. Matriptase activation is likely to be mediated by autoactivation or by other trypsin-like proteases. Recent studies revealed that matriptase autoactivation is promoted by an acidic environment. Given the mildly acidic extracellular environment of solid tumors, matriptase activation may, thus, be accelerated in the tumor microenvironment. HGFA and matriptase activities are regulated by HGFA inhibitor (HAI)-1 (HAI-1) and/or HAI-2 in the pericellular microenvironment. HAIs may have an important role in cancer cell biology by regulating HGF/SF-activating proteases. PMID:25268161

Factors Affecting EWS-FLI1 Activity in Ewing's Sarcoma

PubMed Central

Herrero-Martin, David; Fourtouna, Argyro; Niedan, Stephan; Riedmann, Lucia T.; Schwentner, Raphaela; Aryee, Dave N. T.

2011-01-01

Ewing's sarcoma family tumors (ESFT) are characterized by specific chromosomal translocations, which give rise to EWS-ETS chimeric proteins. These aberrant transcription factors are the main pathogenic drivers of ESFT. Elucidation of the factors influencing EWS-ETS expression and/or activity will guide the development of novel therapeutic agents against this fatal disease. PMID:22135504

Demographic factors, workplace factors and active transportation use in the USA: a secondary analysis of 2009 NHTS data.

PubMed

Quinn, Tyler D; Jakicic, John M; Fertman, Carl I; Barone Gibbs, Bethany

2017-05-01

While active transportation has health, economic and environmental benefits, participation within the USA is low. The purpose of this study is to examine relationships of demographic and workplace factors with health-enhancing active transportation and commuting. Participants in the 2009 National Household Travel Survey reported demographics, workplace factors (time/distance to work, flextime availability, option to work from home and work start time) and active transportation (for any purpose) or commuting (to and from work, workers only) as walking or biking (≥10 min bouts only). Multiple logistic regression examined cross-sectional relationships between demographics and workplace factors with active transportation and commuting. Among 152 573 participants, active transportation was reported by 1.11% by biking and 11.74% by walking. Among 111 808 working participants, active commuting was reported by 0.80% by biking and 2.76% by walking. Increased odds (p<0.05) of active commuting and transportation were associated with younger age, lower income, urban dwelling, and the highest and lowest education categories. Males had greater odds of commuting and transporting by bike but decreased odds of walk transporting. Inconsistent patterns were observed by race, but whites had greater odds of any biking (p<0.05). Odds of active commuting were higher with a flexible schedule (p<0.001), the option to work from home (p<0.05), shorter time and distance to work (both p<0.001), and work arrival time between 11:00 and 15:59 (walking only, p=0.001). Active transportation differed across demographic and workplace factors. These relationships could inform infrastructure policy decisions and workplace wellness programming targeting increased active transportation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

TALE factors poise promoters for activation by Hox proteins.

PubMed

Choe, Seong-Kyu; Ladam, Franck; Sagerström, Charles G

2014-01-27

Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we find maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription. Copyright © 2014 Elsevier Inc. All rights reserved.

Psychosocial factors underlying physical activity.

PubMed

Zhang, Juan; Middlestadt, Susan E; Ji, Cheng-Ye

2007-09-19

factors beyond physical health; barriers of not having enough time and having too many assignments perceived to hinder frequent physical activity; and parental approval. More rigorous research on psychosocial determinants with close-ended items developed from these open-ended data and with larger sample sizes of students is necessary. Research with parents and school staff will be needed to understand the perceptions of these stakeholder groups key to creating the students' social environment.

NCEP ENSEMBLE PRODUCTS

Science.gov Websites

://www.emc.ncep.noaa.gov/GEFS/ The links to the various Forecast Plots are listed under Experimental Data on the new GEFS page. This NCEP Ensemble Home Page is a collection of experimental analysis and forecast products

Nuclear factor erythroid 2-related factor-2 activity controls 4-hydroxynonenal metabolism and activity in prostate cancer cells.

PubMed

Pettazzoni, Piergiorgio; Ciamporcero, Eric; Medana, Claudio; Pizzimenti, Stefania; Dal Bello, Federica; Minero, Valerio Giacomo; Toaldo, Cristina; Minelli, Rosalba; Uchida, Koji; Dianzani, Mario Umberto; Pili, Roberto; Barrera, Giuseppina

2011-10-15

4-Hydroxynonenal (HNE) is an end product of lipoperoxidation with antiproliferative and proapoptotic properties in various tumors. Here we report a greater sensitivity to HNE in PC3 and LNCaP cells compared to DU145 cells. In contrast to PC3 and LNCaP cells, HNE-treated DU145 cells showed a smaller reduction in growth and did not undergo apoptosis. In DU145 cells, HNE did not induce ROS production and DNA damage and generated a lower amount of HNE-protein adducts. DU145 cells had a greater GSH and GST A4 content and GSH/GST-mediated HNE detoxification. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a regulator of the antioxidant response. Nrf2 protein content and nuclear accumulation were higher in DU145 cells compared to PC3 and LNCaP cells, whereas the expression of KEAP1, the main negative regulator of Nrf2 activity, was lower. Inhibition of Nrf2 expression with specific siRNA resulted in a reduction in GST A4 expression and GS-HNE formation, indicating that Nrf2 controls HNE metabolism. In addition, Nrf2 knockdown sensitized DU145 cells to HNE-mediated antiproliferative and proapoptotic activity. In conclusion, we demonstrated that increased Nrf2 activity resulted in a reduction in HNE sensitivity in prostate cancer cells, suggesting a potential mechanism of resistance to pro-oxidant therapy. Copyright © 2011 Elsevier Inc. All rights reserved.

Positive Feedback Loops for Factor V and Factor VII Activation Supply Sensitivity to Local Surface Tissue Factor Density During Blood Coagulation

PubMed Central

Balandina, A.N.; Shibeko, A.M.; Kireev, D.A.; Novikova, A.A.; Shmirev, I.I.; Panteleev, M.A.; Ataullakhanov, F.I.

2011-01-01

Blood coagulation is triggered not only by surface tissue factor (TF) density but also by surface TF distribution. We investigated recognition of surface TF distribution patterns during blood coagulation and identified the underlying molecular mechanisms. For these investigations, we employed 1), an in vitro reaction-diffusion experimental model of coagulation; and 2), numerical simulations using a mathematical model of coagulation in a three-dimensional space. When TF was uniformly immobilized over the activating surface, the clotting initiation time in normal plasma increased from 4 min to >120 min, with a decrease in TF density from 100 to 0.7 pmol/m2. In contrast, surface-immobilized fibroblasts initiated clotting within 3–7 min, independently of fibroblast quantity and despite a change in average surface TF density from 0.5 to 130 pmol/m2. Experiments using factor V-, VII-, and VIII-deficient plasma and computer simulations demonstrated that different responses to these two TF distributions are caused by two positive feedback loops in the blood coagulation network: activation of the TF–VII complex by factor Xa, and activation of factor V by thrombin. This finding suggests a new role for these reactions: to supply sensitivity to local TF density during blood coagulation. PMID:22004734

Human Factors in Railroad Operations : Activities in Fiscal Year 1973

DOT National Transportation Integrated Search

1974-02-01

This is an interim report covering human factors services rendered by TSC to the FRA under the project: "Human Factors in Railroad Operations," during fiscal year 1973. It reviews all activities briefly and contains more detailed reports on a researc...

Glioma-secreted soluble factors stimulate microglial activation: The role of interleukin-1β and tumor necrosis factor-α.

PubMed

Hwang, Ji-Sun; Jung, Eun-Hye; Kwon, Mi-Youn; Han, Inn-Oc

2016-09-15

We aimed to elucidate the effect of soluble factors secreted by glioma on microglial activation. Conditioned medium (CM) from glioma cells, CRT-MG and C6, significantly induced nitric oxide (NO) production and stimulated the mRNA expression of inducible NO synthase (iNOS), interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNF-α) and cyclooxygenase 2 (COX-2) in BV2 cells. Glioma CM stimulated p38 mitogen-activated protein kinase (MAPK) phosphorylation, and a p38 MAPK inhibitor, SB203580, suppressed CM-induced NO production in BV2 cells. In addition, CM stimulated nuclear factor-kappaB (NF-κB) DNA binding and transcriptional activity, which was repressed by SB203580. Gliomas displayed higher mRNA expression and release of TNF-α and IL-1β than primary astrocyte cells. Neutralization of TNF-α and IL-1β in C6-CM using a neutralizing antibody inhibited NO/iNOS expression in BV-2 cells. These results indicate potential contribution of diffusible tumor-derived factors to regulate microglial activation and subsequent tumor microenvironment. Copyright © 2016. Published by Elsevier B.V.

Rsr1 Focuses Cdc42 Activity at Hyphal Tips and Promotes Maintenance of Hyphal Development in Candida albicans

PubMed Central

Pulver, Rebecca; Heisel, Timothy; Gonia, Sara; Robins, Robert; Norton, Jennifer; Haynes, Paula

2013-01-01

The extremely elongated morphology of fungal hyphae is dependent on the cell's ability to assemble and maintain polarized growth machinery over multiple cell cycles. The different morphologies of the fungus Candida albicans make it an excellent model organism in which to study the spatiotemporal requirements for constitutive polarized growth and the generation of different cell shapes. In C. albicans, deletion of the landmark protein Rsr1 causes defects in morphogenesis that are not predicted from study of the orthologous protein in the related yeast Saccharomyces cerevisiae, thus suggesting that Rsr1 has expanded functions during polarized growth in C. albicans. Here, we show that Rsr1 activity localizes to hyphal tips by the differential localization of the Rsr1 GTPase-activating protein (GAP), Bud2, and guanine nucleotide exchange factor (GEF), Bud5. In addition, we find that Rsr1 is needed to maintain the focused localization of hyphal polarity structures and proteins, including Bem1, a marker of the active GTP-bound form of the Rho GTPase, Cdc42. Further, our results indicate that tip-localized Cdc42 clusters are associated with the cell's ability to express a hyphal transcriptional program and that the ability to generate a focused Cdc42 cluster in early hyphae (germ tubes) is needed to maintain hyphal morphogenesis over time. We propose that in C. albicans, Rsr1 “fine-tunes” the distribution of Cdc42 activity and that self-organizing (Rsr1-independent) mechanisms of polarized growth are not sufficient to generate narrow cell shapes or to provide feedback to the transcriptional program during hyphal morphogenesis. PMID:23223038

Chemoenzymatic Synthesis of Heparin Oligosaccharides with both Anti-factor Xa and Anti-factor IIa Activities*

PubMed Central

Xu, Yongmei; Pempe, Elizabeth H.; Liu, Jian

2012-01-01

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides. Heparin is a commonly used anticoagulant drug that inhibits the activities of factors Xa and IIa (also known as thrombin) to prevent blood clot formation. Here, we report the synthesis of a series of size-defined oligosaccharides to probe the minimum size requirement for an oligosaccharide with anti-IIa activity. The synthesis was completed by a chemoenzymatic approach involving glycosyltransferases, HS sulfotransferases, and C5-epimerase. We demonstrate the ability to synthesize highly purified N-sulfo-oligosaccharides having up to 21 saccharide residues. The results from anti-Xa and anti-IIa activity measurements revealed that an oligosaccharide longer than 19 saccharide residues is necessary to display anti-IIa activity. The oligosaccharides also exhibit low binding toward platelet factor 4, raising the possibility of preparing a synthetic heparin with a reduced effect of heparin-induced thrombocytopenia. The results from this study demonstrate the ability to synthesize large HS oligosaccharides and provide a unique tool to probe the structure and function relationships of HS that require the use of large HS fragments. PMID:22773834

Socioeconomic Factors Influence Physical Activity and Sport in Quebec Schools.

PubMed

Morin, Pascale; Lebel, Alexandre; Robitaille, Éric; Bisset, Sherri

2016-11-01

School environments providing a wide selection of physical activities and sufficient facilities are both essential and formative to ensure young people adopt active lifestyles. We describe the association between school opportunities for physical activity and socioeconomic factors measured by low-income cutoff index, school size (number of students), and neighborhood population density. A cross-sectional survey using a 2-stage stratified sampling method built a representative sample of 143 French-speaking public schools in Quebec, Canada. Self-administered questionnaires collected data describing the physical activities offered and schools' sports facilities. Descriptive and bivariate analyses were performed separately for primary and secondary schools. In primary schools, school size was positively associated with more intramural and extracurricular activities, more diverse interior facilities, and activities promoting active transportation. Low-income primary schools were more likely to offer a single gym. Low-income secondary schools offered lower diversity of intramural activities and fewer exterior sporting facilities. High-income secondary schools with a large school size provided a greater number of opportunities, larger infrastructures, and a wider selection of physical activities than smaller low-income schools. Results reveal an overall positive association between school availability of physical and sport activity and socioeconomic factors. © 2016, American School Health Association.

Topical application of recombinant activated factor VII during cesarean delivery for placenta previa.

PubMed

Schjoldager, Birgit T B G; Mikkelsen, Emmeli; Lykke, Malene R; Præst, Jørgen; Hvas, Anne-Mette; Heslet, Lars; Secher, Niels J; Salvig, Jannie D; Uldbjerg, Niels

2017-06-01

During cesarean delivery in patients with placenta previa, hemorrhaging after removal of the placenta is often challenging. In this condition, the extraordinarily high concentration of tissue factor at the placenta site may constitute a principle of treatment as it activates coagulation very effectively. The presumption, however, is that tissue factor is bound to activated factor VII. We hypothesized that topical application of recombinant activated factor VII at the placenta site reduces bleeding without affecting intravascular coagulation. We included 5 cases with planned cesarean delivery for placenta previa. After removal of the placenta, the surgeon applied a swab soaked in recombinant activated factor VII containing saline (1 mg in 246 mL) to the placenta site for 2 minutes; this treatment was repeated once if the bleeding did not decrease sufficiently. We documented the treatment on video recordings and measured blood loss. Furthermore, we determined hemoglobin concentration, platelet count, international normalized ratio, activated partial thrombin time, fibrinogen (functional), factor VII:clot, and thrombin generation in peripheral blood prior to and 15 minutes after removal of the placenta. We also tested these blood coagulation variables in 5 women with cesarean delivery planned for other reasons. Mann-Whitney test was used for unpaired data. In all 5 cases, the uterotomy was closed under practically dry conditions and the median blood loss was 490 (range 300-800) mL. There were no adverse effects of recombinant activated factor VII and we did not measure factor VII to enter the circulation. Neither did we observe changes in thrombin generation, fibrinogen, activated partial thrombin time, international normalized ratio, and platelet count in the peripheral circulation (all P values >.20). This study indicates that in patients with placenta previa, topical recombinant activated factor VII may diminish bleeding from the placenta site without initiation

Time-activity relationships to VOC personal exposure factors

NASA Astrophysics Data System (ADS)

Edwards, Rufus D.; Schweizer, Christian; Llacqua, Vito; Lai, Hak Kan; Jantunen, Matti; Bayer-Oglesby, Lucy; Künzli, Nino

Social and demographic factors have been found to play a significant role in differences between time-activity patterns of population subgroups. Since time-activity patterns largely influence personal exposure to compounds as individuals move across microenvironments, exposure subgroups within the population may be defined by factors that influence daily activity patterns. Socio-demographic and environmental factors that define time-activity subgroups also define quantifiable differences in VOC personal exposures to different sources and individual compounds in the Expolis study. Significant differences in exposures to traffic-related compounds ethylbenzene, m- and p-xylene and o-xylene were observed in relation to gender, number of children and living alone. Categorization of exposures further indicated time exposed to traffic at work and time in a car as important determinants. Increased exposures to decane, nonane and undecane were observed for males, housewives and self-employed. Categorization of exposures indicated exposure subgroups related to workshop use and living downtown. Higher exposures to 3-carene and α-pinene commonly found in household cleaning products and fragrances were associated with more children, while exposures to traffic compounds ethylbenzene, m- and p-xylene and o-xylene were reduced with more children. Considerable unexplained variation remained in categorization of exposures associated with home product use and fragrances, due to individual behavior and product choice. More targeted data collection methods in VOC exposure studies for these sources should be used. Living alone was associated with decreased exposures to 2-methyl-1-propanol and 1-butanol, and traffic-related compounds. Identification of these subgroups may help to reduce the large amount of unexplained variation in VOC exposure studies. Further they may help in assessing impacts of urban planning that result in changes in behavior of individuals, resulting in shifts in

Model of a ternary complex between activated factor VII, tissue factor and factor IX.

PubMed

Chen, Shu-wen W; Pellequer, Jean-Luc; Schved, Jean-François; Giansily-Blaizot, Muriel

2002-07-01

Upon binding to tissue factor, FVIIa triggers coagulation by activating vitamin K-dependent zymogens, factor IX (FIX) and factor X (FX). To understand recognition mechanisms in the initiation step of the coagulation cascade, we present a three-dimensional model of the ternary complex between FVIIa:TF:FIX. This model was built using a full-space search algorithm in combination with computational graphics. With the known crystallographic complex FVIIa:TF kept fixed, the FIX docking was performed first with FIX Gla-EGF1 domains, followed by the FIX protease/EGF2 domains. Because the FIXa crystal structure lacks electron density for the Gla domain, we constructed a chimeric FIX molecule that contains the Gla-EGF1 domains of FVIIa and the EGF2-protease domains of FIXa. The FVIIa:TF:FIX complex has been extensively challenged against experimental data including site-directed mutagenesis, inhibitory peptide data, haemophilia B database mutations, inhibitor antibodies and a novel exosite binding inhibitor peptide. This FVIIa:TF:FIX complex provides a powerful tool to study the regulation of FVIIa production and presents new avenues for developing therapeutic inhibitory compounds of FVIIa:TF:substrate complex.

Biofuel consumption, biodiversity, and the environmental Kuznets curve: trivariate analysis in a panel of biofuel consuming countries.

PubMed

Zaman, Khalid

2017-11-01

This study examined the relationship between biofuel consumption, forest biodiversity, and a set of national scale indicators of per capita income, foreign direct investment (FDI) inflows, trade openness, and population density with a panel data of 12 biofuels consuming countries for a period of 2000 to 2013. The study used Global Environmental Facility (GEF) biodiversity benefits index and forest biodiversity index in an environmental Kuznets curve (EKC) framework. The results confirmed an inverted U-shaped relationship between GEF biodiversity index and per capita income, while there is flat/no relationship between carbon emissions and economic growth, and between forest biodiversity and economic growth models. FDI inflows and trade openness both reduce carbon emissions while population density and biofuel consumption increase carbon emissions and decrease GEF biodiversity index. Trade openness supports to increases GEF biodiversity index while it decreases forest biodiversity index and biofuel consumption in a region.

Role of Abl kinase and the Wave2 signaling complex in HIV-1 entry at a post-hemifusion step.

PubMed

Harmon, Brooke; Campbell, Nancy; Ratner, Lee

2010-06-17

Entry of human immunodeficiency virus type 1 (HIV-1) commences with binding of the envelope glycoprotein (Env) to the receptor CD4, and one of two coreceptors, CXCR4 or CCR5. Env-mediated signaling through coreceptor results in Galphaq-mediated Rac activation and actin cytoskeleton rearrangements necessary for fusion. Guanine nucleotide exchange factors (GEFs) activate Rac and regulate its downstream protein effectors. In this study we show that Env-induced Rac activation is mediated by the Rac GEF Tiam-1, which associates with the adaptor protein IRSp53 to link Rac to the Wave2 complex. Rac and the tyrosine kinase Abl then activate the Wave2 complex and promote Arp2/3-dependent actin polymerization. Env-mediated cell-cell fusion, virus-cell fusion and HIV-1 infection are dependent on Tiam-1, Abl, IRSp53, Wave2, and Arp3 as shown by attenuation of fusion and infection in cells expressing siRNA targeted to these signaling components. HIV-1 Env-dependent cell-cell fusion, virus-cell fusion and infection were also inhibited by Abl kinase inhibitors, imatinib, nilotinib, and dasatinib. Treatment of cells with Abl kinase inhibitors did not affect cell viability or surface expression of CD4 and CCR5. Similar results with inhibitors and siRNAs were obtained when Env-dependent cell-cell fusion, virus-cell fusion or infection was measured, and when cell lines or primary cells were the target. Using membrane curving agents and fluorescence microscopy, we showed that inhibition of Abl kinase activity arrests fusion at the hemifusion (lipid mixing) step, suggesting a role for Abl-mediated actin remodeling in pore formation and expansion. These results suggest a potential utility of Abl kinase inhibitors to treat HIV-1 infected patients.

Symbiotic activity of pea (Pisum sativum) after application of Nod factors under field conditions.

PubMed

Siczek, Anna; Lipiec, Jerzy; Wielbo, Jerzy; Kidaj, Dominika; Szarlip, Paweł

2014-04-29

Growth and symbiotic activity of legumes are mediated by Nod factors (LCO, lipo-chitooligosaccharides). To assess the effects of application of Nod factors on symbiotic activity and yield of pea, a two-year field experiment was conducted on a Haplic Luvisol developed from loess. Nod factors were isolated from Rhizobium leguminosarum bv. viciae strain GR09. Pea seeds were treated with the Nod factors (10⁻¹¹ M) or water (control) before planting. Symbiotic activity was evaluated by measurements of nitrogenase activity (acetylene reduction assay), nodule number and mass, and top growth by shoot mass, leaf area, and seed and protein yield. Nod factors generally improved pea yield and nitrogenase activity in the relatively dry growing season 2012, but not in the wet growing season in 2013 due to different weather conditions.

Transcription Factor Arabidopsis Activating Factor1 Integrates Carbon Starvation Responses with Trehalose Metabolism1[OPEN

PubMed Central

Garapati, Prashanth; Feil, Regina; Lunn, John Edward; Van Dijck, Patrick; Balazadeh, Salma; Mueller-Roeber, Bernd

2015-01-01

Plants respond to low carbon supply by massive reprogramming of the transcriptome and metabolome. We show here that the carbon starvation-induced NAC (for NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON) transcription factor Arabidopsis (Arabidopsis thaliana) Transcription Activation Factor1 (ATAF1) plays an important role in this physiological process. We identified TREHALASE1, the only trehalase-encoding gene in Arabidopsis, as a direct downstream target of ATAF1. Overexpression of ATAF1 activates TREHALASE1 expression and leads to reduced trehalose-6-phosphate levels and a sugar starvation metabolome. In accordance with changes in expression of starch biosynthesis- and breakdown-related genes, starch levels are generally reduced in ATAF1 overexpressors but elevated in ataf1 knockout plants. At the global transcriptome level, genes affected by ATAF1 are broadly associated with energy and carbon starvation responses. Furthermore, transcriptional responses triggered by ATAF1 largely overlap with expression patterns observed in plants starved for carbon or energy supply. Collectively, our data highlight the existence of a positively acting feedforward loop between ATAF1 expression, which is induced by carbon starvation, and the depletion of cellular carbon/energy pools that is triggered by the transcriptional regulation of downstream gene regulatory networks by ATAF1. PMID:26149570

Interaction of Leptospira Elongation Factor Tu with Plasminogen and Complement Factor H: A Metabolic Leptospiral Protein with Moonlighting Activities

PubMed Central

Abe, Cecília M.; Monaris, Denize; Morais, Zenaide M.; Souza, Gisele O.; Vasconcellos, Sílvio A.; Isaac, Lourdes; Abreu, Patrícia A. E.; Barbosa, Angela S.

2013-01-01

The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities. PMID:24312361

Understanding Cytokine and Growth Factor Receptor Activation Mechanisms

PubMed Central

Atanasova, Mariya; Whitty, Adrian

2012-01-01

Our understanding of the detailed mechanism of action of cytokine and growth factor receptors – and particularly our quantitative understanding of the link between structure, mechanism and function – lags significantly behind our knowledge of comparable functional protein classes such as enzymes, G protein-coupled receptors, and ion channels. In particular, it remains controversial whether such receptors are activated by a mechanism of ligand-induced oligomerization, versus a mechanism in which the ligand binds to a pre-associated receptor dimer or oligomer that becomes activated through subsequent conformational rearrangement. A major limitation to progress has been the relative paucity of methods for performing quantitative mechanistic experiments on unmodified receptors expressed at endogenous levels on live cells. In this article we review the current state of knowledge on the activation mechanisms of cytokine and growth factor receptors, critically evaluate the evidence for and against the different proposed mechanisms, and highlight other key questions that remain unanswered. New approaches and techniques have led to rapid recent progress in this area, and the field is poised for major advances in the coming years, which promises to revolutionize our understanding of this large and biologically and medically important class of receptors. PMID:23046381

Giving Children a Voice: Exploring Qualitative Perspectives on Factors Influencing Recess Physical Activity

ERIC Educational Resources Information Center

Pawlowski, Charlotte Skau; Schipperijn, Jasper; Tjørnhøj-Thomsen, Tine; Troelsen, Jens

2018-01-01

Facilitators and barriers to recess physical activity are not well understood. To date, research on recess physical activity has predominantly focused on quantitative measures typically focusing on a narrow set of predefined factors, often constructed by adults. To really understand the factors affecting recess physical activity it is crucial to…

Structural diversities induced by cation sizes in a series of fluorogermanophosphates: A2[GeF2(HPO4)2] (A = Na, K, Rb, NH4, and Cs).

PubMed

Chen, Zhang-Gai; Huang, Xia; Zhuang, Rong-Chuan; Zhang, Yu; Liu, Xin; Shi, Tao; Wang, Shuai-Hua; Wu, Shao-Fan; Mi, Jin-Xiao; Huang, Ya-Xi

2017-09-12

Germanophosphates, in comparison with other metal phosphates, have been less studied but potentially exhibit more diverse structural chemistry with wide applications. Herein we applied a hydro-/solvo-fluorothermal route to make use of both the "tailor effect" of fluoride for the formation of low dimensional anionic clusters and the presence of alkali cations of different sizes to align the anionic clusters to control the overall crystal symmetries of germanophosphates. The synergetic effects of fluoride and alkali cations led to structural changes from chain-like structures to layered structures in a series of five novel fluorogermanophosphates: A 2 [GeF 2 (HPO 4 ) 2 ] (A = Na, K, Rb, NH 4 , and Cs, denoted as Na, K, Rb, NH4, and Cs). Although these fluorogermanophosphates have stoichiometrically equivalent formulas, they feature different anionic clusters, diverse structural dimensionalities, and contrasting crystal symmetries. Chain-like structures were observed for the compounds with the smaller sized alkali ions (Na + , K + , and Rb + ), whereas layered structures were found for those containing the larger sized cations ((NH 4 ) + and Cs + ). Specifically, monoclinic space groups were observed for the Na, K, Rb, and NH4 compounds, whereas a tetragonal space group P4/mbm was found for the Cs compound. These compounds provide new insights into the effects of cation sizes on the anionic clusters built from GeO 4 F 2 octahedra and HPO 4 tetrahedra as well as their influences on the overall structural symmetries in germanophosphates. Further characterization including IR spectroscopy and thermal analyses for all five compounds is also presented.

Factors Associated with Nursing Activities in Humanitarian Aid and Disaster Relief

PubMed Central

Noguchi, Norihito; Inoue, Satoshi; Shimanoe, Chisato; Shibayama, Kaoru; Shinchi, Koichi

2016-01-01

Background Although nurses play an important role in humanitarian aid and disaster relief (HA/DR), little is known about the nursing activities that are performed in HA/DR. We aimed to clarify the nursing activities performed by Japanese nurses in HA/DR and to examine the factors associated with the frequency of nursing activities. Methods A self-administered questionnaire survey was completed by 147 nurses with HA/DR experience. The survey extracted information on demographic characteristics, past experience (e.g., disaster medical training experience, HA/DR experience), circumstances surrounding their dispatched to HA/DR (e.g., team size, disaster type, post-disaster phase, mission term), and the frequency of nursing activities performed under HA/DR. The frequency of nursing activities was rated on a 5-point Likert scale. Evaluation of nursing activities was conducted based on the “nursing activity score”, which represents the frequency of each nursing activity. Factors related to the nursing activity score were evaluated by multiple logistic regression analysis. Results Nurses were involved in 27 nursing activities in HA/DR, 10 of which were performed frequently. On analysis, factors significantly associated with nursing activity score were nursing license as a registered nurse (OR 7.79, 95% CI 2.95–20.57), two or more experiences with disaster medical training (OR 2.90 95%, CI 1.12–7.49) and a post-disaster phase of three weeks or longer (OR 8.77, 95% CI 2.59–29.67). Conclusions These results will contribute to the design of evidence-based disaster medical training that improves the quality of nursing activities. PMID:26959351

P2Y5 is a Gαi, Gα12/13 G protein-coupled receptor activated by lysophosphatidic acid that reduces intestinal cell adhesion

PubMed Central

Lee, Mike; Choi, Sungwon; Halldén, Gunnel; Yo, Sek Jin; Schichnes, Denise

2009-01-01

P2Y5 is a G protein-coupled receptor that binds and is activated by lysophosphatidic acid (LPA). We determined that P2Y5 transcript is expressed along the intestinal mucosa and investigated the intracellular pathways induced by P2Y5 activation, which could contribute to LPA effects on intestinal cell adhesion. P2Y5 heterologously expressed in CHO and small intestinal hBRIE 380i cells was activated by LPA resulting in an increase in intracellular calcium ([Ca2+]i) when the cells concurrently expressed GαΔ6qi5myr. P2Y5 activation also increased the phosphorylation of ERK1/2 that was sensitive to pertussis toxin. Together these indicate that P2Y5 activation by LPA induces an increase in [Ca2+]i and ERK1/2 phosphorylation through Gαi. We discovered that P2Y5 was activated by farnesyl pyrophosphate (FPP) without a detectable change in [Ca2+]i. The activation of P2Y5 by LPA or FPP induced the activity of a serum response element (SRE)-linked luciferase reporter that was inhibited by the RGS domain of p115RhoGEF, C3 exotoxin, and Y-27632, suggesting the involvement of Gα12/13, Rho GTPase, and ROCK, respectively. However, only LPA-mediated induction of SRE reporter activity was sensitive to inhibitors targeting p38 MAPK, PI3K, PLC, and PKC. In addition, only LPA transactivated the epidermal growth factor receptor, leading to an induction of ERK1/2 phosphorylation. These observations correlate with our subsequent finding that P2Y5 activation by LPA, and not FPP, reduced intestinal cell adhesion. This study elucidates a mechanism whereby LPA can act as a luminal and/or serosal cue to alter mucosal integrity. PMID:19679818

Mechanism of action of recombinant activated factor VII: an update.

PubMed

Hedner, Ulla

2006-01-01

Bleeding episodes in patients with hemophilia and inhibitors must be managed using agents that are hemostatically active in the absence of factor VIII or IX. Activated prothrombin complex concentrates have long been used in this context. However, the search for safer and more effective agents has led to the development of recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark). This paper presents an update on the mechanism of action of rFVIIa, and describes how pharmacologic doses of this agent enhance thrombin production and thus contribute to the development of a stable, lysis-resistant fibrin plug at the site of vessel damage. This mechanism explains the reported efficacy of rFVIIa in a range of clinical situations characterized by impaired thrombin generation.

Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

PubMed Central

Hsu, FoSheng; Spannl, Stephanie; Ferguson, Charles; Hyman, Anthony A; Parton, Robert G

2018-01-01

Mitochondrial stress response is essential for cell survival, and damaged mitochondria are a hallmark of neurodegenerative diseases. Thus, it is fundamental to understand how mitochondria relay information within the cell. Here, by investigating mitochondrial-endosomal contact sites we made the surprising observation that the small GTPase Rab5 translocates from early endosomes to mitochondria upon oxidative stress. This process is reversible and accompanied by an increase in Rab5-positive endosomes in contact with mitochondria. Interestingly, activation of Rab5 on mitochondria depends on the Rab5-GEF ALS2/Alsin, encoded by a gene mutated in amyotrophic lateral sclerosis (ALS). Alsin-deficient human-induced pluripotent stem cell-derived spinal motor neurons are defective in relocating Rab5 to mitochondria and display increased susceptibility to oxidative stress. These findings define a novel pathway whereby Alsin catalyzes the assembly of the Rab5 endocytic machinery on mitochondria. Defects in stress-sensing by endosomes could be crucial for mitochondrial quality control during the onset of ALS. PMID:29469808

Science of Integrated Approaches to Natural Resources Management

NASA Astrophysics Data System (ADS)

Tengberg, Anna; Valencia, Sandra

2017-04-01

To meet multiple environmental objectives, integrated programming is becoming increasingly important for the Global Environmental Facility (GEF), the financial mechanism of the multilateral environmental agreements, including the United Nations Convention to Combat Desertification (UNCCD). Integration of multiple environmental, social and economic objectives also contributes to the achievement of the Sustainable Development Goals (SDGs) in a timely and cost-effective way. However, integration is often not well defined. This paper therefore focuses on identifying key aspects of integration and assessing their implementation in natural resources management (NRM) projects. To that end, we draw on systems thinking literature, and carry out an analysis of a random sample of GEF integrated projects and in-depth case studies demonstrating lessons learned and good practices in addressing land degradation and other NRM challenges. We identify numerous challenges and opportunities of integrated approaches that need to be addressed in order to maximise the catalytic impact of the GEF during problem diagnosis, project design, implementation and governance. We highlight the need for projects to identify clearer system boundaries and main feedback mechanisms within those boundaries, in order to effectively address drivers of environmental change. We propose a theory of change for Integrated Natural Resources Management (INRM) projects, where short-term environmental and socio-economic benefits will first accrue at the local level. Implementation of improved INRM technologies and practices at the local level can be extended through spatial planning, strengthening of innovation systems, and financing and incentive mechanisms at the watershed and/or landscape/seascape level to sustain and enhance ecosystem services at larger scales and longer time spans. We conclude that the evolving scientific understanding of factors influencing social, technical and institutional innovations and

SU-E-T-385: Evaluation of DVH Change for PTV Due to Patient Weight Loss in Prostate VMAT Using Gaussian Error Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Viraganathan, H; Jiang, R; Chow, J

Purpose: We proposed a method to predict the change of dose-volume histogram (DVH) for PTV due to patient weight loss in prostate volumetric modulated arc therapy (VMAT). This method is based on a pre-calculated patient dataset and DVH curve fitting using the Gaussian error function (GEF). Methods: Pre-calculated dose-volume data from patients having weight loss in prostate VMAT was employed to predict the change of PTV coverage due to reduced depth in external contour. The effect of patient weight loss in treatment was described by a prostate dose-volume factor (PDVF), which was evaluated by the prostate PTV. Along with themore » PDVF, the GEF was used to fit into the DVH curve for the PTV. To predict a new DVH due to weight loss, parameters from the GEF describing the shape of DVH curve were determined. Since the parameters were related to the PDVF as per the specific reduced depth, we could first predict the PDVF at a reduced depth based on the prostate size from the pre-calculated dataset. Then parameters of the GEF could be determined from the PDVF to plot the new DVH for the PTV corresponding to the reduced depth. Results: A MATLAB program was built basing on the patient dataset with different prostate sizes. We input data of the prostate size and reduced depth of the patient into the program. The program then calculated the PDVF and DVH for the PTV considering the patient weight loss. The program was verified by different patient cases with various reduced depths. Conclusion: Our method can estimate the change of DVH for the PTV due to patient weight loss quickly without CT rescan and replan. This would help the radiation staff to predict the change of PTV coverage, when patient’s external contour reduced in prostate VMAT.« less

Work factors are associated with workplace activity limitations in systemic lupus erythematosus.

PubMed

Al Dhanhani, Ali M; Gignac, Monique A M; Beaton, Dorcas E; Su, Jiandong; Fortin, Paul R

2014-11-01

The objective of this study was to examine the extent of workplace activity limitations among persons with lupus and to identify factors associated with activity limitations among those employed. We conducted a cross-sectional study using a mailed survey and clinical data of persons with lupus who attended a large lupus outpatient clinic. Data were collected on demographics, health, work factors and psychosocial measures. The workplace activity limitations scale (WALS) was used to measure difficulty related to different activities at work. Multivariable analysis examined the association of health, work context, psychosocial and demographic variables with workplace activity limitations. We received 362 responses from 604 (60%) mailed surveys. Among those not employed, 52% reported not working because of lupus. A range of physical and mental tasks were reported as difficult. Each of the physical, cognitive and energy work activities was cited as difficult by more than one-third of participants. Among employed participants, 40% had medium to high WALS difficulty scores. In the multivariable analysis, factors significantly associated with workplace activity limitations were older age, greater disease activity, fatigue, poorer health status measured by the 36-item Short Form Health Survey, lower job control, greater job strain and working more than 40 h/week. People with lupus experience limitations and difficulty at work. Determinants of workplace activity limitations are mainly those related to workplace and health factors. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Norepinephrine activates NF-κB transcription factor in cultured rat pineal gland.

PubMed

Villela, Darine; de Sá Lima, Larissa; Peres, Rafael; Peliciari-Garcia, Rodrigo Antonio; do Amaral, Fernanda Gaspar; Cipolla-Neto, José; Scavone, Cristóforo; Afeche, Solange Castro

2014-01-17

The circadian rhythm in mammalian pineal melatonin secretion is modulated by norepinephrine (NE) released at night. NE interaction with β1-adrenoceptors activates PKA that phosphorylates the transcription factor CREB, leading to the transcription and translation of the arylalkylamine-N-acetyltransferase (AANAT) enzyme. Several studies have reported the interplay between CREB and the nuclear factor-κB (NF-κB) and a circadian rhythm for this transcription factor was recently described in the rat pineal gland. In this work we studied a direct effect of NE on NF-κB activation and the role played by this factor on melatonin synthesis and Aanat transcription and activity. Cultured rat pineal glands were incubated in the presence of two different NF-κB inhibitors, pyrrolidine-dithiocarbamate or sodium salicylate, and stimulated with NE. Melatonin content was quantified by HPLC with electrochemical detection. AANAT activity was measured by a radiometric assay and the expression of Aanat mRNA was analyzed by real-time PCR. Gel shift assay was performed to study the NF-κB activation in cultured rat pineal glands stimulated by NE. Our results showed that the p50/p50 homodimer of NF-κB is activated by NE and that it has a role in melatonin synthesis, acting on Aanat transcription and activity. Here we present evidence that NF-κB is an important transcription factor that acts, directly or indirectly, on Aanat transcription and activity leading to a modulation of melatonin synthesis. NE plays a role in the translocation of NF-κB p50/p50 homodimer to the nucleus of pinealocytes, thus probably influencing the nocturnal pineal melatonin synthesis. Copyright © 2013 Elsevier Inc. All rights reserved.

Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization, Pathogen Vacuole Motility and Infection

PubMed Central

Rothmeier, Eva; Pfaffinger, Gudrun; Hoffmann, Christine; Harrison, Christopher F.; Grabmayr, Heinrich; Repnik, Urska; Hannemann, Mandy; Wölke, Stefan; Bausch, Andreas; Griffiths, Gareth; Müller-Taubenberger, Annette; Itzen, Aymelt; Hilbi, Hubert

2013-01-01

The causative agent of Legionnaires' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS) to form in phagocytes a distinct “Legionella-containing vacuole” (LCV), which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF) domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP) in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED) fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila. PMID:24068924

Prediction of Pathway Activation by Xenobiotic-Responsive Transcription Factors in the Mouse Liver

EPA Science Inventory

Many drugs and environmentally-relevant chemicals activate xenobioticresponsive transcription factors (TF). Identification of target genes of these factors would be useful in predicting pathway activation in in vitro chemical screening. Starting with a large compendium of Affymet...

Two distinct forms of Factor VIII coagulant protein in human plasma. Cleavage by thrombin, and differences in coagulant activity and association with von Willebrand factor.

PubMed Central

Weinstein, M J; Chute, L E

1984-01-01

We have characterized Factor VIII coagulant protein, present in normal human plasma, that reacts with a specific human 125I-labeled anti-human VIII:C antigen Fab antibody fragment. Two major Factor VIII coagulant antigen populations were present. The first, approximately 85% of the total antigen, was bound to von Willebrand factor and when tested in a standard one-stage assay had Factor VIII coagulant activity. The second antigenic population, eluting near fibrinogen when plasma was gel filtered, was not bound to von Willebrand protein, did not have Factor VIII coagulant activity unless activated, but did block anti-VIII:C Fab neutralization of clotting activity. The two antigenic populations were separable by cryoprecipitation and agarose gel electrophoresis. Although the two antigenic populations differed in their Factor VIII coagulant activity and in their binding to von Willebrand factor, the principal member of both populations is of mol wt 2.4 X 10(5). Both antigens, when proteolyzed by thrombin, were quickly converted to a 1 X 10(5)-mol wt form in association with the appearance of VIII:C activity. The 1 X 10(5)-mol wt antigen was further slowly degraded to an 8 X 10(4)-mol wt form while Factor VIII coagulant activity declined. These results demonstrate the presence of an inactive Factor VIII coagulant protein in plasma, not associated with von Willebrand factor, that can react with thrombin to yield Factor VIII coagulant activity. Images PMID:6421875

Environmental, policy, and cultural factors related to physical activity among rural, African American women.

PubMed

Sanderson, Bonnie; Littleton, MaryAnn; Pulley, LeaVonne

2002-01-01

Sixty-one African American women (ages 20-50 years) from a rural community in Alabama participated in six focus groups. Barriers to and enablers of physical activity were identified and grouped into personal, environmental (social and physical), policy, and cultural themes for qualitative analyses. Personal factors included motivation, perceived health, feeling tired, and lack of time; social environmental factors included support from friends, family, and issues related to child care; physical environmental factors included weather, access to facilities, availability of sidewalks or other places to walk; policy factors included personal safety concerns (loose dogs, traffic, etc.) and inflexible work environments. Some, but not all, women perceived cultural differences as a factor affecting physical activity levels. Differences in socioeconomic levels and time demands among women of different cultures were identified as factors that may influence physical activity. Participants provided suggestions for community-based physical activity interventions using an environmental approach.

Induction of endothelial cell proliferation by angiogenic factors released by activated monocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pakala, Rajbabu; Watanabe, Takuya; Benedict, Claude R

2002-06-01

Introduction: Cell-cell interaction is an essential component of atherosclerotic plaque development. Activated monocytes appear to play a central role in the development of atherosclerosis, not only through foam cell formation but also via the production of various growth factors that induce proliferation of different cell types that are involved in the plaque development. Using serum free co-culture method, we determined the effect of monocytes on endothelial cell proliferation. Methods: Endothelial cell proliferation is determined by the amount of [{sup 3}H]thymidine incorporated in to the DNA. Basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) levels inmore » the conditioned medium were determined by ELISA. Results: Conditioned medium from unactivated monocytes partially inhibited endothelial cell proliferation, whereas conditioned medium from activated monocytes promoted endothelial cell proliferation. The mitogenic effect of conditioned medium derived from activated monocytes is due to the presence of b-FGF, VEGF and IL-8. Neutralizing antibodies against b-FGF, VEGF and IL-8 partially reversed the mitogenic effect of conditioned medium derived from activated monocytes. When b-FGF, VEGF and IL-8 were immunoprecipitated from conditioned medium derived from activated monocytes, it is less mitogenic to endothelial cells. Conclusion: Activated monocytes may play an important role in the development of atherosclerotic plaque by producing endothelial cell growth factors.« less

Factors associated with physical activity in Australians with hip or knee osteoarthritis.

PubMed

Heesch, Kristiann Corbusier; Ng, Norman; Brown, Wendy

2011-03-01

Physical activity (PA) is recommended for managing osteoarthritis (OA). However, few people with OA are physically active. Understanding the factors associated with PA is necessary to increase PA in this population. This cross-sectional study examined factors associated with leisure-time PA, stretching exercises, and strengthening exercises in people with OA. For a mail survey, 485 individuals, aged 68.0 years (SD = 10.6) with hip or knee OA, were asked about factors that may influence PA participation, including use ofnon-PAOA management strategies and both psychological and physical health-related factors. Associations between factors and each PA outcome were examined in multivariable logistic regression models. Non-PA management strategies were the main factors associated with the outcomes. Information/education courses, heat/cold treatments, and paracetamol were associated with stretching and strengthening exercises (P < .05). Hydrotherapy and magnet therapy were associated with leisure-time PA; using orthotics and massage therapy, with stretching exercises; and occupational therapy, with strengthening exercises (P < .05). Few psychological or health-related factors were associated with the outcomes. Some management strategies may make it easier for people with OA to be physically active, and could be promoted to encourage PA. Providers of strategies are potential avenues for recruiting people with OA into PA programs.

Biphasic and synergistic activation of p44mapk (ERK1) by growth factors: correlation between late phase activation and mitogenicity.

PubMed

Meloche, S; Seuwen, K; Pagès, G; Pouysségur, J

1992-05-01

We have examined the phosphorylation and protein kinase activity of p44 mitogen-activated protein kinase (p44mapk) in growth factor-stimulated hamster fibroblasts using a specific antiserum. The activity of p44mapk was stimulated both by receptor tyrosine kinases and G protein-coupled receptors. Detailed kinetics revealed that alpha-thrombin induces a biphasic activation of p44mapk in CCL39 cells: a rapid phase appearing at 5-10 min was followed by a late and sustained phase still elevated after 4 h. Inactivation of alpha-thrombin with hirudin after 30 sec, which prevented DNA synthesis, did not alter the early p44mapk response but completely abolished the late phase. Pretreatment of the cells with pertussis toxin, which inhibits by more than 95% alpha-thrombin-induced mitogenicity, resulted in the complete loss of late phase activity, while the early peak was partially attenuated. Treatment of CCL39 cells with basic fibroblast growth factor also induced a strong activation of p44mapk. Serotonin, which is not a mitogen by its own, had no effect on late phase p44mapk activity, but synergized with basic fibroblast growth factor to induce late kinase response and DNA synthesis. Both early and late phase activation of p44mapk were accompanied by tyrosine phosphorylation of the enzyme. Together, the results indicate that there is a very close correlation between the ability of a growth factor to induce late and sustained p44mapk activation and its mitogenic potential. Therefore, we propose that sustained p44mapk activation is an obligatory event for growth factor-induced cell cycle progression.

Enhancer Activation Requires Trans-Recruitment of a Mega Transcription Factor Complex

PubMed Central

Liu, Zhijie; Merkurjev, Daria; Yang, Feng; Li, Wenbo; Oh, Soohwan; Friedman, Meyer J.; Song, Xiaoyuan; Zhang, Feng; Ma, Qi; Ohgi, Kenneth; Krones, Anna; Rosenfeld, Michael G.

2014-01-01

Summary Enhancers provide critical information directing cell-type specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. Here we report that the most strongly activated estrogen (E2)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ERα at ERE-containing enhancers. We refer to enhancers recruiting these factors as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome. PMID:25303530

National Centers for Environmental Prediction

Science.gov Websites

/ VISION | About EMC EMC > GEFS > OPERATIONAL PRODUCTS Home Operational Products Experimental Data Global Ensemble products posted by the GEFS model team are experimental analysis and forecast products , and you will find them listed under the "Experimental Data" section of our web site. The

UV-B radiation induces macrophage migration inhibitory factor-mediated melanogenesis through activation of protease-activated receptor-2 and stem cell factor in keratinocytes.

PubMed

Enomoto, Akiko; Yoshihisa, Yoko; Yamakoshi, Takako; Ur Rehman, Mati; Norisugi, Osamu; Hara, Hiroshi; Matsunaga, Kenji; Makino, Teruhiko; Nishihira, Jun; Shimizu, Tadamichi

2011-02-01

UV radiation indirectly regulates melanogenesis in melanocytes through a paracrine regulatory mechanism involving keratinocytes. Protease-activated receptor (PAR)-2 activation induces melanosome transfer by increasing phagocytosis of melanosomes by keratinocytes. This study demonstrated that macrophage migration inhibitory factor (MIF) stimulated PAR-2 expression in human keratinocytes. In addition, we showed that MIF stimulated stem cell factor (SCF) release in keratinocytes; however, MIF had no effect on the release of endothelin-1 or prostaglandin E2 in keratinocytes. In addition, MIF had no direct effect on melanin and tyrosinase synthesis in cultured human melanocytes. The effect of MIF on melanogenesis was also examined using a three-dimensional reconstituted human epidermal culture model, which is a novel, commercially available, cultured human epidermis containing functional melanocytes. Migration inhibitory factor induced an increase in melanin content in the epidermis after a 9-day culture period. Moreover, melanin synthesis induced by UV-B stimulation was significantly down-regulated by anti-MIF antibody treatment. An in vivo study showed that the back skin of MIF transgenic mice had a higher melanin content than that of wild-type mice after 12 weeks of UV-B exposure. Therefore, MIF-mediated melanogenesis occurs mainly through the activation of PAR-2 and SCF expression in keratinocytes after exposure to UV-B radiation. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Factors Influencing Teachers' Engagement in Informal Learning Activities

ERIC Educational Resources Information Center

Lohman, Margaret C.

2006-01-01

Purpose: The purpose of this study is to examine factors influencing the engagement of public school teachers in informal learning activities. Design/methodology/approach: This study used a survey research design. Findings: Analysis of the data found that teachers rely to a greater degree on interactive than on independent informal learning…

The RhoGEF Net1 Is Required for Normal Mammary Gland Development

PubMed Central

Zuo, Yan; Berdeaux, Rebecca

2014-01-01

Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily-specific guanine nucleotide exchange factor that is overexpressed in human breast cancer and is required for breast cancer cell migration and invasion. However, the role of Net1 in normal mammary gland development or function has never been assessed. To understand the role of Net1 in the mammary gland, we have created a conditional Net1 knockout mouse model. Whole-body deletion of Net1 results in delayed mammary gland development during puberty characterized by slowed of ductal extension and reduced ductal branching. Epithelial cells within the developing ducts show reduced proliferation that is accompanied by diminished estrogen receptor-α expression and activity. Net1-deficient mammary glands also exhibit reduced phosphorylation of regulatory subunits of myosin light chain and myosin light-chain phosphatase, indicating that RhoA-dependent actomyosin contraction is compromised. Net1 deficiency also leads to disorganization of myoepithelial and ductal epithelial cells and increased periductal collagen deposition. Mammary epithelial cell transplantation experiments indicate that reduced ductal branching and disorganization are cell autonomous. These data identify for the first time a role for NET1 in vivo and indicate that NET1 expression is essential for the proliferation and differentiation of mammary epithelial cells in the developing mammary gland. PMID:25321414

Mediation of mouse natural cytotoxic activity by tumour necrosis factor

NASA Astrophysics Data System (ADS)

Ortaldo, John R.; Mason, Llewellyn H.; Mathieson, Bonnie J.; Liang, Shu-Mei; Flick, David A.; Herberman, Ronald B.

1986-06-01

Natural cell-mediated cytotoxic activity in the mouse has been associated with two types of effector cells, the natural killer (NK) cell and the natural cytotoxic (NC) cell, which seem to differ with regard to their patterns of target selectivity, cell surface characteristics and susceptibility to regulatory factors1. During studies on the mechanism of action of cytotoxic molecules, it became evident that WEHI-164, the prototype NC target cell, was highly susceptible to direct lysis by both human and mouse recombinant tumour necrosis factor (TNF). Here we show that NC, but not NK activity mediated by normal splenocytes, is abrogated by rabbit antibodies to recombinant and natural TNF, respectively. Thus, the cell-mediated activity defined as NC is due to release of TNF by normal spleen cells and does not represent a unique natural effector mechanism.

Reliable and Affordable Control Systems Active Combustor Pattern Factor Control

NASA Technical Reports Server (NTRS)

McCarty, Bob; Tomondi, Chris; McGinley, Ray

2004-01-01

Active, closed-loop control of combustor pattern factor is a cooperative effort between Honeywell (formerly AlliedSignal) Engines and Systems and the NASA Glenn Research Center to reduce emissions and turbine-stator vane temperature variations, thereby enhancing engine performance and life, and reducing direct operating costs. Total fuel flow supplied to the engine is established by the speed/power control, but the distribution to individual atomizers will be controlled by the Active Combustor Pattern Factor Control (ACPFC). This system consist of three major components: multiple, thin-film sensors located on the turbine-stator vanes; fuel-flow modulators for individual atomizers; and control logic and algorithms within the electronic control.

An increase in circulating B cell-activating factor in childhood-onset ocular myasthenia gravis.

PubMed

Motobayashi, Mitsuo; Inaba, Yuji; Nishimura, Takafumi; Kobayashi, Norimoto; Nakazawa, Yozo; Koike, Kenichi

2015-04-01

Myasthenia gravis is a B cell-mediated autoimmune disorder. The pathophysiology of childhood-onset ocular myasthenia gravis remains unclear. We investigated serum B cell-activating factor levels and other immunological parameters in child patients with ocular myasthenia gravis. Blood samples were obtained from 9 children with ocular myasthenia gravis and 20 age-matched controls. We assayed serum concentrations of B cell-activating factor, anti-acetylcholine receptor antibody titers, 7 types of cytokines (interleukins-2, -4, -6, -10, and -17A; interferon-γ; tumor necrosis factor-α) as well as the percentages of peripheral blood CD4+, CD8+, and CD19+ cells. Serum B cell-activating factor levels were significantly higher before immunosuppressive therapy in patients with childhood-onset ocular myasthenia gravis than in controls and decreased after immunosuppressive therapy. A significant positive correlation was observed between serum B cell-activating factor levels and anti-acetylcholine receptor antibody titers in patients with myasthenia gravis. Serum B cell-activating factor concentrations did not correlate with the percentages of CD4+, CD8+, and CD19+ cells or the CD4+/CD8+ ratio. No significant differences were observed in the levels of the 7 different types of cytokines examined, including interleukin-17A, between preimmunosuppressive therapy myasthenia gravis patients and controls. Circulating B cell-activating factor may play a key role in the pathophysiology of childhood-onset ocular myasthenia gravis. Copyright © 2015 Elsevier Inc. All rights reserved.

Factors affecting the behavior of unburned carbon upon steam activation

NASA Astrophysics Data System (ADS)

Lu, Zhe

The main objective of this study is to investigate the factors that could affect the behavior of unburned carbon samples upon steam activation. Through this work, the relationships among the factors that could influence the carbon-steam reaction with the surface area of the produced activated carbon were explored. Statistical analysis was used to relate the chemical and physical properties of the unburned carbon to the surface area of the activated carbon. Six unburned carbons were selected as feedstocks for activated carbon, and marked as UCA through UCF. The unburned carbons were activated using steam at 850°C for 90 minutes, and the surface areas of their activated counterparts were measured using N2 adsorption isotherms at 77K. The activated carbons produced from different unburned carbon precursors presented different surface areas at similar carbon burn-off levels. Moreover, in different carbon burn-off regions, the sequences for surface area of activated carbons from different unburned carbon samples were different. The factors that may affect the carbon-steam gasification reactions, including the concentration of carbon active sites, the crystallite size of the carbon, the intrinsic porous structure of carbon, and the inorganic impurities, were investigated. All unburned carbons investigated in this study were similar in that they showed the very broad (002) and (10 ) carbon peaks, which are characteristic of highly disordered carbonaceous materials. In this study, the unburned carbon samples contained about 17--48% of inorganic impurities. Compared to coals, the unburned carbon samples contain a larger amount of inorganic impurities as a result of the burn-off, or at lease part, of the carbon during the combustion process. These inorganic particles were divided into two groups in terms of the way they are associated with carbon particles: free single particles, and particles combined with carbon particles. As indicated from the present work, unburned

Resveratrol regulates gene transcription via activation of stimulus-responsive transcription factors.

PubMed

Thiel, Gerald; Rössler, Oliver G

2017-03-01

Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin of grapes and other fruits and plants, is a common constituent of our diet and of dietary supplements. Many health-promoting benefits have been connected with resveratrol in the treatment of cardiovascular diseases, cancer, diabetes, inflammation, neurodegeneration, and diseases connected with aging. To explain the pleiotropic effects of resveratrol, the molecular targets of this compound have to be identified on the cellular level. Resveratrol induces intracellular signal transduction pathways which ultimately lead to changes in the gene expression pattern of the cells. Here, we review the effect of resveratrol on the activation of the stimulus-responsive transcription factors CREB, AP-1, Egr-1, Elk-1, and Nrf2. Following activation, these transcription factors induce transcription of delayed response genes. The gene products of these delayed response genes are ultimately responsible for the changes in the biochemistry and physiology of resveratrol-treated cells. The activation of stimulus-responsive transcription factors may explain many of the intracellular activities of resveratrol. However, results obtained in vitro may not easily be transferred to in vivo systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mechanisms of integrin-vascular endothelial growth factor receptor cross-activation in angiogenesis.

PubMed

Mahabeleshwar, Ganapati H; Feng, Weiyi; Reddy, Kumar; Plow, Edward F; Byzova, Tatiana V

2007-09-14

The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.

Hoverfly locomotor activity is resilient to external influence and intrinsic factors.

PubMed

Thyselius, Malin; Nordström, Karin

2016-01-01

Hoverflies are found across the globe, with approximately 6000 species described worldwide. Many hoverflies are being used in agriculture and some are emerging as model species for laboratory experiments. As such it is valuable to know more about their activity. Like many other dipteran flies, Eristalis hoverflies have been suggested to be strongly diurnal, but this is based on qualitative visualization by human observers. To quantify how hoverfly activity depends on internal and external factors, we here utilize a locomotor activity monitoring system. We show that Eristalis hoverflies are active during the entire light period when exposed to a 12 h light:12 h dark cycle, with a lower activity if exposed to light during the night. We show that the hoverflies' locomotor activity is stable over their lifetime and that it does not depend on the diet provided. Surprisingly, we find no difference in activity between males and females, but the activity is significantly affected by the sex of an accompanying conspecific. Finally, we show that female hoverflies are more resilient to starvation than males. In summary, Eristalis hoverflies are resilient to a range of internal and external factors, supporting their use in long-term laboratory experiments.

[Family factors influence active commuting to school in Spanish children].

PubMed

Rodríguez-López, Carlos; Villa-González, Emilio; Pérez-López, Isaac J; Delgado-Fernández, Manuel; Ruiz, Jonatan R; Chillón, Palma

2013-01-01

Active commuting to school is associated to higher levels of physical activity among children. Family factors may influence on this behaviour. The objective was to analyze the association between parents' occupational activity and parents' mode of commuting to work with the mode of commuting of their children. A total of 721 families from 4 primary schools in the province of Granada participated in this study. Families reported a questionnaire about mode of commuting of their children, parents' occupational activity and mode of commuting to work, distance and travel time to school. Associations between family's occupational activity and mode of commuting to work with mode of commuting to school of their children were examined using binary logistic regression analysis adjusting for age and children's distance to school. Children whose parents did not work used to engage in higher levels of active commuting to school than those whose parents worked (p = 0,023; OR: 2,67; 95% CI: 1,14-6,23). Children whose parents used to commute actively to work used to engage in higher levels of active commuting to school than those whose parents both used passive modes of commuting to work (p = 0,014; OR: 6,30; 95% CI: 1,45-27,26). Family factors are related to mode of commuting to school in children: Unemployed families and employed families where parent are active commuters to work are more used to have children that commuted to school using active modes. Copyright © AULA MEDICA EDICIONES 2013. Published by AULA MEDICA. All rights reserved.

Basic Fibroblast Growth Factor Activates Serum Response Factor Gene Expression by Multiple Distinct Signaling Mechanisms

PubMed Central

Spencer, Jeffrey A.; Major, Michael L.; Misra, Ravi P.

1999-01-01

Serum response factor (SRF) plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. It is a key regulator of many cellular early response genes which are believed to be involved in cell growth and differentiation. The mechanism by which SRF activates transcription in response to mitogenic agents has been extensively studied; however, significantly less is known about regulation of the SRF gene itself. Previously, we identified distinct regulatory elements in the SRF promoter that play a role in activation, including a consensus ETS domain binding site, a consensus overlapping Sp/Egr-1 binding site, and two SRF binding sites. We further showed that serum induces SRF by a mechanism that requires an intact SRF binding site, also termed a CArG box. In the present study we demonstrate that in response to stimulation of cells by a purified growth factor, basic fibroblast growth factor (bFGF), the SRF promoter is upregulated by a complex pathway that involves at least two independent mechanisms: a CArG box-independent mechanism that is mediated by an ETS binding site, and a novel CArG box-dependent mechanism that requires both an Sp factor binding site and the CArG motifs for maximal stimulation. Our analysis indicates that the CArG/Sp element activation mechanism is mediated by distinct signaling pathways. The CArG box-dependent component is targeted by a Rho-mediated pathway, and the Sp binding site-dependent component is targeted by a Ras-mediated pathway. Both SRF and bFGF have been implicated in playing an important role in mediating cardiogenesis during development. The implications of our findings for SRF expression during development are discussed. PMID:10330138

[Physical activity and cardiovascular risk factors among Chilean young men and women].

PubMed

Arteaga, Antonio; Bustos, Patricia; Soto, Rodrigo; Velasco, Nicolás; Amigo, Hugo

2010-10-01

Physical activity (PA) has a protective role in cardiovascular diseases. To quantify PA in young adults and to correlate it with cardiovascular risk factors. A cross-sectional study was performed employing the international physical activity questionnaire (IPAQ), to measure the PA of 983 randomly selected young adults from Valparaiso region born between 1974 and 1978. Its results were associated with levels of obesity, insulin resistance and cardiovascular risk factors defined by the National Cholesterol Education Program-Adult Treatment Panel III (NCEP- ATP III) RESULTS: Mean physical activity among men and women was 3731 ± 3923 and 1360 ± 2303 METs-minutes/week, respectively (p < 0.001). Fifty percent of women and 21.5% of men had an insufficient level of physical activity (p < 0.001). Sixty percent of men and 23.4% of women had an intense level of physical activity (p < 0.001). There was an inverse association of physical activity and insulin resistance. A high physical activity was protective, specially among men, against a low HDL cholesterol level and high triglyceride levels with Odds Ratios of 0.59 (confidence interval (CI): 0,35-0.98) and 0.49 (CI: 0,27-0,87) respectively, after adjusting for body mass index and age. In this sample, men had higher levels of physical activity, that was protective against insulin resistance and the presence of cardiovascular risk factors.

Laterality of Brain Activation for Risk Factors of Addiction.

PubMed

Gordon, Harold W

2016-01-01

Laterality of brain activation is reported for tests of risk factors of addiction- impulsivity and craving-but authors rarely address the potential significance of those asymmetries. The purpose of this study is to demonstrate this laterality and discuss its relevance to cognitive and neurophysiological asymmetries associated with drug abuse vulnerability in order to provide new insights for future research in drug abuse. From published reports, brain areas of activation for two tests of response inhibition or craving for drugs of abuse were compiled from fMRI activation peaks and were tabulated for eight sections (octants) in each hemisphere. Percent asymmetries were calculated (R-L/R+L) across studies for each area. For impulsivity, most activation peaks favored the right hemisphere. Overall, the percent difference was 32% (Χ2 = 16.026; p < 0.0001) with the greater asymmetry for anterior peaks (46.8%; Χ(2) = 17.329; p < 0.0001). The asymmetries for cue-induced craving were opposite, favoring the left hemisphere by 6.7% (Χ(2) = 4.028; p < 0.05). The consistency of left asymmetry was found for almost all drugs. For nicotine, studies where subjects were not allowed to smoke (deprived) prior to measurement had the same left hemisphere activation but those who smoked (satiated) before the fMRI measure showed right asymmetry. Brain activation studies demonstrate different left/right hemispheric contributions for impulsivity versus craving-factors related to addiction. Failure to take laterality into consideration is a missed opportunity in designing studies and gaining insight into the etiology of drug abuse and pathways for treatment.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Jinqi; Cook, Aaron A.; Bergmeier, Wolfgang

The dynamic regulation of ERK1 and -2 (ERK1/2) is required for precise signal transduction controlling cell proliferation, differentiation, and survival. However, the underlying mechanisms regulating the activation of ERK1/2 are not completely understood. In this study, we show that phosphorylation of RasGRP2, a guanine nucleotide exchange factor (GEF), inhibits its ability to activate the small GTPase Rap1 that ultimately leads to decreased activation of ERK1/2 in cells. ERK2 phosphorylates RasGRP2 at Ser394 located in the linker region implicated in its autoinhibition. These studies identify RasGRP2 as a novel substrate of ERK1/2 and define a negative-feedback loop that regulates the BRaf–MEK–ERKmore » signaling cascade. This negative-feedback loop determines the amplitude and duration of active ERK1/2. -- Highlights: •ERK2 phosphorylates the guanine nucleotide exchange factor RasGRP2 at Ser394. •Phosphorylated RasGRP2 has decreased capacity to active Rap1b in vitro and in cells. •Phosphorylation of RasGRP2 by ERK1/2 introduces a negative-feedback loop into the BRaf-MEK-ERK pathway.« less

Analysis of the restricting factors of laser countermeasure active detection technology

NASA Astrophysics Data System (ADS)

Zhang, Yufa; Sun, Xiaoquan

2016-07-01

The detection effect of laser active detection system is affected by various kinds of factors. In view of the application requirement of laser active detection, the influence factors for laser active detection are analyzed. The mathematical model of cat eye target detection distance has been built, influence of the parameters of laser detection system and the environment on detection range and the detection efficiency are analyzed. Various parameters constraint detection performance is simulated. The results show that the discovery distance of laser active detection is affected by the laser divergence angle, the incident angle and the visibility of the atmosphere. For a given detection range, the laser divergence angle and the detection efficiency are mutually restricted. Therefore, in view of specific application environment, it is necessary to select appropriate laser detection parameters to achieve optimal detection effect.

Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease

PubMed Central

Naranjo, José R.; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M.; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C.; Arrabal, María D.; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt

2016-01-01

Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD. PMID:26752648

78 FR 46418 - Proposed Information Collection (Obligation To Report Factors Affecting Entitlement) Activity...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-07-31

... (Obligation To Report Factors Affecting Entitlement) Activity; Comment Request AGENCY: Veterans Benefits... use of other forms of information technology. Title: Obligation to Report Factors Affecting... entitlement factors. Individual factors such as income, marital status, and the beneficiary's number of...

75 FR 62634 - Proposed Information Collection (Obligation to Report Factors Affecting Entitlement) Activity...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-12

... (Obligation to Report Factors Affecting Entitlement) Activity: Comment Request AGENCY: Veterans Benefits... use of other forms of information technology. Title: Obligation to Report Factors Affecting... entitlement factors. Individual factors such as income, marital status, and the beneficiary's number of...

In vitro effects of recombinant activated factor VII on thrombin generation and coagulation following inhibition of platelet procoagulant activity by prasugrel.

PubMed

Mazzeffi, Michael; Szlam, Fania; Jakubowski, Joseph A; Tanaka, Kenichi A; Sugidachi, Atsuhiro; Levy, Jerrold H

2013-07-01

Prasugrel is a thienopyridyl P2Y12 antagonist with potent antiplatelet effects. At present, little is known about its effects on thrombin generation or what strategies may emergently reverse its anticoagulant effects. In the current study we evaluated whether recombinant activated factor VII may reverse prasugrel induced effects and increase thrombin generation in an in vitro model. The effect of prasugrel active metabolite, PAM (R-138727), was evaluated on platelet aggregation, thrombin generation, and rotational thromboelastometry parameters using blood from 20 healthy volunteers. Additionally, we evaluated the effects of adenosine diphosphate (ADP) and recombinant activated factor VII on restoring these parameters towards baseline values. PAM reduced maximum platelet aggregation and led to platelet disaggregation. It also decreased peak thrombin, increased lag time, and increased time to peak thrombin. Treatment with recombinant activated factor VII restored all three parameters of thrombin generation towards baseline. ADP decreased lag time and time to peak thrombin, but had no effect on peak thrombin. When recombinant activated factor VII and ADP were combined they had a greater effect on thrombin parameters than either drug alone. PAM also increased thromboelastometric clotting time and clot formation time, but had no effect on maximum clot firmness. Treatment with either recombinant activated factor VII or ADP restored these values towards baseline. Recombinant activated factor VII restores thrombin generation in the presence of PAM. In patients taking prasugrel with life-threatening refractory bleeding it has the potential to be a useful therapeutic approach. Additional clinical studies are needed to validate our findings. Copyright © 2013 Elsevier Ltd. All rights reserved.

Krüppel-like factor 4, a novel transcription factor regulates microglial activation and subsequent neuroinflammation.

PubMed

Kaushik, Deepak K; Gupta, Malvika; Das, Sulagna; Basu, Anirban

2010-10-15

Activation of microglia, the resident macrophages of the central nervous system (CNS), is the hallmark of neuroinflammation in neurodegenerative diseases and other pathological conditions associated with CNS infection. The activation of microglia is often associated with bystander neuronal death. Nuclear factor-κB (NF-κB) is one of the important transcription factors known to be associated with microglial activation which upregulates the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2) and other pro-inflammatory cytokines. Recent studies have focused on the role of Krüppel-like factor 4 (Klf4), one of the zinc-finger transcription factors, in mediating inflammation. However, these studies were limited to peripheral system and its role in CNS is not understood. Our studies focused on the possible role of Klf4 in mediating CNS inflammation. For in vitro studies, mouse microglial BV-2 cell lines were treated with 500 ng/ml Salmonella enterica lipopolysacchride (LPS). Brain tissues were isolated from BALB/c mice administered with 5 mg/kg body weight of LPS. Expressions of Klf4, Cox-2, iNOS and pNF-κB were evaluated using western blotting, quantitative real time PCR, and reverse transcriptase polymerase chain reactions (RT-PCRs). Klf4 knockdown was carried out using SiRNA specific for Klf4 mRNA and luciferase assays and electromobility shift assay (EMSA) were performed to study the interaction of Klf4 to iNOS promoter elements in vitro. Co-immunoprecipitation of Klf4 and pNF-κB was done in order to study a possible interaction between the two transcription factors. LPS stimulation increased Klf4 expression in microglial cells in a time- and dose-dependent manner. Knockdown of Klf4 resulted in decreased levels of the pro-inflammatory cytokines TNF-α, MCP-1 and IL-6, along with a significant decrease in iNOS and Cox-2 expression. NO production also decreased as a result of Klf4 knockdown. We found that Klf4 can potentially interact with

The world bank and the environment, exercise 1992. Annual report. Banco mundial y el medio ambiente, ejercicio de 1992

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1992-01-01

The report describes the Bank's activities in assisting countries with environmental management, such as programs to reduce poverty without exhausting natural resources. Included are assessments of the environmental impacts of projects supported by World Bank loans, and accounts of efforts to meet global environmental challenges with coordinated international action through the Global Environment Facility (GEF). Among the events in the period covered by the report are the affirmation of the World Bank's participation with United Nations organizations in the GEF and the Bank's administration of pilot programs aimed at finding solutions to such global environmental problems as pollution of internationalmore » waters, global warming, depletion of the Earth's ozone layer, and dwindling biological diversity; the United Nations Conference on Environment and Development; and the publication of the World Development Report 1992, with its theme of development and the environment.« less

Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morrison, W.J.

1988-01-01

The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF ormore » thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).« less

Cell cycle entry triggers a switch between two modes of Cdc42 activation during yeast polarization

PubMed Central

Witte, Kristen; Strickland, Devin; Glotzer, Michael

2017-01-01

Cell polarization underlies many cellular and organismal functions. The GTPase Cdc42 orchestrates polarization in many contexts. In budding yeast, polarization is associated with a focus of Cdc42•GTP which is thought to self sustain by recruiting a complex containing Cla4, a Cdc42-binding effector, Bem1, a scaffold, and Cdc24, a Cdc42 GEF. Using optogenetics, we probe yeast polarization and find that local recruitment of Cdc24 or Bem1 is sufficient to induce polarization by triggering self-sustaining Cdc42 activity. However, the response to these perturbations depends on the recruited molecule, the cell cycle stage, and existing polarization sites. Before cell cycle entry, recruitment of Cdc24, but not Bem1, induces a metastable pool of Cdc42 that is sustained by positive feedback. Upon Cdk1 activation, recruitment of either Cdc24 or Bem1 creates a stable site of polarization that induces budding and inhibits formation of competing sites. Local perturbations have therefore revealed unexpected features of polarity establishment. DOI: http://dx.doi.org/10.7554/eLife.26722.001 PMID:28682236

Motivational factors and stages of change for physical activity among college students in Amman, Jordan.

PubMed

Madanat, Hala; Merrill, Ray M

2006-01-01

The purpose of this study was to investigate physical activity levels across the five stages of change for physical activity and to identify motivational factors for physical activity according to these stages of change among college students in Amman, Jordan. Analyses were based on a cross-sectional survey of 431 students, with a mean age of 21.1 (SD=0.16) and 67.5% female. Based on the recommendation that physical activity requires at least 30 minutes of physical activity 3 or more days per week, men were more likely than women to classify themselves in later stages: 7.3% vs. 9.5% in the precontemplation stage, 17.4% vs. 14.7% in the contemplation stage, 50.0% vs. 63.5% in the preparation stage, 9.4% vs. 5.6% in the action stage, and 15.9% vs. 6.7% in the maintenance stage [X2(4) = 14.04, p = 0.0072]. Seven potential motivational items for physical activity were assessed using factor analysis: experience better self-worth, prevent chronic disease, relieve stress, stay in shape, longevity, recreation/fun, and social benefits. Two factor groupings were identified from these items. The first factor included the first five items, labeled as "Physical and Mental". The second factor included the last two items, labeled as "Social and Recreational." "Physical and Mental" items compared with "Social and Recreational" items were most likely to motivate physical activity across the stages of change for physical activity. The strongest motivator of physical activity was to stay in shape. The weakest motivator of physical activity was for social reasons. The influence of the intermediate motivational factors was slightly affected by the students' stage of change for physical activity. Motivators for physical activity did not differ according to sex. These results provide important information about the motivational factors for physical activity for college-aged students in Jordan that can be useful in developing effective physical activity intervention programs.

Psychosocial factors and theory in physical activity studies in minorities.

PubMed

Mama, Scherezade K; McNeill, Lorna H; McCurdy, Sheryl A; Evans, Alexandra E; Diamond, Pamela M; Adamus-Leach, Heather J; Lee, Rebecca E

2015-01-01

To summarize the effectiveness of interventions targeting psychosocial factors to increase physical activity (PA) among ethnic minority adults and explore theory use in PA interventions. Studies (N = 11) were identified through a systematic review and targeted African American/Hispanic adults, specific psychosocial factors, and PA. Data were extracted using a standard code sheet and the Theory Coding Scheme. Social support was the most common psychosocial factor reported, followed by motivational readiness, and self-efficacy, as being associated with increased PA. Only 7 studies explicitly reported using a theoretical framework. Future efforts should explore theory use in PA interventions and how integration of theoretical constructs, including psychosocial factors, increases PA.

Cross-Species Analyses Identify the BNIP-2 and Cdc42GAP Homology (BCH) Domain as a Distinct Functional Subclass of the CRAL_TRIO/Sec14 Superfamily

PubMed Central

Gupta, Anjali Bansal; Wee, Liang En; Zhou, Yi Ting; Hortsch, Michael; Low, Boon Chuan

2012-01-01

The CRAL_TRIO protein domain, which is unique to the Sec14 protein superfamily, binds to a diverse set of small lipophilic ligands. Similar domains are found in a range of different proteins including neurofibromatosis type-1, a Ras GTPase-activating Protein (RasGAP) and Rho guanine nucleotide exchange factors (RhoGEFs). Proteins containing this structural protein domain exhibit a low sequence similarity and ligand specificity while maintaining an overall characteristic three-dimensional structure. We have previously demonstrated that the BNIP-2 and Cdc42GAP Homology (BCH) protein domain, which shares a low sequence homology with the CRAL_TRIO domain, can serve as a regulatory scaffold that binds to Rho, RhoGEFs and RhoGAPs to control various cell signalling processes. In this work, we investigate 175 BCH domain-containing proteins from a wide range of different organisms. A phylogenetic analysis with ∼100 CRAL_TRIO and similar domains from eight representative species indicates a clear distinction of BCH-containing proteins as a novel subclass within the CRAL_TRIO/Sec14 superfamily. BCH-containing proteins contain a hallmark sequence motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs (‘h’ is large and hydrophobic residue and ‘s’ is small and weekly polar residue) and can be further subdivided into three unique subtypes associated with BNIP-2-N, macro- and RhoGAP-type protein domains. A previously unknown group of genes encoding ‘BCH-only’ domains is also identified in plants and arthropod species. Based on an analysis of their gene-structure and their protein domain context we hypothesize that BCH domain-containing genes evolved through gene duplication, intron insertions and domain swapping events. Furthermore, we explore the point of divergence between BCH and CRAL-TRIO proteins in relation to their ability to bind small GTPases, GAPs and GEFs and lipid ligands. Our study suggests a need for a more extensive analysis of previously uncharacterized BCH, �

Up-regulation of the RhoA/Rho-kinase signaling pathway in corpus cavernosum from endothelial nitric-oxide synthase (NOS), but not neuronal NOS, null mice.

PubMed

Priviero, Fernanda B M; Jin, Li-Ming; Ying, Zhekang; Teixeira, Cleber E; Webb, R Clinton

2010-04-01

We tested the hypothesis that the basal release of nitric oxide (NO) from endothelial cells modulates contractile activity in the corpus cavernosum (CC) via inhibition of the RhoA/Rho-kinase signaling pathway. Cavernosal strips from wild-type (WT), endothelial nitric-oxide synthase knockout [eNOS(-/-)], and neuronal nitric-oxide synthase knockout [nNOS(-/-)] mice were mounted in myographs, and isometric force was recorded. mRNA and protein expression of key molecules in the RhoA/Rho-kinase pathway were analyzed by real-time polymerase chain reaction and Western blot, respectively. The cGMP levels were determined. The Rho-kinase inhibitors (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl] homopiperazine (H-1152) reduced cavernosal contractions evoked by phenylephrine or electrical field stimulation (EFS) in a concentration-dependent manner, although this inhibition was less effective in tissues from eNOS(-/-) mice. Y-27632 enhanced relaxations induced by sodium nitroprusside, EFS, and NO (administered as acidified NaNO2) without affecting the cGMP content of the cavernosal strips. This enhancement was less prominent in CC from eNOS(-/-). The protein expression of RhoA, Rho-guanine dissociation inhibitor, and Rho-kinase beta did not differ among the strains. However, in eNOS(-/-) CC, the protein expression of Rho-kinase alpha and both mRNA and protein expression of p115-Rho-associated guanine exchange factor (RhoGEF), PDZ-RhoGEF, and leukemia-associated RhoGEF were up-regulated. Phosphorylation of MYPT1 at Thr696 was higher in tissues from eNOS(-/-) mice. A high concentration of Y-27632 significantly enhanced NO release in CC stimulated by EFS. These results suggest a basal release of NO from endothelial cells, which inhibits contractions mediated by the RhoA/Rho-kinase pathway and modulates the expression of proteins related to this pathway in mouse CC. It indicates that

[Factors influencing research activity of Andalusian nurses and improvement strategies].

PubMed

López Alonso, Sergio R; Gálvez González, María; Amezcua, Manuel

2013-04-01

To identify factors influencing research activity of Andalusian nurses and to find improvement strategies. Qualitative research using SWOT analysis (weaknesses, threats, strengths, opportunities). Nurses were selected deliberately in eight groups according to predetermined criteria. Analysis included categorization and relationship of factors and strategies. 81 participants were included in groups of 7-12 range. 45 categories were identified with 212 factors: 12 weaknesses (50 factors), 10 strengths (44 factors), 12 threats (68 factors) and 11 opportunities (50 factors). In addition, 32 categories were identified with 53 strategies: 14 categories of W-T strategies (42 strategies), 3 categories of S-T strategies (11 strategies), 5 categories of W-O strategies (13 strategies) and 10 categories of S-O strategies (41 strategies). Nurses identified numerous factors, mainly threats. The strategies are focused on W-T but they also suggest many but weak 5-0 strategies due to the low potential of the opportunities and strengths perceived.

Factors related to physical activity adherence in women: review and suggestions for future research.

PubMed

White, Jennifer L; Ransdell, Lynda B; Vener, Jamie; Flohr, Judith A

2005-01-01

Approximately 50 percent of individuals who start an exercise program withdraw within 6 months. Thus, many individuals withdraw before health benefits have been realized. This is a disconcerting statistic considering the well known benefits of physical activity for decreasing risk of hypokinetic diseases and improving quality of life. The literature has suggested a plethora of factors to increase the number of individuals who initiate a physical activity program. However, little is known about the factors that keep women exercising-otherwise known as exercise adherence. The purpose of this paper is to: (a) systematically review the quantitative literature to discern the major factors contributing to adherence to physical activity in women and men and make recommendations for specific gender-based considerations that are important when designing PA interventions for women, and (b) suggest areas of future research related to increasing adherence to physical activity in women. Key factors reviewed in this paper may be useful in developing efficacious physical activity programs for women.

Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1

PubMed Central

Belov, George A.; Kovtunovych, Gennadiy; Jackson, Catherine L.; Ehrenfeld, Ellie

2010-01-01

Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the Arf GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to BFA, an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism. PMID:20497182

Serum factors and clinical characteristics associated with serum E-Screen activity

PubMed Central

Wang, Jue; Trentham-Dietz, Amy; Hemming, Jocelyn D. C.; Hedman, Curtis J.; Sprague, Brian L.

2013-01-01

Background The E-Screen bioassay can measure the mitogenicity of human serum and thus may be useful as a biomarker in epidemiologic studies of breast cancer. While the assay’s MCF-7 cells are known to proliferate in response to estrogen, the specific determinants of variation in E-Screen activity in human serum samples are poorly understood. We sought to identify serum molecules and patient characteristics associated with serum E-Screen activity among postmenopausal women. Methods Postmenopausal women (N=219) aged 55–70 with no history of postmenopausal hormone use or breast cancer completed a questionnaire and provided a blood sample. Serum was analyzed for E-Screen activity and a variety of molecules including sex hormones, growth factors, and environmental chemicals. Stepwise selection procedures were used to identify correlates of E-Screen activity. Results Serum samples from all women had detectable E-Screen activity, with a median estradiol equivalents value of 0.027 ng/mL and interquartile range of 0.018–0.036 ng/mL. In the final multivariable-adjusted model, serum E-Screen activity was positively associated with serum estradiol, estrone, IGFBP-3, and testosterone levels (p<0.05), as well as body mass index (p=0.03). Serum E-Screen activity was lower among women with higher SHBG (p<0.0001) and progesterone levels (p=0.03). Conclusion Serum E-Screen activity varies according to levels of endogenous estrogens and other serum molecules. Obesity appears to confer additional serum mitogenicity beyond its impact on the measured hormones and growth factors. Impact By capturing mitogenicity due to a variety of patient and serum factors, the E-Screen may provide advantages for use as a biomarker in breast cancer studies. PMID:23588007

Influences of lifestyle factors on cardiac autonomic nervous system activity over time.

PubMed

Hu, Mandy Xian; Lamers, Femke; de Geus, Eco J C; Penninx, Brenda W J H

2017-01-01

Physical activity, alcohol use and smoking might affect cardiovascular disease through modifying autonomic nervous system (ANS) activity. We investigated: 1) whether there are consistent relationships between lifestyle factors and cardiac ANS activity over time, and 2) whether 2-year changes in lifestyle factors relate to 2-year changes in cardiac activity. Baseline (n=2618) and 2-year follow-up (n=2010) data of the Netherlands Study of Depression and Anxiety was combined. Baseline data was collected in the Netherlands from 2004-2007. Lifestyle factors were habitual physical activity, frequency of sport activities, alcohol use, and smoking. Indicators of cardiac activity were heart rate (HR), respiratory sinus arrhythmia (RSA) and pre-ejection period (PEP) (100min of registration). The results showed that high physical activity (-1.8beats/min compared to low activity), high frequency of sport activities ('couple of times/week': -2.5beats/min compared to 'almost never') and mild/moderate alcohol use (-1.2beats/min compared to non-drinking) were related to low HR. Heavy smoking was related to high HR (>30cigarettes/day: +5.1beats/min compared to non-smoking). High frequency of sport activities was associated with high RSA ('couple of times/week':+1.7ms compared to 'almost never') and moderate smoking with longer PEP (11-20cigarettes/day: +2.8ms compared to non-smoking). Associations were consistent across waves. Furthermore, 2-year change in frequency of sport activities and number of smoked cigarettes/day was accompanied by 2-year change in HR (β=-0.076 and β=0.101, respectively) and RSA (β=0.046 and β=-0.040, respectively). Our findings support consistent effects of lifestyle on HR and parasympathetic activity in the expected direction. Cardiac autonomic dysregulation may be partly mediating the relationship between lifestyle and subsequent cardiovascular health. Copyright © 2016 Elsevier Inc. All rights reserved.

Physical activity practice and associated factors in adolescents in Northeastern Brazil.

PubMed

Farias Júnior, José Cazuza de; Lopes, Adair da Silva; Mota, Jorge; Hallal, Pedro Curi

2012-06-01

To estimate the proportion of physically active adolescents and identify associated factors. The sample was composed of 2,874 high school students (public and private schools) aged 14 to 19 years in the city of João Pessoa, Northeastern Brazil. Physical activity level was measured by means of a questionnaire and considered physically active if > 300 minutes/week. Sociodemographic variables were analyzed, as well as nutritional status, sedentary behavior, self-report of health status and participation in physical education classes. The prevalence ratio was used as association measure, estimated by means of Poisson regression. The prevalence of physical activity was 50.2% (95%CI: 47.3;53.1). Male adolescents were more physically active than female adolescents (66.3% vs. 38.5%; p<0.001). The factors directly associated with practice of physical activity were: father's higher level of schooling for the male sex and mother's higher level of schooling for the female sex, positive self-reported health status, and participation in physical education classes. The majority of the adolescents was classified as physically active, particularly the male youths. Adolescents with parents whose level of schooling was higher, with positive health status perception, and who participated in physical education classes were more likely to be physically active.

Assessing public health policy approaches to level-up the gradient in health inequalities: the Gradient Evaluation Framework.

PubMed

Davies, J K; Sherriff, N S

2014-03-01

This paper seeks to introduce and analyse the development of the Gradient Evaluation Framework (GEF) to facilitate evaluation of policy actions for their current or future use in terms of their 'gradient friendliness'. In particular, this means their potential to level-up the gradient in health inequalities by addressing the social determinants of health and thereby reducing decision-makers' chances of error when developing such policy actions. A qualitative developmental study to produce a policy-based evaluation framework. The scientific basis of GEF was developed using a comprehensive consensus-building process. This process followed an initial narrative review, based on realist review principles, which highlighted the need for production of a dedicated evaluation framework. The consensus-building process included expert workshops, a pretesting phase, and external peer review, together with support from the Gradient project Scientific Advisory Group and all Gradient project partners, including its Project Steering Committee. GEF is presented as a flexible policy tool resulting from a consensus-building process involving experts from 13 European countries. The theoretical foundations which underpin GEF are discussed, together with a range of practical challenges. The importance of systematic evaluation at each stage of the policy development and implementation cycle is highlighted, as well as the socio-political context in which policy actions are located. GEF offers potentially a major contribution to the public health field in the form of a practical, policy-relevant and common frame of reference for the evaluation of public health interventions that aim to level-up the social gradient in health inequalities. Further research, including the need for practical field testing of GEF and the exploration of alternative presentational formats, is recommended. Copyright © 2013 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.

The Model of Children's Active Travel (M-CAT): a conceptual framework for examining factors influencing children's active travel.

PubMed

Pont, Karina; Ziviani, Jenny; Wadley, David; Abbott, Rebecca

2011-06-01

The current decline in children's participation in physical activity has attracted the attention of those concerned with children's health and wellbeing. A sustainable approach to ensuring children engage in adequate amounts of physical activity is to support their involvement in incidental activity such as active travel (AT), which includes walking or riding a bicycle to or from local destinations, such as school or a park. Understanding how we can embed physical activity into children's everyday occupational roles is a way in which occupational therapists can contribute to this important health promotion agenda. To present a simple, coherent and comprehensive framework as a means of examining factors influencing children's AT. Based on current literature, this conceptual framework incorporates the observable environment, parents' perceptions and decisions regarding their children's AT, as well as children's own perceptions and decisions regarding AT within their family contexts across time. The Model of Children's Active Travel (M-CAT) highlights the complex and dynamic nature of factors impacting the decision-making process of parents and children in relation to children's AT. The M-CAT offers a way forward for researchers to examine variables influencing active travel in a systematic manner. Future testing of the M-CAT will consolidate understanding of the factors underlying the decision-making process which occurs within families in the context of their communities. © 2010 The Authors. Australian Occupational Therapy Journal © 2010 Australian Association of Occupational Therapists.

Physical activity, psychosocial and perceived environmental factors in adolescents from Northeast Brazil.

PubMed

Farias Júnior, José Cazuza de; Reis, Rodrigo Siqueira; Hallal, Pedro Curi

2014-05-01

The aim of this study was to evaluate the association between levels of physical activity, psychosocial and perceived environmental factors in adolescents from Northeastern Brazil. A cross-sectional epidemiologic study was conducted with 2,859 adolescents enrolled in secondary schools (57.8% females; mean = 16.5 years; SD = 1.2) in the city of João Pessoa, Paraíba State, Brazil. The following physical activity correlates were measured: attitude, self-efficacy, social support from friends and parents, and perceived environmental characteristics. Physical activity was measured using a questionnaire. Multivariable ordinal logistic regression with proportional odds model analysis showed that the following factors are positively related to physical activity levels in adolescents: attitude, self-efficacy, as well as social support from parents and friends. Physical activity intervention programs should increase self-efficacy and social support from parents and friends, as well as a positive attitude toward physical activity.

Factors associated to quality of life in active elderly.

PubMed

Alexandre, Tiago da Silva; Cordeiro, Renata Cereda; Ramos, Luiz Roberto

2009-08-01

To analyze whether quality of life in active, healthy elderly individuals is influenced by functional status and sociodemographic characteristics, as well as psychological parameters. Study conducted in a sample of 120 active elderly subjects recruited from two open universities of the third age in the cities of São Paulo and São José dos Campos (Southeastern Brazil) between May 2005 and April 2006. Quality of life was measured using the abbreviated Brazilian version of the World Health Organization Quality of Live (WHOQOL-bref) questionnaire. Sociodemographic, clinical and functional variables were measured through crossculturally validated assessments by the Mini Mental State Examination, Geriatric Depression Scale, Functional Reach, One-Leg Balance Test, Timed Up and Go Test, Six-Minute Walk Test, Human Activity Profile and a complementary questionnaire. Simple descriptive analyses, Pearson's correlation coefficient, Student's t-test for non-related samples, analyses of variance, linear regression analyses and variance inflation factor were performed. The significance level for all statistical tests was set at 0.05. Linear regression analysis showed an independent correlation without colinearity between depressive symptoms measured by the Geriatric Depression Scale and four domains of the WHOQOL-bref. Not having a conjugal life implied greater perception in the social domain; developing leisure activities and having an income over five minimum wages implied greater perception in the environment domain. Functional status had no influence on the Quality of Life variable in the analysis models in active elderly. In contrast, psychological factors, as assessed by the Geriatric Depression Scale, and sociodemographic characteristics, such as marital status, income and leisure activities, had an impact on quality of life.

Laterality of Brain Activation for Risk Factors of Addiction

PubMed Central

Gordon, Harold W.

2016-01-01

Background: Laterality of brain activation is reported for tests of risk factors of addiction - impulsivity and craving - but authors rarely address the potential significance of those asymmetries. Objective: The purpose of this study is to demonstrate this laterality and discuss its relevance to cognitive and neurophysiological asymmetries associated with drug abuse vulnerability in order to provide new insights for future research in drug abuse. Method: From published reports, brain areas of activation for two tests of response inhibition or craving for drugs of abuse were compiled from fMRI activation peaks and were tabulated for eight sections (octants) in each hemisphere. Percent asymmetries were calculated (R-L/R+L) across studies for each area. Results: For impulsivity, most activation peaks favored the right hemisphere. Overall, the percent difference was 32% (Χ2 = 16.026; p < 0.0001) with the greater asymmetry for anterior peaks (46.8%; Χ2 = 17.329; p < 0.0001). The asymmetries for cue-induced craving were opposite, favoring the left hemisphere by 6.7% (Χ2 = 4.028; p < 0.05). The consistency of left asymmetry was found for almost all drugs. For nicotine, studies where subjects were not allowed to smoke (deprived) prior to measurement had the same left hemisphere activation but those who smoked (satiated) before the fMRI measure showed right asymmetry. Conclusion: Brain activation studies demonstrate different left/right hemispheric contributions for impulsivity versus craving - factors related to addiction. Failure to take laterality into consideration is a missed opportunity in designing studies and gaining insight into the etiology of drug abuse and pathways for treatment. PMID:26674074

Newly diagnosed congenital factor VII deficiency and utilization of recombinant activated factor VII (NovoSeven(®)).

PubMed

Bartosh, Nicole S; Tomlin, Tara; Cable, Christian; Halka, Kathleen

2013-01-01

This case report presents a newly diagnosed congenital factor VII deficiency treated with recombinant activated factor VII (rFVIIa). Congenital factor VII deficiency is a rare autosomal-recessive bleeding disorder that occurs in fewer than 1/500,000 persons. Its presentation can vary from epistaxis to hemarthroses and severe central nervous system bleeding, and correlates poorly with factor VII levels. Our patient had not had a significant hemostatic challenge prior to his presentation and therefore never had any symptomatology suggestive of this disease. He was treated with rFVIIa, and was able to undergo repair of his fractures without bleeding. A 19-year-old African-American male presented to the emergency room after an altercation that resulted in significant trauma. He sustained bilateral mandibular angle fractures and orbital floor fractures, requiring urgent surgical correction. On initial evaluation, he was noted to have a prolonged prothrombin time of 40.1 seconds, with an International Normalized Ratio of 4.0, a normal activated partial thromboplastin time of 29.9 seconds, and a platelet count of 241. After receiving vitamin K and fresh frozen plasma, he was taken to the operating room for a temporary rigid maxillomandibular fixation. A 1:1 mixing study with normal plasma corrected the prothrombin time (decreasing from 40.7 to 14.7 seconds) and a factor VII assay revealed 5% of the normal factor VII level. The patient was diagnosed with congenital factor VII deficiency. Due to his coagulopathy and the extensive surgical correction needed, rFVIIa was administered and surgery was accomplished without hemorrhagic sequelae. This case report and review describes a rare congenital disease, the history of rFVIIa use, and its mechanism. rFVIIA use in our patient provided a treatment option that allowed the necessary surgical correction, but further prospective studies on dose optimization would ensure adequate dosing with minimal risk of severe side effects.

Factors that influence physical activity for pregnant and postpartum women and implications for primary care.

PubMed

Doran, Frances; Davis, Kierrynn

2011-01-01

Many pregnant women and women of child-bearing age do not engage in the recommended levels of physical activity despite the well known benefits. Pregnancy and the postpartum period can be a time when inactivity actually increases. Women who experience gestational diabetes mellitus (GDM) during their pregnancy are often advised to become more active in order to ameliorate their increased risk of developing type 2 diabetes. Health professionals have an influential role in promoting physical activity, which would be enhanced with an understanding of the factors that positively and negatively influence women's participation in physical activity during pregnancy and in the postpartum period. This research sought to explore these factors with pregnant and postpartum women including those who had experienced GDM and the attention given to physical activity during pregnancy. A survey was developed after a critical review of factors identified from previous studies. Women were recruited from the antenatal clinic, community health centres and the local media. Results from 72 women are reported from a predominately well educated, Caucasian population. Overall, the results were confirmatory of factors previously identified. Lack of child care, time constraints, no time and feeling unwell during pregnancy hindered activity and factors that facilitated activity included family support, enjoyment of activity and to prevent later health problems. It was also found that non-GDM women are given minimal advice about exercise during pregnancy. A checklist has been developed for health professionals, in partnership with women, to direct attention to the factors that enable and hinder participation in physical activity during and after pregnancy.

Psychosocial Factors and Theory in Physical Activity Studies in Minorities

PubMed Central

Mama, Scherezade K.; McNeill, Lorna H.; McCurdy, Sheryl A.; Evans, Alexandra E.; Diamond, Pamela M.; Adamus-Leach, Heather J.; Lee, Rebecca E.

2015-01-01

Objectives To summarize the effectiveness of interventions targeting psychosocial factors to increase physical activity (PA) among ethnic minority adults and explore theory use in PA interventions. Methods Studies (N = 11) were identified through a systematic review and targeted African American/Hispanic adults, specific psychosocial factors, and PA. Data were extracted using a standard code sheet and the Theory Coding Scheme. Results Social support was the most common psychosocial factor reported, followed by motivational readiness, and self-efficacy, as being associated with increased PA. Only 7 studies explicitly reported using a theoretical framework. Conclusions Future efforts should explore theory use in PA interventions and how integration of theoretical constructs, including psychosocial factors, increases PA. PMID:25290599

Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase

PubMed Central

Jung, Gyung Ah; Shin, Bong Shik; Jang, Yeon Sue; Sohn, Jae Bum; Woo, Seon Rang; Kim, Jung Eun; Choi, Go; Lee, Kyung-Mi; Min, Bon Hong

2011-01-01

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21Cip/WAF1 activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21Cip/WAF1 short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway. PMID:21778808

Factors Associated with Leisure Activity among Young Adults with Developmental Disabilities

ERIC Educational Resources Information Center

Van Naarden Braun, Kim; Yeargin-Allsopp, Marshalyn; Lollar, Donald

2006-01-01

The framework of the International Classification of Functioning, Disability, and Health (ICF) was applied to examine the factors associated with childhood impairment and leisure activity. Information on leisure activity was obtained using a structured questionnaire from a population-based cohort of young adults with childhood impairment. The…

Activated Monocytes Enhance Platelet-Driven Contraction of Blood Clots via Tissue Factor Expression.

PubMed

Peshkova, Alina D; Le Minh, Giang; Tutwiler, Valerie; Andrianova, Izabella A; Weisel, John W; Litvinov, Rustem I

2017-07-11

Platelet-driven reduction in blood clot volume (clot contraction or retraction) has been implicated to play a role in hemostasis and thrombosis. Although these processes are often linked with inflammation, the role of inflammatory cells in contraction of blood clots and thrombi has not been investigated. The aim of this work was to study the influence of activated monocytes on clot contraction. The effects of monocytes were evaluated using a quantitative optical tracking methodology to follow volume changes in a blood clot formed in vitro. When a physiologically relevant number of isolated human monocytes pre-activated with phorbol-12-myristate-13-acetate (PMA) were added back into whole blood, the extent and rate of clot contraction were increased compared to addition of non-activated cells. Inhibition of tissue factor expression or its inactivation on the surface of PMA-treated monocytes reduced the extent and rate of clot contraction back to control levels with non-activated monocytes. On the contrary, addition of tissue factor enhanced clot contraction, mimicking the effects of tissue factor expressed on the activated monocytes. These data suggest that the inflammatory cells through their expression of tissue factor can directly affect hemostasis and thrombosis by modulating the size and density of intra- and extravascular clots and thrombi.

Framework for power and activity factor allocation in a multiclass CDMA system

NASA Astrophysics Data System (ADS)

Wu, Xinzhou; Srikant, Rayadurgam

2001-07-01

We consider a multimedia CDMA uplink where there are multiple classes of users with different Quality-of-Service (QoS) requirements. Each user is modeled as an ON-OFF source, where in the ON state, the user transmits a fixed number of bits in each time slot and in the OFF state, the user is silent. The probability of being in the ON state, known as the activity factor, could be different for different users. Assuming a constant channel gain, we first characterize the set of transmit power levels, activity factors and number of users in each class that can be supported by a system with a given spreading gain under the constraint that each user's QoS requirement must be met. Using this characterization, we then present a utility function-based algorithm for choosing the activity factors of elastic users in the network.

Factors influencing hemolytic activity of venom from the jellyfish Rhopilema esculentum Kishinouye.

PubMed

Yu, Huahua; Li, Cuiping; Li, Ronggui; Xing, Ronge; Liu, Song; Li, Pengcheng

2007-07-01

In this study, hemolytic activity of venom from the jellyfish Rhopilema esculentum Kishinouye and some factors affecting it were assayed. The HU(50) of R. esculentum full venom (RFV) against chicken erythrocytes was 3.40 microg/ml and a Hill coefficient value was 1.73 suggesting at least two molecules participated in hemolytic activity. The hemolytic activity of RFV was affected by some chemical and physical factors such as divalent cations, EDTA, (NH(4))(2)SO(4), pH and temperature. In the presence of Mg(2+), Cu(2+), Zn(2+), Fe(2+), Ca(2+) (>or=2 mM), Mn(2+) ((>or=1 mM), EDTA ((>or=2 mM) and (NH(4))(2)SO(4), the hemolytic activity of RFV was reduced. RFV had strong hemolytic activity at the pH 6-10 and the hemolytic ratios were 0.95-1.19. Hemolytic activity was temperature-sensitive and when RFV was pre-incubated at temperatures over 40 degrees C, it was sharply reduced.

A specific colorimetric assay for measuring transglutaminase 1 and factor XIII activities.

PubMed

Hitomi, Kiyotaka; Kitamura, Miyako; Alea, Mileidys Perez; Ceylan, Ismail; Thomas, Vincent; El Alaoui, Saïd

2009-11-15

Transglutaminase (TGase) is an enzyme that catalyzes both isopeptide cross-linking and incorporation of primary amines into proteins. Eight TGases have been identified in humans, and each of these TGases has a unique tissue distribution and physiological significance. Although several assays for TGase enzymatic activity have been reported, it has been difficult to establish an assay for discriminating each of these different TGase activities. Using a random peptide library, we recently identified the preferred substrate sequences for three major TGases: TGase 1, TGase 2, and factor XIII. In this study, we use these substrates in specific tests for measuring the activities of TGase 1 and factor XIII.

Salicylates inhibit flavivirus replication independently of blocking nuclear factor kappa B activation.

PubMed

Liao, C L; Lin, Y L; Wu, B C; Tsao, C H; Wang, M C; Liu, C I; Huang, Y L; Chen, J H; Wang, J P; Chen, L K

2001-09-01

Flaviviruses comprise a positive-sense RNA genome that replicates exclusively in the cytoplasm of infected cells. Whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. Flavivirus infections quickly activate nuclear factor kappa B (NF-kappaB), and salicylates have been shown to inhibit NF-kappaB activation. In this study, we investigated whether salicylates suppress flavivirus replication and virus-induced apoptosis in cultured cells. In a dose-dependent inhibition, we found salicylates within a range of 1 to 5 mM not only restricted flavivirus replication but also abrogated flavivirus-triggered apoptosis. However, flavivirus replication was not affected by a specific NF-kappaB peptide inhibitor, SN50, and a proteosome inhibitor, lactacystin. Flaviviruses also replicated and triggered apoptosis in cells stably expressing IkappaBalpha-DeltaN, a dominant-negative mutant that antagonizes NF-kappaB activation, as readily as in wild-type BHK-21 cells, suggesting that NF-kappaB activation is not essential for either flavivirus replication or flavivirus-induced apoptosis. Salicylates still diminished flavivirus replication and blocked apoptosis in the same IkappaBalpha-DeltaN cells. This inhibition of flaviviruses by salicylates could be partially reversed by a specific p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Together, these results show that the mechanism by which salicylates suppress flavivirus infection may involve p38 MAP kinase activity but is independent of blocking the NF-kappaB pathway.

Activation of Rho GTPases by Cytotoxic Necrotizing Factor 1 Induces Macropinocytosis and Scavenging Activity in Epithelial Cells

PubMed Central

Fiorentini, Carla; Falzano, Loredana; Fabbri, Alessia; Stringaro, Annarita; Logozzi, Mariaantonia; Travaglione, Sara; Contamin, Stéphanette; Arancia, Giuseppe; Malorni, Walter; Fais, Stefano

2001-01-01

Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function. It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors. In this case, the internalized material recycles back to the cell surface. We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes. In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation. Taken together, these findings indicate that CNF1-induced “switching on” of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes. The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages. PMID:11452003

Platelet-Derived Short-Chain Polyphosphates Enhance the Inactivation of Tissue Factor Pathway Inhibitor by Activated Coagulation Factor XI.

PubMed

Puy, Cristina; Tucker, Erik I; Ivanov, Ivan S; Gailani, David; Smith, Stephanie A; Morrissey, James H; Gruber, András; McCarty, Owen J T

2016-01-01

Factor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin. Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma. Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP.

Constitutive activation of NF-kappa B and secretion of interleukin-8 induced by the G protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus involve G alpha(13) and RhoA.

PubMed

Shepard, L W; Yang, M; Xie, P; Browning, D D; Voyno-Yasenetskaya, T; Kozasa, T; Ye, R D

2001-12-07

The Kaposi's sarcoma herpesvirus (KSHV) open reading frame 74 encodes a G protein-coupled receptor (GPCR) for chemokines. Exogenous expression of this constitutively active GPCR leads to cell transformation and vascular overgrowth characteristic of Kaposi's sarcoma. We show here that expression of KSHV-GPCR in transfected cells results in constitutive transactivation of nuclear factor kappa B (NF-kappa B) and secretion of interleukin-8, and this response involves activation of G alpha(13) and RhoA. The induced expression of a NF-kappa B luciferase reporter was partially reduced by pertussis toxin and the G beta gamma scavenger transducin, and enhanced by co-expression of G alpha(13) and to a lesser extent, G alpha(q). These results indicate coupling of KSHV-GPCR to multiple G proteins for NF-kappa B activation. Expression of KSHV-GPCR led to stress fiber formation in NIH 3T3 cells. To examine the involvement of the G alpha(13)-RhoA pathway in KSHV-GPCR-mediated NF-kappa B activation, HeLa cells were transfected with KSHV-GPCR alone and in combination with the regulator of G protein signaling (RGS) from p115RhoGEF or a dominant negative RhoA(T19N). Both constructs, as well as the C3 exoenzyme from Clostritium botulinum, partially reduced NF-kappa B activation by KSHV-GPCR, and by a constitutively active G alpha(13)(Q226L). KSHV-GPCR-induced NF-kappa B activation is accompanied by increased secretion of IL-8, a function mimicked by the activated G alpha(13) but not by an activated G alpha(q)(Q209L). These results suggest coupling of KSHV-GPCR to the G alpha(13)-RhoA pathway in addition to other G proteins.

Elevated circulating soluble thrombomodulin activity, tissue factor activity and circulating procoagulant phospholipids: new and useful markers for pre-eclampsia?

PubMed

Rousseau, Aurélie; Favier, Rémi; Van Dreden, Patrick

2009-09-01

One of the most frequently proposed mechanisms for pre-eclampsia refers to uteroplacental thrombosis. However, the contribution of classical thrombotic risk factors remains questionable. The aims of this study were to investigate the activities of thrombomodulin, tissue factor and procoagulant phospholipids to assess endothelial cell injury in pregnant women with pre-eclampsia and to compare them with other classical markers of vascular injury and thrombotic risk. Using three new functional assays we studied the plasma levels of these new markers in 35 healthy women, 30 healthy pregnant women, and 35 women with pre-eclampsia. We found that plasma levels of thrombomodulin activity, tissue factor activity and procoagulant phospholipids were significantly elevated in women with pre-eclampsia versus normal pregnant and non-pregnant women. It is thus suggested that elevated levels of these parameters in pre-eclampsia may reflect vascular endothelium damage, and may be a more valuable biomarker than antigen for the assessment of endothelial damage in pre-eclampsia. The high increased levels of procoagulant phospholipids and tissue factor activities in pre-eclampsia could suggest that the procoagulant potential may be implicated in this complication and makes these markers very promising for the understanding, follow-up and therapeutic handling of complicated pregnancy.

Factors of physical activity among Chinese children and adolescents: a systematic review.

PubMed

Lu, Congchao; Stolk, Ronald P; Sauer, Pieter J J; Sijtsma, Anna; Wiersma, Rikstje; Huang, Guowei; Corpeleijn, Eva

2017-03-21

Lack of physical activity is a growing problem in China, due to the fast economic development and changing living environment over the past two decades. The aim of this review is to summarize the factors related to physical activity in Chinese children and adolescents during this distinct period of development. A systematic search was finished on Jan 10 th , 2017, and identified 2200 hits through PubMed and Web of Science. English-language published studies were included if they reported statistical associations between factors and physical activity. Adapted criteria from the Strengthening The Reporting of OBservational studies in Epidemiology (STROBE) statement and evaluation of the quality of prognosis studies in systematic reviews (QUIPS) were used to assess the risk of bias of the included studies. Related factors that were reported in at least three studies were summarized separately for children and adolescents using a semi-quantitative method. Forty two papers (published 2002-2016) were included. Most designs were cross-sectional (79%), and most studies used questionnaires to assess physical activity. Sample size was above 1000 in 18 papers (43%). Thirty seven studies (88%) showed acceptable quality by methodological quality assessment. Most studies reported a low level of physical activity. Boys were consistently more active than girls, the parental physical activity was positively associated with children and adolescents' physical activity, children in suburban/rural regions showed less activity than in urban regions, and, specifically in adolescents, self-efficacy was positively associated with physical activity. Family socioeconomic status and parental education were not associated with physical activity in children and adolescents. The studies included in this review were large but mostly of low quality in terms of study design (cross-sectional) and methods (questionnaires). Parental physical activity and self-efficacy are promising targets for future

Increased active von Willebrand factor during disease development in the aging diabetic patient population.

PubMed

Chen, Shuang Feng; Xia, Zuo Li; Han, Ji Ju; Wang, Yi Ting; Wang, Ji Yue; Pan, Shao Dong; Wu, Ya Ping; Zhang, Bin; Li, Guang Yao; Du, Jing Wei; Gao, Hen Qiang; de Groot, Philip G; de Laat, Bas; Hollestelle, Martine J

2013-02-01

Type 2 diabetes is known to cause endothelial activation resulting in the secretion of von Willebrand factor (VWF). We have shown that levels of VWF in a glycoprotein Ib-binding conformation are increased in specific clinical settings. The aim of the current study is to investigate whether active VWF levels increase during aging and the development of diabetes within the population of patients suffering from type 2 diabetes. Patients and controls were divided into two groups based on age: older and younger than 60 years of age. VWF antigen, VWF propeptide, VWF activation factor and total active VWF were measured. Patients older than 60 years of age had increased levels of total active VWF, VWF activation factor and VWF propeptide compared to younger patients and controls. All measured VWF parameters were associated with age in diabetic patients. Total active VWF and VWF propeptide correlated with the period of being diagnosed with diabetes. Regression analyses showed that especially the VWF activation factor was strongly associated with diabetes in patients older than 60 years of age. In conclusion, we found that the conformation of VWF could be involved in the disease process of diabetes and that the VWF in a glycoprotein Ib-binding conformation could play a role as risk marker during the development of diabetes in combination with an increase in age. Our study shows that the active quality of VWF was more important than the quantity.

Activation of Hypoxia-Inducible Factors Prevents Diabetic Nephropathy

PubMed Central

Nordquist, Lina; Friederich-Persson, Malou; Fasching, Angelica; Liss, Per; Shoji, Kumi; Nangaku, Masaomi; Hansell, Peter

2015-01-01

Hyperglycemia results in increased oxygen consumption and decreased oxygen tension in the kidney. We tested the hypothesis that activation of hypoxia-inducible factors (HIFs) protects against diabetes-induced alterations in oxygen metabolism and kidney function. Experimental groups consisted of control and streptozotocin-induced diabetic rats treated with or without chronic cobalt chloride to activate HIFs. We elucidated the involvement of oxidative stress by studying the effects of acute administration of the superoxide dismutase mimetic tempol. Compared with controls, diabetic rats displayed tissue hypoxia throughout the kidney, glomerular hyperfiltration, increased oxygen consumption, increased total mitochondrial leak respiration, and decreased tubular sodium transport efficiency. Diabetic kidneys showed proteinuria and tubulointerstitial damage. Cobalt chloride activated HIFs, prevented the diabetes-induced alterations in oxygen metabolism, mitochondrial leak respiration, and kidney function, and reduced proteinuria and tubulointerstitial damage. The beneficial effects of tempol were less pronounced after activation of HIFs, indicating improved oxidative stress status. In conclusion, activation of HIFs prevents diabetes-induced alteration in kidney oxygen metabolism by normalizing glomerular filtration, which reduces tubular electrolyte load, preventing mitochondrial leak respiration and improving tubular transport efficiency. These improvements could be related to reduced oxidative stress and account for the reduced proteinuria and tubulointerstitial damage. Thus, pharmacologic activation of the HIF system may prevent development of diabetic nephropathy. PMID:25183809

EPI64B Acts as a GTPase-activating Protein for Rab27B in Pancreatic Acinar Cells*

PubMed Central

Hou, Yanan; Chen, Xuequn; Tolmachova, Tatyana; Ernst, Stephen A.; Williams, John A.

2013-01-01

The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells. PMID:23671284

Parental Factors in Children’s Active Transport to School

PubMed Central

Henne, Heather M.; Tandon, Pooja S.; Frank, Larry D.; Saelens, Brian E.

2014-01-01

Objective Identify non-distance factors related to children’s active transport (AT) to school, including parental, home, and environment characteristics. Understanding the factors related to children’s AT to school, beyond distance to school, could inform interventions to increase AT and children’s overall physical activity. Study Design Participants were in the Neighborhood Impact on Kids Study, a longitudinal, observational cohort study of children aged 6 - 11 and their parents in King County, WA and San Diego County, CA between 2007-2009. Parents reported frequency and mode of child transport to school, perceived neighborhood, home and family environments, parental travel behaviors, and sociodemographics. Methods Children living less than a 20 minute walk to school were in this analysis. Children classified as active transporters (walked/bicycled to or from school at least once per week) were compared with those not using AT as often. Results Children using AT were older and had parents who reported themselves using active transport. Having a family rule that restricts the child to stay within sight of the parent or home and more parent working hours was related to lower odds of a child using AT. Conclusions Children’s AT to school is associated with parental AT to work and other locations. Interventions should be considered that enable whole family AT, ameliorate safety concerns and decrease the need for parental supervision, such as walking school buses. PMID:24999161

Carbohydrates and activity of natural and recombinant tissue factor.

PubMed

Krudysz-Amblo, Jolanta; Jennings, Mark E; Mann, Kenneth G; Butenas, Saulius

2010-01-29

The effect of glycosylation on tissue factor (TF) activity was evaluated, and site-specific glycosylation of full-length recombinant TF (rTF) and that of natural TF from human placenta (pTF) were studied by liquid chromatography-tandem mass spectrometry. The amidolytic activity of the TF.factor VIIa (FVIIa) complex toward a fluorogenic substrate showed that the catalytic efficiency (V(max)) of the complex increased in the order rTF(1-243) (Escherichia coli) < rTF(1-263) (Sf9 insect cells) < pTF for the glycosylated and deglycosylated forms. Substrate hydrolysis was unaltered by deglycosylation. In FXase, the K(m) of FX for rTF(1-263)-FVIIa remained unchanged after deglycosylation, whereas the k(cat) decreased slightly. A pronounced decrease, 4-fold, in k(cat) was observed for pTF.FVIIa upon deglycosylation, whereas the K(m) was minimally altered. The parameters of FX activation by both rTF(1-263D)-FVIIa and pTF(D)-FVIIa were identical and similar to those for rTF(1-243)-FVIIa. In conclusion, carbohydrates significantly influence the activity of TF proteins. Carbohydrate analysis revealed glycosylation on asparagines 11, 124, and 137 in both rTF(1-263) and pTF. The carbohydrates of rTF(1-263) contain high mannose, hybrid, and fucosylated glycans. Natural pTF contains no high mannose glycans but is modified with hybrid, highly fucosylated, and sialylated sugars.

Cycles of Ubiquitination and Deubiquitination Critically Regulate Growth Factor-Mediated Activation of Akt Signaling

PubMed Central

Yang, Wei-Lei; Jin, Guoxiang; Li, Chien-Feng; Jeong, Yun Seong; Moten, Asad; Xu, Dazhi; Feng, Zizhen; Chen, Wei; Cai, Zhen; Darnay, Bryant; Gu, Wei; Lin, Hui-Kuan

2013-01-01

K63-linked ubiquitination of Akt is a posttranslational modification that plays a critical role in growth factor-mediated membrane recruitment and activation of Akt. Although E3 ligases involved in growth factor-induced Akt ubiquitination have been defined, the deubiquitinating enzyme (DUB) that triggers deubiquitination of Akt and the function of Akt deubiquitination remain largely unclear. Here, we showed that CYLD was a DUB for Akt and suppressed growth factor-mediated Akt ubiquitination and activation. CYLD directly removed ubiquitin moieties on Akt under serum-starved conditions. CYLD dissociated from Akt upon growth factor stimulation, thereby allowing E3 ligases to induce ubiquitination and activation of Akt. CYLD deficiency also promoted cancer cell proliferation, survival, glucose uptake and growth of prostate tumors. Our findings reveal the crucial role of cycles of ubiquitination and deubiquitination of Akt in its membrane recruitment and activation, and further identifies CYLD as a molecular switch for these processes. PMID:23300340

Activation of protein kinase C by mycobacterial cord factor, trehalose 6-monomycolate, resulting in tumor necrosis factor-alpha release in mouse lung tissues.

PubMed

Sueoka, E; Nishiwaki, S; Okabe, S; Iida, N; Suganuma, M; Yano, I; Aoki, K; Fujiki, H

1995-08-01

Cord factors are mycoloyl glycolipids in cell walls of bacteria belonging to Actinomycetales, such as Mycobacterium, Nocardia and Rhodococcus. They induce granuloma formation in the lung and interstitial pneumonitis, associated with production of macrophage-derived cytokines. We studied how cord factors induce biological activities in the cells. Cord factors isolated from M. tuberculosis, trehalose 6-monomycolate (mTMM) and trehalose 6,6'-dimycolate (mTDM), enhanced protein kinase C (PKC) activation in the presence of phosphatidylserine (PtdSer), diacylglycerol and Ca2+, and mTMM activated PKC alpha more strongly than PKC beta or gamma under the same assay conditions. Kinetic studies of mTMM in response to PKC activation revealed that mTMM increased the apparent affinity of PKC to Ca2+ in the presence of both PtdSer and diolein. Although this is similar to observations with unsaturated fatty acids, such as arachidonic acid, mTMM was synergistic with PtdSer for PKC activation, but arachidonic acid was not. mTMM was also different as regards PKC activation, as phorbol ester was. A single i.p. administration of mTMM to mouse induced tumor necrosis factor-alpha (TNF-alpha) in serum and in the lung, which is a unique target tissue of cord factors. Based on our recent finding that TNF-alpha is an endogenous tumor promoter, the correlation between lung cancer and pulmonary tuberculosis is discussed.

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation

PubMed Central

Lim, Chinten James; Spiegelman, George B.; Weeks, Gerald

2001-01-01

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC– cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC– cells stimulated by 2′-deoxy-cAMP, but is produced in response to GTPγS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC– cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC– cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC– cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC– cells, suggesting that AleA may activate RasC. PMID:11500376

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

PubMed

Lim, C J; Spiegelman, G B; Weeks, G

2001-08-15

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

Activated platelet-derived growth factor β receptor and Ras-mitogen-activated protein kinase pathway in natural bovine urinary bladder carcinomas.

PubMed

Corteggio, Annunziata; Di Geronimo, Ornella; Roperto, Sante; Roperto, Franco; Borzacchiello, Giuseppe

2012-03-01

Bovine papillomavirus types 1 or 2 (BPV-1/2) are involved in the aetiopathogenesis of bovine urinary bladder cancer. BPV-1/2 E5 activates the platelet-derived growth factor β receptor (PDGFβR). The aim of this study was to analyse the Ras/mitogen-activated protein kinase (MAPK) pathway in relation to activation of PDGFβR in natural bovine urinary bladder carcinomas. Co-immunoprecipitation and Western blot analysis demonstrated that recruitment of growth factor receptor bound protein 2 (GRB-2) and Sos-1 to the activated PDGFβR was increased in carcinomas compared to normal tissues. Higher grade bovine urinary bladder carcinomas were associated with activation of Ras, but not with activation of downstream mitogen-activated protein kinase/extracellular signal-regulated kinase (Mek 1/2) or extracellular signal-regulated kinase (Erk 1/2). Copyright © 2011 Elsevier Ltd. All rights reserved.

Nuclear Factor Kappa B Activation and Peroxisome Proliferator-activated Receptor Transactivational Effects of Chemical Components of the Roots of Polygonum multiflorum.

PubMed

Sun, Ya Nan; Li, Wei; Song, Seok Bean; Yan, Xi Tao; Yang, Seo Young; Kim, Young Ho

2016-01-01

Polygonum multiflorum is well-known as "Heshouwu" in traditional Chinese herbal medicine. In Northeast Asia, it is often used as a tonic to prevent premature aging of the kidney and liver, tendons, and bones and strengthening of the lower back and knees. To research the anti-inflammatory activities of components from P. multiflorum. The compounds were isolated by a combination of silica gel and YMC R-18 column chromatography, and their structures were identified by analysis of spectroscopic data (1D, 2D-nuclear magnetic resonance, and mass spectrometry). The anti-inflammatory activities of the isolated compounds 1-15 were evaluated by luciferase reporter gene assays. Fifteen compounds (1-15) were isolated from the roots of P. multiflorum. Compounds 1-5 and 14-15 significantly inhibited tumor necrosis factor-α-induced nuclear factor kappa B-luciferase activity, with IC50 values of 24.16-37.56 μM. Compounds 1-5 also greatly enhanced peroxisome proliferator-activated receptors transcriptional activity with EC50 values of 18.26-31.45 μM. The anthraquinone derivatives were the active components from the roots of P. multiflorum as an inhibitor on inflammation-related factors in human hepatoma cells. Therefore, we suggest that the roots of P. multiflorum can be used to treat natural inflammatory diseases. This study presented that fifteen compounds (1-15) isolated from the roots of Polygonum multiflrum exert signifiant anti inflmmatory effects by inhibiting TNF α induced NF κB activation and PPARs transcription. Abbreviation used: NF κB: Nuclear factor kappa B, PPARs: Peroxisome proliferator activated receptors, PPREs: Peroxisome proliferator response elements, TNF α: Tumor necrosis factor α, ESI-MS: Electrospray ionization mass spectrometry, HepG2: Human hepatoma cells.

The Contribution of Home, Neighbourhood and School Environmental Factors in Explaining Physical Activity among Adolescents

PubMed Central

Haerens, Leen; Craeynest, Mietje; Deforche, Benedicte; Maes, Lea; Cardon, Greet; De Bourdeaudhuij, Ilse

2009-01-01

The present study aimed at investigating the influence of home, neighbourhood and school environmental factors on adolescents' engagement in self-reported extracurricular physical activity and leisure time sports and on MVPA objectively measured by accelerometers. Environmental factors were assessed using questionnaires. Gender specific hierarchical regression analyses were conducted, with demographic variables entered in the first block, and environmental, psychosocial factors and interactions terms entered in the second block. Participation in extracurricular activities at school was positively related to the number of organized activities and the provision of supervision. Perceived accessibility of neighborhood facilities was not related to engagement in leisure time sports, whereas the availability of sedentary and physical activity equipment was. Findings were generally supportive of ecological theories stating that behaviors are influenced by personal and environmental factors that are constantly interacting. PMID:20041023

Evolution of Osteocrin as an activity-regulated factor in the primate brain

PubMed Central

Ataman, Bulent; Boulting, Gabriella L.; Harmin, David A.; Yang, Marty G.; Baker-Salisbury, Mollie; Yap, Ee-Lynn; Malik, Athar N.; Mei, Kevin; Rubin, Alex A.; Spiegel, Ivo; Durresi, Ershela; Sharma, Nikhil; Hu, Linda S.; Pletikos, Mihovil; Griffith, Eric C.; Partlow, Jennifer N.; Stevens, Christine R.; Adli, Mazhar; Chahrour, Maria; Sestan, Nenad; Walsh, Christopher A.; Berezovskii, Vladimir K.; Livingstone, Margaret S.; Greenberg, Michael E.

2017-01-01

Sensory stimuli drive the maturation and function of the mammalian nervous system in part through the activation of gene expression networks that regulate synapse development and plasticity. These networks have primarily been studied in mice, and it is not known whether there are species- or clade-specific activity-regulated genes that control features of brain development and function. Here we use transcriptional profiling of human fetal brain cultures to identify an activity-dependent secreted factor, Osteocrin (OSTN), that is induced by membrane depolarization of human but not mouse neurons. We find that OSTN has been repurposed in primates through the evolutionary acquisition of DNA regulatory elements that bind the activity-regulated transcription factor MEF2. In addition, we demonstrate that OSTN is expressed in primate neocortex and restricts activity-dependent dendritic growth in human neurons. These findings suggest that, in response to sensory input, OSTN regulates features of neuronal structure and function that are unique to primates. PMID:27830782

Role of Bioavailable Iron in Coal Dust-Induced Activation of Activator Protein-1 and Nuclear Factor of Activated T Cells

PubMed Central

Huang, Chuanshu; Li, Jingxia; Zhang, Qi; Huang, Xi

2010-01-01

Activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) are two important transcription factors responsible for the regulation of cytokines, which are involved in cell proliferation and inflammation. Coal workers’ pneumoconiosis (CWP) is an occupational lung disease that may be related to chronic inflammation caused by coal dust exposure. In the present study, we demonstrate that coal from the Pennsylvania (PA) coalmine region, which has a high prevalence of CWP, can activate both AP-1 and NFAT in JB6 mouse epidermal cells. In contrast, coal from the Utah (UT) coalmine region, which has a low prevalence of CWP, has no such effects. The PA coal stimulates mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-Jun-NH2-terminal kinases, as determined by the phosphorylation assay. The increase in AP-1 by the PA coal was completely eliminated by the pretreatment of cells with PD98059, a specific MAPK kinase inhibitor, and SB202190, a p38 kinase inhibitor, further confirming that the PA coal-induced AP-1 activation is mediated through ERKs and p38 MAPK pathways. Deferoxamine (DFO), an iron chelator, synergistically enhanced the PA coal-induced AP-1 activity, but inhibited NFAT activity. For comparison, cells were treated with ferrous sulfate and/or DFO. We have found that iron transactivated both AP-1 and NFAT, and DFO further enhanced iron-induced AP-1 activation but inhibited NFAT. These results indicate that activation of AP-1 and NFAT by the PA coal is through bioavailable iron present in the coal. These data are in agreement with our previous findings that the prevalence of CWP correlates well with levels of bioavailable iron in coals from various mining regions. PMID:12397016

Newly diagnosed congenital factor VII deficiency and utilization of recombinant activated factor VII (NovoSeven®)

PubMed Central

Bartosh, Nicole S; Tomlin, Tara; Cable, Christian; Halka, Kathleen

2013-01-01

This case report presents a newly diagnosed congenital factor VII deficiency treated with recombinant activated factor VII (rFVIIa). Congenital factor VII deficiency is a rare autosomal-recessive bleeding disorder that occurs in fewer than 1/500,000 persons. Its presentation can vary from epistaxis to hemarthroses and severe central nervous system bleeding, and correlates poorly with factor VII levels. Our patient had not had a significant hemostatic challenge prior to his presentation and therefore never had any symptomatology suggestive of this disease. He was treated with rFVIIa, and was able to undergo repair of his fractures without bleeding. Case report A 19-year-old African-American male presented to the emergency room after an altercation that resulted in significant trauma. He sustained bilateral mandibular angle fractures and orbital floor fractures, requiring urgent surgical correction. On initial evaluation, he was noted to have a prolonged prothrombin time of 40.1 seconds, with an International Normalized Ratio of 4.0, a normal activated partial thromboplastin time of 29.9 seconds, and a platelet count of 241. After receiving vitamin K and fresh frozen plasma, he was taken to the operating room for a temporary rigid maxillomandibular fixation. A 1:1 mixing study with normal plasma corrected the prothrombin time (decreasing from 40.7 to 14.7 seconds) and a factor VII assay revealed 5% of the normal factor VII level. The patient was diagnosed with congenital factor VII deficiency. Due to his coagulopathy and the extensive surgical correction needed, rFVIIa was administered and surgery was accomplished without hemorrhagic sequelae. Conclusion This case report and review describes a rare congenital disease, the history of rFVIIa use, and its mechanism. rFVIIA use in our patient provided a treatment option that allowed the necessary surgical correction, but further prospective studies on dose optimization would ensure adequate dosing with minimal risk of

Alkyl isothiocyanates suppress epidermal growth factor receptor kinase activity but augment tyrosine kinase activity.

PubMed

Nomura, Takahiro; Uehara, Yoshimasa; Kawajiri, Hiroo; Ryoyama, Kazuo; Yamori, Takao; Fuke, Yoko

2009-10-01

We have reported the in vitro and in vivo anticancer activities of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) derived from a Japanese spice, wasabi. In order to obtain some clues about the mechanism of the anticancer activity, we have studied the effect of alkyl isothiocyanates (MITCs) on protein kinase activities. The anti-autophosphorylation activity of MITCs with respect to the epidermal growth factor (EGF)-stimulated receptor kinase of A431 epidermoid carcinoma cells was examined by incorporation of radioactive ATP into an acid-insoluble fraction. Their anti-phosphorylation activity with respect to the non-receptor protein kinase was analyzed by a standard SDS-PAGE method. All the tested MITCs interfered with the EGF-stimulated receptor kinase activity in a dose-dependent manner, although their effects were less than 1/10 of that of erbstatin in microg/ml. On the other hand, the MITCs did not interfere with non-receptor kinases (kinase A, kinase C, tyrosine kinase and calmodulin dependent kinase III), but enhanced non-receptor tyrosine kinase. A possible anticancer mechanism of MITCs may involve the suppression of EGF receptor kinase activity and augmentation of non-receptor PTK.

Perceived influence of intrinsic/extrinsic factors on participation in life activities after spinal cord injury.

PubMed

Cobb, John E; Leblond, Jean; Dumont, Frédéric S; Noreau, Luc

2018-04-03

Various types of limitations on community participation are experienced by people with spinal cord injury (SCI). To determine: 1) the perceived influence of six intrinsic/extrinsic factors (i.e. physical impairment, emotional condition, thinking skills, environment, lack of assistance, discrimination) on participation in 26 life activities, 2) if this influence varied based on extent of participation, and 3) if personal or environmental characteristics influenced perceptions. Secondary analysis of a cohort (SCI Community Survey, n = 1508) using the SCI Person-Perceived Participation in Daily Activities Questionnaire. Frequency tables, Fisher's exact tests and correspondence analyses. Respectively, 79.6% and 38.5% of respondents perceived that their physical impairment and the natural and/or built environment were the main factors that limited participation across all activities. Considering participation between three groups (no participation; less than wanted; as much as wanted), significant differences (p < 0.001) of perceptions were observed in 65% of the combinations (26 activities x 6 factors). The hypothesis that respondents who did not participate would perceive the highest proportion of limitations was confirmed in 41% of the combinations. Perceived influence of the intrinsic/extrinsic factors on participation was not significantly influenced by other personal or environmental characteristics. A majority of people with SCI perceived that their participation is limited by one or more of intrinsic/extrinsic factors. Perceptions regarding which factors influence participation differ between activities and these perceptions appear related to the extent of participation suggesting that those who actively participate could be the most sensitive to limitations in certain activities. Copyright © 2018 Elsevier Inc. All rights reserved.

FACTORS AFFECTING TIME-ACTIVITY BUDGETS OF BUFFLEHEAD WINTERING IN NARRAGANSETT, BAY, RI

EPA Science Inventory

Daily activities of wintering waterfowl can be influenced by the physical environment and by habitat factors such as prey abundance and availability. We examined variability in diurnal activity budgets of Bufflehead (Bucephala albeola) wintering at seven locations within Narragan...

Physical and psychosocial factors associated with physical activity in patients with chronic obstructive pulmonary disease.

PubMed

Hartman, Jorine E; Boezen, H Marike; de Greef, Mathieu H; Ten Hacken, Nick H

2013-12-01

To assess physical activity and sitting time in patients with chronic obstructive pulmonary disease (COPD) and to investigate which physical and psychosocial factors are associated with physical activity and sitting time. Cross-sectional study. Patients were recruited at outpatient clinics of general hospitals and from general practitioners. Patients (N=113) with mild to very severe COPD. Not applicable. Physical activity and sitting time were measured with a triaxial accelerometer (24h/d). Mean locomotion time per 24 hours was 6.8% (range, 0.7%-20.4%). Elevated physical activity was independently associated with higher self-efficacy, higher functional exercise capacity, and lower lung hyperinflation. Decreased physical activity was strongest in more severe stages of COPD, in which the patients were mainly limited by physical disease-specific factors (higher lung hyperinflation, worse dyspnea severity, worse leg muscle function, and oxygen use). In less severe patients, physical activity was independently associated with more generic factors (higher self-efficacy and the spring/summer season). Sitting time did not differ between severity stages, and longer sitting time in the total group was independently associated with more positive perception of treatment control, less autonomous motivation to exercise, not using sleep medication, and oxygen use. Both physical and psychosocial factors were associated with physical activity in patients with COPD. The factors associated with physical activity differed between disease severity stages, raising the question of whether physical activity enhancement programs should differ as well. Sitting time should be investigated further. Copyright © 2013 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.

Role of exchange protein directly activated by cAMP (EPAC1) in breast cancer cell migration and apoptosis.

PubMed

Kumar, Naveen; Gupta, Sonal; Dabral, Surbhi; Singh, Shailja; Sehrawat, Seema

2017-06-01

Despite the current progress in cancer research and therapy, breast cancer remains the leading cause of mortality among half a million women worldwide. Migration and invasion of cancer cells are associated with prevalent tumor metastasis as well as high mortality. Extensive studies have powerfully established the role of prototypic second messenger cAMP and its two ubiquitously expressed intracellular cAMP receptors namely the classic protein kinaseA/cAMP-dependent protein kinase (PKA) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) in cell migration, cell cycle regulation, and cell death. Herein, we performed the analysis of the Cancer Genome Atlas (TCGA) dataset to evaluate the essential role of cAMP molecular network in breast cancer. We report that EPAC1, PKA, and AKAP9 along with other molecular partners are amplified in breast cancer patients, indicating the importance of this signaling network. To evaluate the functional role of few of these proteins, we used pharmacological modulators and analyzed their effect on cell migration and cell death in breast cancer cells. Hence, we report that inhibition of EPAC1 activity using pharmacological modulators leads to inhibition of cell migration and induces cell death. Additionally, we also observed that the inhibition of EPAC1 resulted in disruption of its association with the microtubule cytoskeleton and delocalization of AKAP9 from the centrosome as analyzed by in vitro imaging. Finally, this study suggests for the first time the mechanistic insights of mode of action of a primary cAMP-dependent sensor, Exchange protein activated by cAMP 1 (EPAC1), via its interaction with A-kinase anchoring protein 9 (AKAP9). This study provides a new cell signaling cAMP-EPAC1-AKAP9 direction to the development of additional biotherapeutics for breast cancer.

A structural study of the complex between neuroepithelial cell transforming gene 1 (Net1) and RhoA reveals a potential anticancer drug hot spot.

PubMed

Petit, Alain-Pierre; Garcia-Petit, Christel; Bueren-Calabuig, Juan A; Vuillard, Laurent M; Ferry, Gilles; Boutin, Jean A

2018-06-08

The GTPase RhoA is a major player in many different regulatory pathways. RhoA catalyzes GTP hydrolysis, and its catalysis is accelerated when RhoA forms heterodimers with proteins of the guanine nucleotide exchange factor (GEF) family. Neuroepithelial cell transforming gene 1 (Net1) is a RhoA-interacting GEF implicated in cancer, but the structural features supporting the RhoA/Net1 interaction are unknown. Taking advantage of a simple production and purification process, here we solved the structure of a RhoA/Net1 heterodimer with X-ray crystallography at 2-Å resolution. Using a panel of several techniques, including molecular dynamics simulations, we characterized the RhoA/Net1 interface. Moreover, deploying an extremely simple peptide-based scanning approach, we found that short peptides (penta- to nonapeptides) derived from the protein/protein interaction region of RhoA could disrupt the RhoA/Net1 interaction and thereby diminish the rate of nucleotide exchange. The most inhibitory peptide, EVKHF, spanning residues 102-106 in the RhoA sequence, displayed an IC 50 of ∼100 μm without further modifications. The peptides identified here could be useful in further investigations of the RhoA/Net1 interaction region. We propose that our structural and functional insights might inform chemical approaches for transforming the pentapeptide into an optimized pseudopeptide that antagonizes Net1-mediated RhoA activation with therapeutic anticancer potential. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Theoretical and vibrational study of N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)-quinazolin-4-amine (gefitinib)

NASA Astrophysics Data System (ADS)

Mıhçıokur, Özlem; Özpozan, Talat

2015-12-01

N-(3-chloro-4fluoro-phenyl)-7-methoxy-6-(3-morpholin-4ylpropoxy)-quinazolin-4-amine (GEF), a quinalizoline derivative used as new anti-cancer agent, designed to target activity of epidermal growth factor receptor (EGFR) promoting the growth, division and spread of cancer cells, was examined from the vibrational and theoretical point of view. All calculations have been carried out both in gaseous and aqueous phases. In the calculations of both phases, the molecule has been optimized through conformer analysis beginning with the x-ray data. The conformer analyses have been carried out in each phases and the geometrical differences between the most stable structures in gaseous and in aqueous phases have been discussed. The solvent effect for GEF in aqueous solution was simulated by using self-consistent reaction field (SCRF) calculations employing the integral equation formalism variant (IEFPCM) model. NBO analysis has been performed to indicate the presence of intramolecular charge transfer. The complete assignments of the vibrational spectra (IR&Raman) were made with the aid of calculated spectra both in gaseous and aqueous phases. The observed spectral data of the title compound were compared with the calculated spectra obtained by DFT/B3LYP and DFT/B3PW91 methods using 6-31G(d,p) basis set. The theoretical results were found to be in good agreement with the measured experimental data especially for the interpretation of intra molecular interactions.

Fibroblast growth factor represses Smad-mediated myofibroblast activation in aortic valvular interstitial cells

PubMed Central

Cushing, Melinda C.; Mariner, Peter D.; Liao, Jo-Tsu; Sims, Evan A.; Anseth, Kristi S.

2008-01-01

This study aimed to identify signaling pathways that oppose connective tissue fibrosis in the aortic valve. Using valvular interstitial cells (VICs) isolated from porcine aortic valve leaflets, we show that basic fibroblast growth factor (FGF-2) effectively blocks transforming growth factor-β1 (TGF-β1)-mediated myofibroblast activation. FGF-2 prevents the induction of α-smooth muscle actin (αSMA) expression and the exit of VICs from the cell cycle, both of which are hallmarks of myofibroblast activation. By blocking the activity of the Smad transcription factors that serve as the downstream nuclear effectors of TGF-β1, FGF-2 treatment inhibits fibrosis in VICs. Using an exogenous Smad-responsive transcriptional promoter reporter, we show that Smad activity is repressed by FGF-2, likely an effect of the fact that FGF-2 treatment prevents the nuclear localization of Smads in these cells. This appears to be a direct effect of FGF signaling through mitogen-activated protein kinase (MAPK) cascades as the treatment of VICs with the MAPK/extracellular regulated kinase (MEK) inhibitor U0126 acted to induce fibrosis and blocked the ability of FGF-2 to inhibit TGF-β1 signaling. Furthermore, FGF-2 treatment of VICs blocks the development of pathological contractile and calcifying phenotypes, suggesting that these pathways may be utilized in the engineering of effective treatments for valvular disease.—Cushing, M. C., Mariner, P. D., Liao, J. T., Sims, E. A., Anseth, K. S. Fibroblast growth factor represses Smad-mediated myofibroblast activation in aortic valvular interstitial cells. PMID:18218921

The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3 and 5 and mediates nuclear factor-kappaB activation.

PubMed Central

Brown, Sharron A N; Richards, Christine M; Hanscom, Heather N; Feng, Sheau-Line Y; Winkles, Jeffrey A

2003-01-01

Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory kappaBalpha phosphorylation and transcriptional activation of a nuclear factor-kappaB (NF-kappaB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-kappaB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-kappaB transcription factor signalling pathway. PMID:12529173

Synthesis and characterization of (18)F-labeled active site inhibited factor VII (ASIS).

PubMed

Erlandsson, Maria; Nielsen, Carsten H; Jeppesen, Troels E; Kristensen, Jesper B; Petersen, Lars C; Madsen, Jacob; Kjaer, Andreas

2015-05-15

Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an (18)F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[(18)F]fluorobenzoate, and the [(18)F]ASIS was purified on a PD-10 desalting column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [(18)F]ASIS to TF and to a specific anti-factor VII antibody (F1A2-mAb). No significant difference in binding efficacy between [(18)F]ASIS and ASIS could be detected. Furthermore, [(18)F]ASIS was relatively stable in vitro and in vivo in mice. In conclusion, [(18)F]ASIS has for the first time been successfully synthesized as a possible positron emission tomography tracer to image TF expression levels. In vivo positron emission tomography studies to evaluate the full potential of [(18)F]ASIS are in progress. Copyright © 2015 John Wiley & Sons, Ltd.

Polymorphisms of the factor VII gene associated with the low activities of vitamin K-dependent coagulation factors in one-month-old infants.

PubMed

Ito, Koichi; Goto, Kenji; Sugiura, Tokio; Muramatsu, Kanji; Ando, Toshihiro; Maniwa, Hiroko; Yokoyama, Takao; Sugiyama, Kohachiro; Togari, Hajime

2007-01-01

Despite administration of vitamin K (VK), some infants show lower activity of VK-dependent coagulation factors and they could develop intracranial hemorrhage. For preventing VK deficiency bleeding (VKDB) in infants, oral administration of VK and a screening test for VK deficiency are carried out in Japan. For the screening, the total activity of VK-dependent coagulation factors is measured using a commercial product, Normotest. This study was undertaken to clarify the importance of the following genetic and environmental factors on the coagulation status in one-month-old infants: two polymorphisms in the factor VII gene, -323P0/10 (a 10-bp insertion in the promoter region at position -323) and R353Q (the replacement of arginine [R] with glutamine [Q] at residue 353) and sex, age, gestational age, birth weight, and feeding regimen. Two hundred Japanese infants (34.6 +/- 4.0 days old) were screened for VK-dependent coagulation activity with Normotest and were genotyped for the two polymorphisms. Among the subjects screened, 18 infants (9%) carried the P10 allele and 26 (13%) carried the R353Q allele. Multiple regression analysis showed that the 10-bp inserted (P10) allele or the Q allele was associated with the lower coagulation activities. The coagulation activities for the R/Q genotype were significantly lower than those for the R/R genotype and those for the P0/P10 genotype were significantly lower than those for the P0/P0 genotype. Therefore, infants who carry the P10 allele or the Q allele show lower activity of VK-dependent coagulation factors. These infants may have a higher risk of VKDB manifestation.

Using avian radar to examine relationships among avian activity, bird strikes, and meteorological factors